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Stewart Kim, M., Racaniello VR. Barral PM, Morrison, JM, Drahos J, Gupta P, Sarkar D, Fisher, PB, Racaniello VR. Kauder S, Kan S, Racaniello VR. Rosenfeld AB, Racaniello VR Harris
JR, Racaniello VR Kauder,
S.E., Racaniello, V.R. Brown DM, Kauder SE, Cornell CT, Jang GM, Racaniello
VR, Semler BL. Hughes,
S.A., Thaker, H.M., Racaniello, V.R. Flint, S.J., Enquist, L., Racaniello,
V.R., and Skalka, A.M. Béatrice
Baury, B., D. Masson, B. M. McDermott Jr., A. Jarry, H. M. Blottière,
C L. Laboisse, P. Lustenberger, V. R. Racaniello, M.G. Denis. Harris
JR, Racaniello VR Tsang, SK, McDermott, BM, Racaniello, VR, Hogle, JM Racaniello, V.R. Racaniello VR. McDermott BM Jr, Rux AH, Eisenberg RJ, Cohen GH, Racaniello VR. Bouchard MJ, Dong Y, McDermott BM Jr, Lam DH, Brown KR, Shelanski M, Bellve AR, Racaniello VR. Dove AW, Racaniello VR. Belnap DM, McDermott BM Jr, Filman DJ, Cheng N, Trus BL, Zuccola HJ, Racaniello VR, Hogle JM, Steven AC. Flint, S.J., Enquist, L., Krug, R.M., Racaniello, V.R., and Skalka, A.M. Shukla D, Rowe CL, Dong Y, Racaniello VR, Spear PG. Deatly AM, Taffs RE, McAuliffe JM, Nawoschik SP, Coleman JW, McMullen G, Weeks-Levy C, Johnson AJ, Racaniello VR. Zhang S, Racaniello VR. Dove AW, Racaniello VR. Zhang S, Racaniello VR. Dove AW, Racaniello VR. Gutierrez AL, Denova-Ocampo M, Racaniello VR, del Angel RM. Bouchard MJ, Racaniello VR. Gutierrez-Escolano AL, Medina F, Racaniello VR, Del Angel RM. Racaniello VR. Racaniello VR. Racaniello VR, Ren R. Bouchard MJ, Lam DH, Racaniello VR. Colston EM, Racaniello VR. Weeks-Levy, C., E.J. Gorgacz and V. Racaniello Racaniello, V.R. Colston E, Racaniello VR. Morrison ME, He YJ, Wien MW, Hogle JM, Racaniello VR. Shepley MP, Racaniello VR. Racaniello VR, Ren R. Racaniello VR. Gomez Yafal A, Kaplan G, Racaniello VR, Hogle JM. Pipkin PA, Wood DJ, Racaniello VR, Minor PD. Racaniello VR, Ren R, Bouchard M. Racaniello, V.R. Ren R, Racaniello VR. Morrison ME, Racaniello VR. Tatem JM, Weeks-Levy C, Georgiu A, DiMichele SJ, Gorgacz EJ, Racaniello VR, Cano FR, Mento SJ. Blackburn RV, Racaniello VR, Righthand VF. Ren R, Racaniello VR. Racaniello, V.R. Racaniello VR. Freistadt MS, Racaniello VR. Racaniello VR. Moss EG, Racaniello VR. Kaplan G, Racaniello VR. Ren RB, Moss EG, Racaniello VR. Racaniello VR. Racaniello, V.R. Kaplan G, Peters D, Racaniello VR. Freistadt MS, Kaplan G, Racaniello VR. Ren RB, Costantini F, Gorgacz EJ, Lee JJ, Racaniello VR. Kaplan G, Freistadt MS, Racaniello VR. Racaniello VR. O'Neill RE, Racaniello VR. del Angel RM, Papavassiliou AG, Fernandez-Tomas C, Silverstein SJ, Racaniello VR. Skinner MA, Racaniello VR, Dunn G, Cooper J, Minor PD, Almond JW. La Monica N, Racaniello VR. Moss EG, O'Neill RE, Racaniello VR. Mendelsohn CL, Wimmer E, Racaniello VR. Kaplan G, Levy A, Racaniello VR. Murray MG, Bradley J, Yang XF, Wimmer E, Moss EG, Racaniello VR. Pelletier J, Kaplan G, Racaniello VR, Sonenberg N. Kaplan G, Racaniello VR. Pelletier J, Kaplan G, Racaniello VR, Sonenberg N. Racaniello VR. La Monica N, Kupsky WJ, Racaniello VR. La Monica N, Almond JW, Racaniello VR. Racaniello VR. Racaniello VR, Meriam C. Mendelsohn C, Johnson B, Lionetti KA, Nobis P, Wimmer E, Racaniello VR. Lubinski JM, Kaplan G, Racaniello VR, Dasgupta A. La Monica N, Meriam C, Racaniello VR. Kaplan G, Lubinski J, Dasgupta A, Racaniello VR. Postdoctoral Publications of V. Racaniello: Levenson R, Racaniello VR, Albritton L, Housman D. Racaniello VR. Ticehurst JR, Racaniello VR, Baroudy BM, Baltimore D, Purcell RH, Feinstone SM. Racaniello VR, Baltimore D. Lemischka IR, Farmer S, Racaniello VR, Sharp PA. Racaniello VR, Baltimore D. Ph.D. Thesis Publications of V. Racaniello: Palese P, Racaniello VR, Desselberger U, Young J, Baez M. Desselberger U, Racaniello VR, Zazra JJ, Palese P. Racaniello VR, Palese P. Racaniello VR, Palese P. Racaniello, V.R., and P. Palese. J.
Virol. 79:10126-10137. Amino Acid Changes in Proteins 2B and 3A Mediate Rhinovirus
Type 39 Growth in Mouse Cells J
Clin Invest. 2004 113:1743-53. Poliovirus tropism and attenuation are determined after internal
ribosome entry. Cell-dependent role for the poliovirus 3' noncoding region
in positive-strand RNA synthesis. Proc. Natl. Acad. Sci. USA 2003 100(26):15906-11. Identification of CD155 isoforms. Changes in rhinovirus protein 2C allow efficient replication
in mouse cells Kinetic Analysis of the Effect of Poliovirus Receptor on Viral Uncoating: the Receptor as a Catalyst We
examined the role of soluble poliovirus receptor on the transition
of native poliovirus (160S or N particle) to an infectious intermediate
(135S or A particle). The viral receptor behaves as a classic
transition
state theory catalyst, facilitating the N-to-A conversion by
lowering the activation energy for the process by 50 kcal/mol.
In contrast to earlier studies which demonstrated that capsid-binding
drugs inhibit
thermally mediated N-to-A conversion through entropic stabilization
alone, capsid-binding drugs are shown to inhibit receptor-mediated
N-to-A conversion through a combination of enthalpic and entropic effects.
J Biol Chem 2000 Jul 28;275(30):23089-96 Two distinct binding affinities of poliovirus for its cellular receptor. To study the kinetics and equilibrium of poliovirus binding to the poliovirus receptor, we used surface plasmon resonance to examine the interaction of a soluble form of the receptor with poliovirus. Soluble receptor purified from mammalian cells is able to bind poliovirus, neutralize viral infectivity, and induce structural changes in the virus particle. Binding studies revealed that there are two binding sites for the receptor on the poliovirus type 1 capsid, with affinity constants at 20 degrees C of K(D)(1) = 0.67 microm and K(D)(2) = 0.11 microm. The relative abundance of the two binding sites varies with temperature. At 20 degrees C, the K(D)(2) site constitutes approximately 46% of the total binding sites on the sensor chip, and its relative abundance decreased with decreasing temperature such that at 5 degrees C, the relative abundance of the K(D)(2) site is only 12% of the total binding sites. Absolute levels of the K(D)(1) site remained relatively constant at all temperatures tested. The two binding sites may correspond to docking sites for domain 1 of the receptor on the viral capsid, as predicted by a model of the poliovirus-receptor complex. Alternatively, the binding sites may be a consequence of structural breathing, or could result from receptor-induced conformational changes in the virus.
Mol Cell Biol 2000 Apr;20(8):2865-73 Defects in nuclear and cytoskeletal morphology and mitochondrial localization in spermatozoa of mice lacking nectin-2, a component of cell-cell adherens junctions. Nectin-2 is a cell adhesion molecule encoded by a member of the poliovirus receptor gene family. This family consists of human, monkey, rat, and murine genes that are members of the immunoglobulin gene superfamily. Nectin-2 is a component of cell-cell adherens junctions and interacts with l-afadin, an F-actin-binding protein. Disruption of both alleles of the murine nectin-2 gene resulted in morphologically aberrant spermatozoa with defects in nuclear and cytoskeletal morphology and mitochondrial localization. Homozygous null males are sterile, while homozygous null females, as well as heterozygous males and females, are fertile. The production by nectin-2(-/-) mice of normal numbers of spermatozoa containing wild-type levels of DNA suggests that Nectin-2 functions at a late stage of germ cell development. Consistent with such a role, Nectin-2 is expressed in the testes only during the later stages of spermatogenesis. The structural defects observed in spermatozoa of nectin-2(-/-) mice suggest a role for this protein in organization and reorganization of the cytoskeleton during spermiogenesis.
J Virol 2000 Apr;74(8):3929-31 An antiviral compound that blocks structural transitions of poliovirus prevents receptor binding at low temperatures. Drugs such as WIN51711 that inhibit picornavirus replication are thought to block poliovirus infectivity by binding to the capsid and preventing structural transitions required for uncoating. We examined the activity of WIN51711 at temperatures where capsid flexibility is thought to be decreased. Below 37 degrees C, WIN51711 inhibits the binding of wild-type poliovirus to cells but does not affect the binding of a poliovirus mutant which is believed to undergo structural transitions more readily. These results suggest that the poliovirus capsid must undergo structural changes to bind to its cellular receptor.
Proc Natl Acad Sci U S A 2000 Jan 4;97(1):73-8 Three-dimensional structure of poliovirus receptor bound to poliovirus. Poliovirus
initiates infection by binding to its cellular receptor (Pvr).
We have studied this interaction by using cryoelectron microscopy to
determine
the structure, at 21-A resolution, of poliovirus complexed
with a soluble form of its receptor (sPvr). This density map aided construction
of
a homology-based model of sPvr and, in conjunction with the
known crystal structure of the virus, allowed delineation of the binding
site. The
virion does not change significantly in structure on binding
sPvr in short incubations at 4 degrees C. We infer that the binding
configuration
visualized represents the initial interaction that is followed
by structural changes in the virion as infection proceeds. sPvr is segmented
into
three well-defined Ig-like domains. The two domains closest
to the virion (domains 1 and 2) are aligned and rigidly connected, whereas
domain
3 diverges at an angle of approximately 60 degrees. Two nodules
of density on domain 2 are identified as glycosylation sites. Domain
1 penetrates
the "canyon" that surrounds the 5-fold protrusion on the capsid
surface, and its binding site involves all three major capsid
proteins. The inferred pattern of virus-sPvr interactions accounts for
most mutations
that affect the binding of Pvr to poliovirus.
The murine homolog (Mph) of human herpesvirus entry protein B (HveB) mediates entry of pseudorabies virus but not herpes simplex virus types 1 and 2. Department of Microbiology-Immunology, Northwestern University Medical School, Chicago, Illinois 60611, USA.
Microb Pathog 1998 Jul;25(1):43-54 Characterization of mouse lines transgenic with the human poliovirus receptor gene. Two mouse lines transgenic with the human poliovirus receptor gene (PVR), TGM-PRG-1 and TGM-PRG-3, were characterized to determine whether transgene copy number and PVR expression levels influence susceptibility to poliovirus. The mouse lines have been bred for more than 10 generations and the transgene was stably transmitted to progeny as determined by Southern blot hybridization and restriction fragment length polymorphism. The transgene copy number is 10 in the TGM-PRG-3 mouse line and one in the TGM-PRG-1 mouse line. Abundance of PVR RNA is on average three-fold higher in TGM-PRG-3 relative to TGM-PRG-1 tissues, and the abundance of the receptor molecule is three-fold higher in TGM-PRG-3 central nervous system tissues compared to TGM-PRG-1 tissues as determined by Western blot analysis. When TGM-PRG-1 and TGM-PRG-3 mice were inoculated intracranially with a neurovirulent type III poliovirus strain, they developed clinical symptoms and CNS lesions characteristic of human poliomyelitis. These results indicate that the PVR gene is expressed as a functional receptor in the CNS of both mouse lines rendering the mice susceptible to poliovirus infection. Even though the two mouse lines have different copy numbers of the transgene and different levels of PVR RNA and protein, they are similar in their susceptibility to poliovirus.
Virology 1997 Sep 1;235(2):293-301 Related Articles, Books, LinkOut Persistent echovirus infection of mouse cells expressing the viral receptor VLA-2. Mouse cells are not susceptible to infection with echovirus 1 (EV-1) because they lack the viral receptor, human VLA-2. Two mouse fibroblast cell lines, L cells and 3T3 cells, were made susceptible to EV-1 infection after transformation with cDNAs of human VLA-2. After EV-1 infection, L cell transformants of human VLA-2 (alpha2beta1 L cells) develop cytopathic effect (CPE) as expected, while 3T3 cell transformants of human VLA-2 (alpha2beta1 3T3 cells) or the alpha2 subunit of human VLA-2 (alpha2 3T3 cells) become persistently infected. The distinct outcome is not a result of differential virus growth on these transformants because one-step growth curve analysis reveals little difference in EV-1 replication in both cell lines. In addition, 3T3 cell transformants expressing the poliovirus receptor (Pvr 3T3 cells) are lysed during poliovirus infection, suggesting that 3T3 cells are not intrinsically resistant to CPE caused by enterovirus infection. The results of limit dilution assays indicate that all EV-1-infected alpha2 3T3 cells produce infectious virus. All EV-1-infected alpha2 3T3 cells remain viable after EV-1 infection, and the kinetics of cell growth were not altered. FACS analysis reveals that receptor down-regulation is not involved in the establishment of persistent infection. Furthermore, inhibition of host protein synthesis was not observed in EV-1-infected alpha2 3T3 or alpha2beta1 L cells. Since alpha2beta1 L cells are lysed by EV-1 infection, these findings suggest that virus-induced translation inhibition is not a determinant of cell killing.
Science. 1997 Aug 8;277(5327):779-80. The polio eradication effort: should vaccine eradication be next? The World Health Organization (WHO) has implemented a plan to eradicate poliovirus that is widely viewed as having made enormous progress. If all goes as planned, polio will be eradicated on this planet by the year 2003. However, there is a debate, as highlighted in a pair of Policy Forums in this issue, over when vaccination against polio can be stopped. Dove and Racaniello believe that the reliance of the WHO on the live Sabin oral poliovirus vaccine (OPV) means that there will be a continuing threat of release of potentially pathogenic virus into the environment. They are also concerned that the planned destruction of all wild-type polio stocks will not be possible. They therefore recommend a switch to the inactivated polio vaccine (IPV). In response, Hull and Aylward explain why a switch from OPV is not necessary and describe the studies being sponsored by the WHO to determine how and when immunization can safely be ended.
J Virol 1997 Jul;71(7):4915-20. Expression of the poliovirus receptor in intestinal epithelial cells is not sufficient to permit poliovirus replication in the mouse gut. Although the initial site of poliovirus replication in humans is the intestine, previously isolated transgenic mice which carry the human poliovirus receptor (PVR) gene (TgPVR mice), which develop poliomyelitis after intracerebral inoculation, are not susceptible to infection by the oral route. The low levels of PVR expressed in the TgPVR mouse intestine might explain the absence of poliovirus replication at that site. To ascertain whether PVR is the sole determinant of poliovirus susceptibility of the mouse intestine, we have generated transgenic mice by using the promoter for rat intestine fatty acid binding protein to direct PVR expression in mouse gut. Pvr was detected by immunohistochemistry in the enterocytes and M cells of transgenic mouse (TgFABP-PVR) small intestine. Upon oral inoculation with poliovirus, no increase in virus titer was detected in the feces of TgFABP-PVR mice, and no virus replication was observed in the small intestine, although poliovirus replicated in the brain after intracerebral inoculation. The failure of poliovirus to replicate in the TgFABP-PVR mouse small intestine was not due to lack of virus binding sites, because poliovirus could attach to fragments of small intestine from these mice. These results indicate that the inability of poliovirus to replicate in the mouse alimentary tract is not solely due to the absence of virus receptor, and other factors are involved in determining the ability of poliovirus to replicate in the mouse gut.
Cold-adapted poliovirus mutants bypass a postentry replication block. In the current model of poliovirus entry, the initial interaction of the native virion with its cellular receptor is followed by a transition to an altered form, which then acts as an intermediate in viral entry. While the native virion sediments at 160S in a sucrose gradient, the altered particle sediments at 135S, has lost the coat protein VP4, and has become more hydrophobic. Altered particles can be found both associated with cells and in the culture medium. It has been hypothesized that the cell-associated 135S particle releases the viral genome into the cell cytoplasm and that nonproductive transitions to the 135S form are responsible for the high particle-to-PFU ratio observed for polioviruses. At 25 degrees C, a temperature at which the transition to 135S particles does not occur, the P1/Mahoney strain of poliovirus was unable to replicate, and cold-adapted (ca) mutants were selected from the population. These mutants have not gained the ability to convert to 135S particles at 25 degrees C, and the block to wild-type (wt) infection at low temperatures is not at the level of cellular entry. The particle-to-PFU ratio of poliovirus does not change at 25 degrees C in the absence of alteration. Three independent amino acid changes in the 2C coding region were identified in ca mutants, at positions 218 (Val to Ile), 241 (Arg to Ala), and 309 (Met to Val). Introduction of any of these mutations individually into wt poliovirus by site-directed mutagenesis confers the ca phenotype. All three serotypes of the Sabin vaccine strains and the P3/Leon strain of poliovirus also exhibit the ca phenotype. These results do not support a model of poliovirus entry into cells that includes an obligatory transition to the 135S particle.
Attenuating mutations in the poliovirus 5' untranslated region alter its interaction with polypyrimidine tract-binding protein. Mutations in the 5' untranslated regions (5'-UTRs) of all three serotypes of the Sabin vaccine strains are known to be major determinants of the attenuation phenotype. To further understand the functional basis of the attenuation phenotype caused by mutations in the 5'-UTR, we studied their effects on viral replication, translation, and the interaction of the viral RNA with cell proteins. A mutation at base 472 (C472U), which attenuates neurovirulence in primates and mice, was previously found to reduce viral replication and translation in neuroblastoma cells but not in HeLa cells. This mutation reduced cross-linking of the poliovirus 5'-UTR to polypyrimidine tract-binding protein (pPTB) in neuroblastoma cells but not in HeLa cells. These defects were absent in a neurovirulent virus with C at nucleotide 472. When C472U and an additional mutation, G482A, were introduced into the 5'-UTR, the resulting virus was more attenuated, had a replication and translation defect in both HeLa cells and neuroblastoma cells, and cross-linked poorly to pPTB from both cell types. A neurovirulent revertant of this virus (carrying U472C, G482A, and C529U) no longer had a replication defect in HeLa and SH-SY5Y cell lines and cross-linked with pPTB to wild-type levels. The results suggest that the attenuating effects of the mutation C472U may result from an impaired interaction of the 5'-UTR with pPTB in neural cells, which reduces viral translation and replication. Introduction of a second mutation, G482A, into the 5'-UTR extends this defect to HeLa cells.
CD44 is not required for poliovirus replication. The identification of a monoclonal antibody, AF3, which recognizes a single isoform of the cell surface protein CD44 and preferentially blocks binding of serotype 2 poliovirus to HeLa cells, suggested that CD44 might be an accessory molecule to Pvr, the cell receptor for poliovirus, and that it could play a role in the function of the poliovirus receptor site. We show here that only AF3 blocks binding of serotype 2 poliovirus to HeLa cells and, in contrast to a previously published report, that the anti-CD44 monoclonal antibodies A3D8 and IM7 are unable to block binding of poliovirus. To determine whether CD44 is involved in poliovirus infection, we analyzed the replication of all three serotypes of poliovirus in human neuroblastoma cells which lack or express CD44 and in mouse neuroblastoma cells which lack Pgp-1, the mouse homolog of human CD44, and which express Pvr. All three poliovirus serotypes replicate with normal kinetics and to normal levels in the absence or presence of CD44 or in the absence of Pgp-1. Furthermore, the binding affinity constants of all three poliovirus serotypes for Pvr are unaffected by the presence or absence of CD44 in the human neuroblastoma cell line. We conclude that CD44 and Pgp-1 are not required for poliovirus replication and are unlikely to be involved in poliovirus pathogenesis.
Virology 1997 Jan 20;227(2):505-8 Differences in the UV-crosslinking patterns of the poliovirus 5'-untranslated region with cell proteins from poliovirus-susceptible and -resistant tissues. The restricted tissue tropism observed in poliovirus infection is not governed solely by the expression of the poliovirus receptor (PVR) gene, but might be controlled at stages beyond virus entry, such as translation, replication, or assembly. Translation of poliovirus RNA by a cap-independent mechanism requires interactions of the 5'-untranslated region (5'UTR) with cell proteins. To determine whether the patterns of these interacting proteins differ in HeLa cells and permissive and nonpermissive tissues, UV-crosslinking assays using the poliovirus 5'UTR and tissue extracts from PVR transgenic mice were performed. The results indicate a correlation between the presence of a 97-kDa UV-crosslinked protein and permissivity to poliovirus infection. Acquired poliovirus susceptibility in in vitro-cultured kidney cells also correlates with the presence of a 97-kDa crosslinked band. The interaction of the 97-kDa protein from HeLa cells and mouse brain with the poliovirus 5'UTR is stable and specific. Whether the 97-kDa protein plays a role in poliovirus translation and tissue susceptibility remains to be determined.
Proc Natl Acad Sci U S A 1996 Oct 15;93(21):11378-81 Early events in poliovirus infection: virus-receptor interactions. The interaction of poliovirus with its cell receptor initiates conformational changes that lead to uncoating of the viral RNA. Three types of genetic analyses have been used to study the poliovirus-receptor interaction: (i) mutagenesis of the poliovirus receptor (PVR), (ii) selection of viral mutants resistant to neutralization with soluble PVR, and (iii) selection of viral variants adapted to use mutant PVRs. The results of these studies show that a small portion of the first immunoglobulin-like domain of PVR contacts viral residues within a deep depression on the surface of the capsid that encircles the fivefold axis of symmetry. Viral capsid residues that influence the interaction with PVR are also found in locations such as the capsid interior that cannot directly contact PVR. These mutations might influence the ability of the capsid to undergo receptor-mediated conformational transitions that are necessary for high-affinity interactions with PVR.
Structure 1996 Jul 15;4(7):769-73 The poliovirus receptor: a hook, or an unzipper? The
cell receptor for poliovirus may be more than a simple "snare'
that attaches virus to cells. Recent results indicate that
receptor binding may cause conformational changes in the virus that
lead to uncoating
of the viral RNA.
Determinants of attenuation and temperature sensitivity in the type 1 poliovirus Sabin vaccine.
Poliovirus variants selected on mutant receptor-expressing cells identify capsid residues that expand receptor recognition. Mutations
in the predicted C'-C"-D edge of the first immunoglobulin-like
domain of the poliovirus receptor were previously shown to
eliminate poliovirus binding. To identify capsid residues that expand
receptor
recognition, 16 poliovirus suppressor mutants were selected
that replicate in three different mutant receptor-expressing cell lines
as well as
in cells expressing the wild-type receptor. Sequence analysis
of the mutant viruses revealed three capsid residues that enable poliovirus
to utilize defective receptors. Two residues are in regions
of the capsid
that are known to regulate receptor binding and receptor-mediated
conformational transitions. A third residue is located in a highly exposed
loop on
the virion surface that controls poliovirus host range in mice
by influencing receptor recognition. One of the suppressor mutations
enables the primate-restricted
P1/Mahoney strain to paralyze mice by enabling the virus to
recognize a receptor in the mouse central nervous system. Capsid mutations
that
suppress receptor defects may exert their effect at the binding
site or may improve receptor binding by regulating structural transitions
of the capsid.
EMBO J 1994 Dec 15;13(24):5855-62 Soluble receptor-resistant poliovirus mutants identify surface and internal capsid residues that control interaction with the cell receptor. Poliovirus initiates infection by binding to its cell receptor and undergoing a receptor-mediated conformational alteration. To identify capsid residues that control these interactions, we have isolated and characterized poliovirus mutants that are resistant to neutralization by a soluble form of the poliovirus receptor. Twenty one soluble receptor-resistant (srr) mutants were identified which still use the poliovirus receptor to infect cells. All but one srr mutant contain a single amino acid change at one of 13 different positions, either on the surface or in the interior of the virion. The results of binding and alteration assays demonstrate that both surface and internal capsid residues regulate attachment to the receptor and conformational change of the virus. Mutations that reduce alteration also affect receptor binding, suggesting a common structural basis for early events in poliovirus infection.
J Virol 1994 Apr;68(4):2578-88 Homolog-scanning mutagenesis reveals poliovirus receptor residues important for virus binding and replication. Poliovirus
initiates infection of primate cells by binding to the poliovirus
receptor, Pvr. Mouse cells do not bind poliovirus but express a Pvr
homolog, Mph,
that does not function as a poliovirus receptor. Previous work
has shown that the first immunoglobulin-like domain of the Pvr protein
contains
the virus binding site. To further identify sequences of Pvr
important for its interaction with poliovirus, stable cell lines expressing
mutated
Pvr molecules were examined for their abilities to bind virus
and support virus replication. Substitution of the amino-terminal domain
of Mph
with that of Pvr yields a molecule that can function as a poliovirus
receptor. Cells expressing this chimeric receptor have normal
binding affinity for poliovirus, yet the kinetics of virus replication
are delayed.
Results of virus alteration assays indicate that this chimeric
receptor is defective in converting native virus to 135S altered particles.
This
defect is not observed with cells expressing receptor recombinants
that include Pvr domains 1 and 2. Because altered particles are believed
to be an intermediate in poliovirus entry, these findings suggest
that
Pvr domains 2 and 3 participate in early stages of infection.
Additional mutants were made by substituting variant Mph residues for
the corresponding
residues in Pvr. The results were interpreted by using a model
of Pvr predicted from the known structures of other immunoglobulin-like
V-type
domains. Analysis of stable cell lines expressing the mutant
proteins revealed that virus binding is influenced by mutations in the
predicted
C'-C" loop, the C" beta-strand, the C"-D loop, and the D-E loop. Mutations in homologous regions of the immunoglobulin-like CD4 molecule alter its interaction with gp120 of human immunodeficiency virus type 1. Cells expressing Pvr mutations on the predicted C" edge
do not develop cytopathic effect during poliovirus infection,
suggesting that poliovirus-induced cytopathic effect may be induced
by the virus-receptor
interaction.
A monoclonal antibody that blocks poliovirus attachment recognizes the lymphocyte homing receptor CD44. A monoclonal antibody, AF3, was previously shown to specifically inhibit poliovirus binding to HeLa cells and to detect a 100-kDa glycoprotein only in cell lines and tissues permissive for poliovirus infection. These results suggested that the 100-kDa protein may be involved in the pathogenesis of poliomyelitis and the cellular function of the poliovirus receptor site. To study further the role of the 100-kDa protein in poliovirus attachment, immunoaffinity purification, amino acid sequencing, and cDNA cloning were undertaken. The results demonstrate that antibody AF3 reacts with the lymphocyte homing receptor CD44, a multifunctional cell surface glycoprotein involved in the homing of circulating lymphocytes to lymph nodes and the modulation of lymphocyte adhesion and activation. Antibody AF3 reacts with a subset of CD44 molecules (AF3CD44H), which appears to be a small fraction of the heterogeneously glycosylated CD44 molecules expressed on hematopoietic and nonhematopoietic cells. Anti-CD44 monoclonal antibodies, previously reported to induce CD44-mediated modulation of lymphocyte activation and adhesion, compete with 125I-AF3 in binding assays, demonstrating functional overlap among the epitopes. The anti-CD44 monoclonal antibody A3D8, which binds to a greater molecular weight range of CD44 than does AF3, inhibits poliovirus binding to a similar degree. CD44 does not act as a poliovirus receptor, since CD44-expressing mouse L-cell transformants did not bind poliovirus. The poliovirus receptor and AF3CD44H may be noncovalently associated, or they may interact through the cytoskeleton or signal transduction pathways.
Transgenic mice and the pathogenesis of poliomyelitis. Transgenic mice expressing the cell receptor for poliovirus have been generated and are susceptible to poliovirus infection. TgPVR mice have been used to answer questions about the pathogenesis of poliovirus infection. Despite the widespread pattern of PVR expression, poliovirus infection in TgPVR mice is restricted to only a few sites, indicating that poliovirus tropism is not controlled solely by the ability of cells to bind virus. After intramuscular inoculation, poliovirus travels to the spinal cord by axonal transport. This route of entry into the central nervous system may play a role in the pathogenesis of poliovirus infections in humans.
Biologicals 1993 Dec;21(4):365-9 Infectious cDNA, cell receptors and transgenic mice in the study of Sabin's poliovirus vaccines. The determination of the nucleotide sequence of the poliovirus genome, the isolation of infectious poliovirus cDNAs, and the identification of the cell receptor for poliovirus and establishment of a transgenic mouse model for poliomyelitis have all contributed to our understanding of the live, attenuated Sabin poliovirus vaccines. These highly effective vaccines have been studied extensively by many laboratories to determine the molecular basis for their attenuation phenotype. For this special issue I would like to review how our research has improved the understanding of Sabin's poliovirus vaccines, and highlight Albert's influence on our work.
Virology 1993 Nov;197(1):501-5 Characterization of poliovirus conformational alteration mediated by soluble cell receptors. Soluble
extracts of Spodoptera frugiperda cells expressing the poliovirus
receptor (PVR) induce the native poliovirus (PV) to "A" particle
conformational change (J. Virol. 64, 4697-4702). We describe the variables
that regulate
this passage and study the suitability of solubilized PVR both
for use as an in vitro system to characterize the receptor-mediated
conformational
alteration and for the production of large amounts of altered
virus for structural analysis. PVR seems to function in a stoichiometric
fashion
and the A particles produced appear as intact, stain excluding,
spherical structures by electron microscopy, regardless of the extensive
proteolysis
of the capsid protein VP1, which takes place during the conversion.
The products obtained, time course, and temperature and ionic
strength dependence of the alteration of PV by the solubilized PVR are
indistinguishable
from those of the alteration that leads to productive infection
in cultured cells. Therefore, solubilized PVR may provide a convenient
in vitro
system for further characterization of the cell entry process.
Poliovirus attenuation and pathogenesis in a transgenic mouse model for poliomyelitis. A transgenic mouse model for poliomyelitis has been used to study aspects of poliovirus attenuation and pathogenesis. Transgenic mice expressing the cell receptor for poliovirus (TgPVR mice) develop poliomyelitis after inoculation with poliovirus by a variety of routes. TgPVR mice have been used to identify genomic sequences responsible for the attenuation phenotype of the P1/Sabin and P2/Sabin vaccine strains. Primary cell cultures derived from TgPVR mice differentiate between neurovirulent and attenuated virus strains, indicating that it may be possible to use these cultures to determine the functional basis of attenuation. Studies in TgPVR mice indicate that poliovirus tropism is not controlled solely by expression of PVR, and that poliovirus spreads from muscle to the central nervous system by neural pathways.
J Infect Dis 1992 Oct;166(4):747-52 Poliovirus spreads from muscle to the central nervous system by neural pathways. A transgenic mouse model was used to address an unsolved question in the pathogenesis of poliomyelitis: how poliovirus invades the central nervous system (CNS). LD50 values for intramuscular and intracerebral inoculation of poliovirus in transgenic mice expressing poliovirus receptors (TgPVR mice) were similar. After intramuscular inoculation with poliovirus, paralysis was observed first in the inoculated limb. In contrast, localization of initial paralysis to the inoculated limb was not observed in normal mice inoculated intramuscularly with the mouse-adapted P2/Lansing poliovirus strain. After intramuscular inoculation, infectious poliovirus was first detected in the inferior segment of the spinal cord, then in the superior spinal cord and the brain. Sciatic nerve transection blocked poliovirus spread to the spinal cord after inoculation into the hindlimb footpad of TgPVR mice. These results demonstrate that in TgPVR mice, poliovirus spreads from muscle to the CNS through nerve pathways and that expression of the poliovirus receptor plays an important role in viral spread by this route.
J Virol 1992 May;66(5):2807-13 Molecular cloning and expression of a murine homolog of the human poliovirus receptor gene. The poliovirus receptor (Pvr) is a member of the immunoglobulin superfamily of proteins, but its function in the cell is not known. Southern blot hybridization analysis indicated that the murine genome contains a sequence homolog of pvr. As a first step toward using the murine pvr homolog (mph) to study the function of Pvr, murine genomic and cDNA clones encoding mph were isolated. mph encodes a polypeptide with extensive sequence similarity to the extracellular domains of the human PVR. mph mRNAs of 2.0 and 3.0 kb are transcribed in the adult mouse brain, the spinal cord, the spleen, the kidney, the heart, and the liver. The Mph protein does not function as a receptor for poliovirus. However, substitution of domain 1 of the Mph protein with the corresponding sequence from pvr produced a chimeric receptor that could bind poliovirus and lead to productive infection. By constructing pvr-mph chimeras, it will be possible to identify the contact points of poliovirus within domain 1 of Pvr. Identification of the ligand and the cellular function of the Mph protein may help us understand the role of Pvr in the cell.
A mutation present in the amino terminus of Sabin 3 poliovirus VP1 protein is attenuating. The attenuated phenotype of Sabin 3 poliovirus compared with its neurovirulent progenitor strain has been largely accounted for by mutations in the genome at positions 472 and 2034 (G. D. Westrop, K. A. Wareham, D. M. A. Evans, G. Dunn, P. D. Minor, D. I. Magrath, F. Taffs, S. Marsden, M. A. Skinner, G. C. Schild, and J. W. Almond, J. Virol. 63:1338-1344, 1989). By sequencing vaccine virus RNA, we recently identified another Sabin 3-specific mutation at position 2493 (U----C), which predicts an Ile----Thr change at the sixth residue of VP1 (C. Weeks-Levy, J. M. Tatem, S. J. DiMichele, W. Waterfield, A. F. Georgiu, and S. J. Mento, Virology 185:934-937, 1991). Viruses generated by using cDNAs which represent the vaccine sequence (LED3) and a derivative (VR318) possessing a single base change to the wild-type nucleotide (U) at 2493 were used to determine the impact of the 2493 mutation on virus phenotype. The VP1 proteins of LED3 and VR318 viruses were distinguishable by denaturing electrophoretic analysis. LED3 produced smaller plaques in Vero cells than VR318 virus did. Neurovirulence testing of these cDNA-derived viruses in monkeys demonstrated that the 2493 mutation in LED3 virus is attenuating.
Construction of an infectious cDNA clone of echovirus 6. A complete cDNA copy of the echovirus 6 genome was constructed. Complementary DNA was reversed transcribed from viral RNA. Subgenomic cDNAs were obtained by direct cloning and polymerase chain reactions. Full length cDNA was constructed into the Bluescript II vector (pBSII) using unique, overlapping, restriction sites of four clones. The cDNA was infectious and produced echovirus 6 particles that behaved in the same manner as the parental virus.
J Virol 1992 Jan;66(1):296-304 Human poliovirus receptor gene expression and poliovirus tissue tropism in transgenic mice. Expression of the human poliovirus receptor (PVR) in transgenic mice results in susceptibility to poliovirus infection. In the primate host, poliovirus infection is characterized by restricted tissue tropism. To determine the pattern of poliovirus tissue tropism in PVR transgenic mice, PVR gene expression and susceptibility to poliovirus infection were examined by in situ hybridization. PVR RNA is expressed in transgenic mice at high levels in neurons of the central and peripheral nervous system, developing T lymphocytes in the thymus, epithelial cells of Bowman's capsule and tubules in the kidney, alveolar cells in the lung, and endocrine cells in the adrenal cortex, and it is expressed at low levels in intestine, spleen, and skeletal muscle. After infection, poliovirus replication was detected only in neurons of the brain and spinal cord and in skeletal muscle. These results demonstrated that poliovirus tissue tropism is not governed solely by expression of the PVR gene nor by accessibility of cells to virus. Although transgenic mouse kidney tissue expressed poliovirus binding sites and was not a site of poliovirus replication, when cultivated in vitro, kidney cells developed susceptibility to infection. Identification of the changes in cultured kidney cells that permit poliovirus infection may provide information on the mechanism of poliovirus tissue tropism.
Mutational analysis of the cellular receptor for poliovirus. To identify sequences of the cellular poliovirus receptor (PVR) involved in viral infection, mutant PVR cDNAs were constructed and assayed for biological activity in mouse L cells. To confirm that mutant PVRs reached the cell surface, an immunological tag, consisting of part of CH3 from human immunoglobulin G1, was engineered into the PVR. Deletion of PVR amino acids 256 to 320 or 385 to the carboxy terminus yielded receptors that were able to support poliovirus infection. PVRs lacking amino acids 40 to 136 or 137 to 256 were expressed at the cell surface but were not active as receptors for poliovirus. The results show that immunoglobulin-type domain 3 and the extreme carboxy terminus of the PVR are not required for viral receptor function, but sequences within the two amino-terminal domains contribute to the initiation of poliovirus infection.
Host range determinants located on the interior of the poliovirus capsid. The inability of certain poliovirus strains to infect mice can be overcome by the expression of human poliovirus receptors in mice or by the presence of a particular amino acid sequence of the B-C loop of the viral capsid protein VP1. We have identified changes in an additional capsid structure that permit host-restricted poliovirus strains to infect mice. Variants of the mouse-virulent P2/Lansing strain were constructed containing amino acid changes, deletions and insertions in the B-C loop of VP1. These variants were attenuated in mice, demonstrating the importance of the B-C loop sequence in host range. Passage of two of the B-C loop variants in mice led to the selection of viruses that were substantially more virulent. The increased neurovirulence of these strains was mapped to two different suppressor mutations in the N-terminus of VP1. Whereas the B-C loop of VP1 is highly exposed on the surface of the capsid, near the five-fold axis of symmetry, the suppressor mutations are in the interior of the virion, near the three-fold axis. Introduction of the suppressor mutations into the genome of the mouse-avirulent P1/Mahoney strain resulted in neurovirulent viruses, demonstrating that the P2/Lansing B-C loop sequence is not required to infect mice. Because the internal host range determinants are in a structure known to be important in conformational transitions of the virion, the host range of poliovirus may be determined by the ability of virions to undergo transitions catalyzed by cell receptors.
J Virol 1991 Apr;65(4):1829-35 Down regulation of poliovirus receptor RNA in HeLa cells resistant to poliovirus infection. A line of HeLa cells (SOFIA) was previously isolated that is resistant to poliovirus infection and does not express functional virus binding sites at the cell surface. The expression of the poliovirus receptor (PVR) gene in SOFIA cells was examined to determine the molecular basis for the failure of these cells to express PVRs. Southern blot analysis of genomic DNA revealed that the PVR gene in SOFIA cells did not contain gross alterations. However, PVR transcripts were not detected in Northern (RNA) blot analysis of SOFIA cell RNA. In vitro nuclear run-on analysis showed that transcription of PVR-specific RNA was reduced in SOFIA cells. Treatment of SOFIA cells with 5-azacytidine restored susceptibility to poliovirus infection, which correlated with the appearance of PVRs at the cell surface, as detected with anti-PVR monoclonal antibody D171. PVR RNA was detected in clones derived from 5-azacytidine-treated SOFIA cells. SOFIA cells were converted to poliovirus sensitivity at a rate of 5 to 7%, suggesting that down regulation of PVR expression involved few cellular targets. Resistance of SOFIA cells to poliovirus infection therefore appears to result from down regulation of PVR RNA, leading to lack of PVR expression at the cell surface. Methylation may play a role in regulating the expression of the PVR gene, which is not essential for survival of HeLa cells.
J Virol 1991 Mar;65(3):1377-82 Identification of two determinants that attenuate vaccine-related type 2 poliovirus. The poliovirus P2/P712 strain is an attenuated virus that is closely related to the type 2 Sabin vaccine strain. By using a mouse model for poliomyelitis, sequences responsible for attenuation of the P2/P712 strain were previously mapped to the 5' noncoding region of the genome and a central region encoding VP1, 2Apro, 2B, and part of 2C. To identify specific determinants that attenuate the P2/P712 strain, recombinants between this virus and the mouse-adapted P2/Lansing were constructed and their neurovirulence in mice was determined. By using this approach, the attenuation determinant in the central region was mapped to capsid protein VP1. Candidate attenuating sequences in VP1 and the 5' noncoding region were identified by comparing the P2/P712 sequence with that of vaccine-associated isolate P2/P117, and the P2/117 sequences were introduced into the P2/Lansing-P2/P712 recombinants by site-directed mutagenesis. Results of neurovirulence assays in mice indicate that an A at nucleotide 481 in the 5' noncoding region and isoleucine (Ile) at position 143 of capsid protein VP1 are the major determinants of attenuation of P2/P712. These determinants also attenuated neurovirulence in transgenic mice expressing human poliovirus receptors, a new model for poliomyelitis in which virulent viruses are not host restricted. These results demonstrate that A-481 and Ile-143 are general determinants of attenuation.
Virus-receptor interaction in poliovirus entry and pathogenesis. Knowledge of the three-dimensional structure of poliovirus has provided insight into many aspects of poliovirus infection. However, the structure alone cannot answer questions about how the virus interacts with the receptor to lead to release of the genome into the cell. Genetic and biochemical analysis of virus mutants are required to understand these steps in infection. The availability of cDNA clones of the poliovirus genome and PVR enables the isolation of virus and PVR mutants that can be used to study early events in infection. The results of these studies, coupled with the resolution of the three-dimensional structure of PVR, and perhaps of a PVR-poliovirus complex, should provide a detailed picture of virus binding, alteration, and uncoating. Viral receptors do not exist solely for the benefit of viruses, but serve a particular function in the cell. The study of virus receptors, therefore, bridges the disciplines of virology and cell biology. Identification of viral receptors may reveal previously unknown cell proteins whose functions can be studied, or might enhance our knowledge of a known protein. As a member of the Ig superfamily of proteins, PVR is likely to have a role in cell adhesion and/or cell-cell communication. It will, therefore, be important to identify a PVR ligand or coreceptor. The study of the expression of PVR and its ligand will provide a basis for understanding the normal function of this protein. Determination of the function of MPH will be facilitated by the analysis of mice containing a targeted disruption of the MPH gene. It is likely that knowledge of the cell function of MPH can also provide information on the interaction of poliovirus with host cells. It has been suggested that virus binding to cell receptors may lead to activation of cell events that lead to disease (69). While there is no evidence that poliovirus affects cells in this way, studying the interaction of MPH with its ligand might provide clues about cell processes that may be activated by poliovirus infection.
Science 1990 Dec 14;250(4987):1596-9 Poliovirus mutants resistant to neutralization with soluble cell receptors. Poliovirus mutants resistant to neutralization with soluble cellular receptor were isolated. Replication of soluble receptor-resistant (srr) mutants was blocked by a monoclonal antibody directed against the HeLa cell receptor for poliovirus, indicating that the mutants use this receptor to enter cells. The srr mutants showed reduced binding to HeLa cells and cell membranes. However, the reduced binding phenotype did not have a major impact on viral replication, as judged by plaque size and one-step growth curves. These results suggest that the use of soluble receptors as antiviral agents could lead to the selection of neutralization-resistant mutants that are able to bind cell surface receptors, replicate, and cause disease.
Mol Cell Biol 1990 Nov;10(11):5700-6 Heterogeneous expression of poliovirus receptor-related proteins in human cells and tissues. Portions of the cellular receptor for poliovirus were expressed in Escherichia coli as fusion proteins with the product of the trpE gene. One of two antireceptor antisera obtained by immunizing rabbits with the fusion proteins blocked poliovirus infection. Western immunoblot analyses demonstrated that poliovirus receptor-related proteins were expressed in HeLa cells and a variety of human tissues, including those that are not sites of poliovirus replication. Tissue-specific variation in electrophoretic mobility, immunoreactivity, and subunit arrangement of poliovirus receptor-related proteins was observed. These results demonstrate that poliovirus tissue tropism cannot be explained by a limited distribution of receptor polypeptide, but may be the result of alternative splicing, posttranslational modifications, or both. In addition, the widespread but heterogeneous expression of the receptor suggests that the protein may have an important endogenous function.
Transgenic mice expressing a human poliovirus receptor: a new model for poliomyelitis. A human poliovirus receptor (PVR) gene was used to generate transgenic mice that express PVR transcripts and poliovirus binding sites in a wide range of tissues. Intracerebral inoculation of PVR transgenic mice with poliovirus type 1, Mahoney strain, resulted in viral replication in the brain and spinal cord and development of paralytic poliomyelitis. P1/Mahoney did not replicate or cause paralysis in nontransgenic mice. PVR transgenic mice failed to develop clinical disease when inoculated intracerebrally with the live attenuated Sabin type 1 vaccine strain. These results demonstrate that the PVR is the major determinant of poliovirus host range in mice. Transgenic mice expressing human PVR should be useful for studying poliovirus neurovirulence, attenuation, and tissue tropism, and for development and testing of poliovirus vaccine strains.
J Virol 1990 Oct;64(10):4697-702 Neutralization of poliovirus by cell receptors expressed in insect cells. To examine the interaction of the poliovirus receptor (PVR) with virus and the role of the PVR in virus entry, the PVR was expressed in insect cells. Poliovirus bound to insect cells infected with a recombinant baculovirus (AcPVR) carrying cDNA encoding the PVR. Antibodies raised against PVR expressed in bacteria immunoprecipitated a 67-kilodalton polypeptide from cytoplasmic extracts of AcPVR-infected cells. Treatment of AcPVR-infected cells with tunicamycin revealed that the PVR is a glycoprotein containing N-glycosidic linkages and that carbohydrate accounts for nearly 50% of its molecular weight as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. When PVR was solubilized from AcPVR-infected insect cells and incubated with poliovirus, viral infectivity was neutralized. Sedimentation analysis revealed that irreversibly altered 135S particles were formed after incubation of poliovirus at 37 degrees C with solubilized extracts of AcPVR-infected insect cells. These results demonstrate that poliovirus eclipse may result from interaction with the cell receptor at neutral pH in the absence of membranes and suggest that soluble receptors may be effective antiviral agents against picornaviruses.
J Virol 1989 Dec;63(12):5069-75 Inhibition of translation in cells infected with a poliovirus 2Apro mutant correlates with phosphorylation of the alpha subunit of eucaryotic initiation factor 2. A poliovirus type 2 Lansing mutant was constructed by inserting 6 base pairs into the 2Apro region of an infectious cDNA clone, resulting in the addition of a leucine and threonine into the polypeptide sequence. The resulting small-plaque mutant, 2A-2, had a reduced viral yield in HeLa cells and synthesized viral proteins inefficiently. Infection with the mutant did not lead to specific inhibition of host cell protein synthesis early in infection, and this defect was attributed to a failure to induce cleavage of the cap-binding complex protein p220. At late times after infection with the mutant virus, both cellular and viral protein syntheses were severely inhibited. To explain this global inhibition of protein synthesis, the phosphorylation state of the alpha subunit of eucaryotic initiation factor 2 (eIF-2 alpha) was examined. eIF-2 alpha was phosphorylated in both R2-2A-2- and wild-type-virus-infected cells, indicating that poliovirus does not encode a function that blocks phosphorylation of eIF-2 alpha. The kinetics and extent of eIF-2 alpha phosphorylation correlated with the production of double-stranded RNA in infected cells, suggesting that eIF-2 alpha is phosphorylated by P1/eIF-2 alpha kinase. When HeLa cells were infected with R2-2A-2 in the presence of 2-aminopurine, a protein kinase inhibitor, much higher virus titers were produced, cleavage of p220 occurred, and host cell protein synthesis was specifically inhibited. Since phosphorylation of eIF-2 alpha was not inhibited by 2-aminopurine, we propose that 2-aminopurine rescues the ability of R2-2A-2 to induce cleavage of p220 by inhibition of a second as yet unidentified kinase.
Proc Natl Acad Sci U S A 1989 Nov;86(21):8299-303 Cell proteins bind to multiple sites within the 5' untranslated region of poliovirus RNA. The 5' noncoding region of poliovirus RNA contains sequences necessary for translation and replication. These functions are probably carried out by recognition of poliovirus RNA by cellular and/or viral proteins. Using a mobility-shift electrophoresis assay and 1,10-phenanthroline/Cu+ footprinting, we demonstrate specific binding of cytoplasmic factors with a sequence from nucleotides 510-629 within the 5' untranslated region (UTR). Complex formation was also observed with a second sequence (nucleotides 97-182) within the 5' UTR. These two regions of the 5' UTR appear to be recognized by distinct cell factors as determined by competition analysis and the effects of ionic strength on complex formation. However, both complexes contain eukaryotic initiation factor 2 alpha, as revealed by their reaction with specific antibody.
J Mol Biol 1989 May 20;207(2):379-92 New model for the secondary structure of the 5' non-coding RNA of poliovirus is supported by biochemical and genetic data that also show that RNA secondary structure is important in neurovirulence. A secondary structure model for the 5' non-coding RNA of poliovirus has been derived by comparing computer-generated folding patterns of equivalent sequences from a number of related enteroviruses and rhinoviruses and identifying compensating mutations that suggest conservation of a common secondary structure. Although certain elements are similar, the new model differs considerably from a previously published minimal energy structure and is consistent with the observed sensitivity of in vitro RNA transcripts of infectious poliovirus cDNA to RNases and modifying chemicals. The sequence of a neurovirulent revertant of an attenuated mutant provides additional evidence for an interaction between a region known to be important for neurovirulence, sequence 471-483, and nucleotides 528 to 538.
J Virol 1989 May;63(5):2357-60 Differences in replication of attenuated and neurovirulent polioviruses in human neuroblastoma cell line SH-SY5Y. A base change from C to U at position 472 of the 5' noncoding region of the poliovirus genome is known to be a major determinant of attenuation in the P3/Sabin vaccine strain. To determine the biochemical basis for the attenuated phenotype imparted by this mutation, a cell line in which replication of neurovirulent and attenuated viruses could be distinguished was identified. A pair of P3/Sabin-P2/Lansing viral recombinants that differ only at position 472 was used; the viruses replicated equally well in HeLa cells, but the virus with a U at base 472 was attenuated in mice. In the human neuroblastoma cell line SH-SY5Y, recombinants with a U at base 472 replicated to approximately 10-fold-lower titers than did neurovirulent viruses with a C at this position. Analysis of viral RNA and protein synthesis indicated that translation of the attenuated viral RNA was specifically reduced in SH-SY5Y cells.
J Virol 1989 May;63(5):1884-90 Mapping of attenuating sequences of an avirulent poliovirus type 2 strain. A mouse model for poliomyelitis was used to identify genomic sequences that attenuate neurovirulence of poliovirus strain P2/P712. This type 2 strain is avirulent in primates and mice yet grows as well as virulent strains in cell culture. The approach used was to exchange portions of the genome of the mouse-virulent P2/Lansing strain with the corresponding region from P2/P712 to identify sequences that could attenuate Lansing neurovirulence in mice. A full-length infectious cDNA of P2/P712 was assembled and used to construct recombinants between P2/P712 and P2/Lansing. The results of neurovirulence testing of 11 recombinants indicated that strong attenuating determinants are located in the 5' noncoding region of P2/P712 and a region encoding capsid protein VP1 and 2Apro, 2B, and part of 2C. An attenuating determinant was further localized to between nucleotides 456 and 628 of P2/P712. A third sequence from P2/P712, nucleotides 752 to 2268, encoding VP4, VP2, and part of VP3, was weakly attenuating. The sequence from nucleotide 4454, approximately halfway through the 2C-coding region, to the end of the P2/P712 genome did not contain attenuating determinants. Nucleotide sequence analysis revealed that P2/P712 differs from the type 2 Sabin vaccine strain by only 22 nucleotides. Six differences lead to amino acid changes in the coding region, and four differences are in the 5' noncoding region. These studies show that, like the type 1 and type 3 Sabin vaccine strains, the attenuated type 2 strain P712 contains multiple attenuating sequences, including strongly attenuating sequences in the 5' noncoding region of the genome.
Cellular receptor for poliovirus: molecular cloning, nucleotide sequence, and expression of a new member of the immunoglobulin superfamily. Restriction of poliovirus replication to a few sites in the infected primate host appears to be controlled by the expression of viral receptors. To learn more about these binding sites and their role in viral tissue tropism, cDNA clones encoding functional poliovirus receptors were isolated. The predicted amino acid sequence reveals that the human poliovirus receptor is an integral membrane protein with the conserved amino acids and domain structure characteristic of members of the immunoglobulin superfamily. Northern hybridization analysis indicates that poliovirus receptor transcripts are expressed in a wide range of human tissues, in contrast to the limited expression of virus binding sites, which suggests that additional factors or modifications of the receptor protein are required to permit poliovirus attachment.
Isolation and characterization of HeLa cell lines blocked at different steps in the poliovirus life cycle. Cotransfection of poliovirus RNA and R1, a poliovirus subgenomic RNA containing a deletion of nearly all of the capsid region, resulted in surviving cells, in contrast to the complete cell death observed after transfection with viral RNA. Cells that survived the cotransfection grew into colonies, produced infectious poliovirus, and underwent cycles of cell lysis (crisis periods) where less than 1% of the cells survived, followed by periods of growth. Poliovirus evolved during the persistent infection as judged by changes in plaque size. After passage for 6 months, a stable line called SOFIA emerged that no longer produced infectious virus and did not contain viral proteins or viral RNA. Cells frozen in liquid N2 while still in crisis and recultured 4 months later (named SOFIA N2) were also stabilized. After infection with poliovirus, SOFIA N2 cells showed a delay in the development of cytopathic effect, viral production, and cellular death when compared with HeLa cells. In contrast, SOFIA cells did not develop cytopathic effect and produced 10,000 times less virus than SOFIA N2 or HeLa cells. Viral production was delayed in SOFIA and SOFIA N2 cells transfected with poliovirus RNA when compared with HeLa cells, suggesting the presence of an intracellular block to poliovirus replication. Analysis of the cellular receptor for poliovirus by virus binding, an enzyme-linked immunosorbent assay, and in situ rosette assays with an antireceptor monoclonal antibody showed that receptors were expressed in SOFIA N2 cells but not in SOFIA cells. Echovirus 6, an enterovirus which uses a different cellular receptor, formed small plaques on SOFIA cells. Vesicular stomatitis virus formed plaques of similar size on SOFIA and HeLa cells, suggesting that the intracellular block was specific for enteroviruses. Cotransfection of the subgenomic replicon R1 with poliovirion RNA therefore resulted in the selection of HeLa cell variants containing blocks to poliovirus replication at the level of receptor and within the cell.
Science 1988 Jul 8;241(4862):213-5 Poliovirus host range is determined by a short amino acid sequence in neutralization antigenic site I. The mouse-adapted strain of poliovirus type 2 (Lansing) induces fatal poliomyelitis in mice after intracerebral inoculation, whereas mice inoculated with poliovirus type 1 (Mahoney) show no signs of disease. Previous work indicated that the adaptation to mouse virulence is associated with the viral capsid proteins and that mutations in neutralization antigenic site I of poliovirus reduce neurovirulence of the Lansing strain in mice. The role of antigenic site I in mouse neurovirulence was further explored by constructing an antigenic hybrid virus. Six amino acids in antigenic site I of the Mahoney strain were replaced with a sequence specific for the Lansing strain by using a mutagenesis cartridge. The hybrid virus was neutralized by polyclonal antisera elicited by the type 1 and type 2 strains of poliovirus and by neutralizing monoclonal antibodies directed against antigenic site I of type 2 virus. The hybrid virus induced paralytic disease in mice, an observation demonstrating that a short sequence of amino acids in antigenic site I is an important determinant of poliovirus host range. Antigenic site I may be involved in attachment of poliovirus to cells of the mouse central nervous system.
J Virol 1988 Jul;62(7):2219-27 Translational efficiency of poliovirus mRNA: mapping inhibitory cis-acting elements within the 5' noncoding region. Poliovirus mRNA contains a long 5' noncoding region of about 750 nucleotides (the exact number varies among the three virus serotypes), which contains several AUG codons upstream of the major initiator AUG. Unlike most eucaryotic mRNAs, poliovirus does not contain a m7GpppX (where X is any nucleotide) cap structure at its 5' end and is translated by a cap-independent mechanism. To study the manner by which poliovirus mRNA is expressed, we examined the translational efficiencies of a series of deletion mutants within the 5' noncoding region of the mRNA. In this paper we report striking translation system-specific differences in the ability of the altered mRNAs to be translated. The results suggest the existence of an inhibitory cis-acting element(s) within the 5' noncoding region of poliovirus (between nucleotides 70 and 381) which restricts mRNA translation in reticulocyte lysate, wheat germ extract, and Xenopus oocytes, but not in HeLa cell extracts. In addition, we show that HeLa cell extracts contain a trans-acting factor(s) that overcomes this restriction.
J Virol 1988 May;62(5):1687-96 Construction and characterization of poliovirus subgenomic replicons. Poliovirus
RNAs containing in-frame deletions within the capsid-coding region were
produced by in vitro transcription of altered poliovirus type 1 cDNA
by using bacteriophage T7 RNA polymerase. Three RNAs were transcribed
that contained deletions of 2,317 nucleotides (bases 747 to 3064), 1,781
nucleotides (bases 1,175 to 2,956), and 1,295 nucleotides (bases 1,175
to 2,470). All three subgenomic RNAs replicated after transfection into
HeLa cells, demonstrating that sequences encoding the capsid polypeptides
are not essential for viral RNA replication in vivo. Viral RNA containing
the largest deletion (R1) replicated approximately three times better
than full-length RNA produced in vitro. Northern blot (RNA blot) hybridization
analysis of total cellular RNA from HeLa cells at different times after
transfection with R1 demonstrated the presence of increasing amounts
of the expected 5.1-kilobase subgenomic RNA. Analysis by immunoprecipitation
of viral proteins induced after transfection of R1 RNA into HeLa cells
revealed the presence of proteins 2Apro, 2C, and 3Dpol and its precursors,
suggesting that the polyprotein cleavages are similar to those occurring
in virus-infected cells. Replication of P2/Lansing virion RNA was inhibited
by cotransfection with the R1 replicon, as demonstrated by hybridization
analysis with a serotype-specific oligonucleotide probe. A higher level
of inhibition of RNA replication was observed when P2/Lansing RNA was
cotransfected into HeLa cells with truncated R1 transcripts (R1-PvuII)
that were missing 395 3' nucleotides and a poly(A) tail. These internally
and terminally deleted RNAs inhibited the replication of subgenomic
replicons R1, R2, and R3 and caused a reduction in plaque size when
cotransfected with P1/Mahoney or P2/Lansing viral RNA, suggesting that
individual cells had received both RNAs. No inhibition of plaque size
was observed when replicon RNAs were used that were missing 1,384 or
1,839 3' nucleotides or contained plasmid-derived sequences downstream
of the 3' poly(A). The trans-acting inhibitory effect of R1-PvuII on
the replication of poliovirus P2/Lansing RNA did not involve entry of
RNA into cells and appeared to reduce viral translation and RNA synthesis
late in the infection cycle.
Mol Cell Biol 1988 Mar;8(3):1103-12 Cap-independent translation of poliovirus mRNA is conferred by sequence elements within the 5' noncoding region. Poliovirus polysomal RNA is naturally uncapped, and as such, its translation must bypass any 5' cap-dependent ribosome recognition event. To elucidate the manner by which poliovirus mRNA is translated, we have determined the translational efficiencies of a series of deletion mutants within the 5' noncoding region of the mRNA. We found striking differences in translatability among the altered mRNAs when assayed in mock-infected and poliovirus-infected HeLa cell extracts. The results identify a functional cis-acting element within the 5' noncoding region of the poliovirus mRNA which enables it to translate in a cap-independent fashion. The major determinant of this element maps between nucleotides 320 and 631 of the 5' end of the poliovirus mRNA. We also show that this region (320 to 631), when fused to a heterologous mRNA, can function in cis to render the mRNA cap independent in translation.
Virology 1987 Dec;161(2):429-37 Reduced mouse neurovirulence of poliovirus type 2 Lansing antigenic variants selected with monoclonal antibodies. The
Lansing strain of poliovirus type 2 is a mouse-adapted virus that induces
a fatal paralytic disease in mice after intracerebral inoculation. Our
previous results indicated that the mouse-adapted phenotype maps to
the Lansing viral capsid. To further define regions of the capsid that
are specifically involved in the infection of mice, antigenic variants
resistant to neutralization with monoclonal antibodies were selected,
and their mouse neurovirulence was studied. The monoclonal antibodies
used were directed against antigenic site 1, an immunodominant loop
of capsid polypeptide VP1 located on the virion surface. Ten of twenty-two
variants selected had lower intracerebral neurovirulence in mice when
compared to the parental virus. Four of the ten antigenic variants with
reduced neurovirulence were temperature sensitive (ts) for replication
in HeLa cells, while the remaining six variants replicated in HeLa cells
as well as the parent virus. Two ts+ variants that were studied had
a reduced ability to replicate in the mouse brain. There was no difference
in the histopathology and pattern of involvement in the central nervous
system of one variant compared to the parent virus. In three variants,
reduction of neurovirulence correlated with specific amino acid substitutions
at positions 100 and 101 of VP1, located within antigenic site 1. The
ts phenotype in three variants was associated with a single amino acid
deletion at position 105. Virus recovered from the brain of paralyzed
mice that had been inoculated with the antigenic variants was characterized
to identify the virus causing disease. In most cases, brain isolates
resembled the inoculated virus in neurovirulence and amino acid sequence
at the antigenic site. Virus recovered from brains of paralyzed mice
that had been inoculated with the ts variants was either ts+ or cold
sensitive, and had become more neurovirulent. These results suggest
that specific amino acid changes within an antigenic site on the virion
surface may result in reduction of mouse neurovirulence without affecting
viral replication in cultured cells.
J Virol 1987 Sep;61(9):2917-20 A mouse model for poliovirus neurovirulence identifies mutations that attenuate the virus for humans. A mutation in the genome of poliovirus type 3 that is known to reduce neurovirulence in humans similarly reduces neurovirulence in mice when incorporated into a mouse-adapted-human poliovirus recombinant. Viral recombinants with a uracil at nucleotide position 472 in the 5'-noncoding regions of their genomes are unable to replicate in the mouse brain. Viral recombinants with a cytosine at this position are neurovirulent in mice. Neurovirulence of poliovirus in mice may therefore prove to be a useful indicator of the genetic stability of new attenuating mutations created by site-directed mutagenesis.
Virology 1986 Dec;155(2):498-507 Poliovirus temperature-sensitive mutant containing a single nucleotide deletion in the 5'-noncoding region of the viral RNA. The effect on viral replication of deleting nucleotide 10 of the poliovirus RNA genome was determined. This deletion, which removes a base pair from a predicted hairpin structure in the viral RNA, was introduced into full-length cDNA. Virus recovered after transfection of HeLa cells with the mutated cDNA contained the expected deletion and was temperature sensitive for plaque formation. Analysis of viral replication by one-step growth experiments indicated that mutant virus production at the nonpermissive temperature was at least 100 times less than that of wild type virus, and release of virus from mutant-infected cells was delayed. The synthesis of positive- and negative-strand viral RNA in mutant virus-infected cells was temperature sensitive. Virus-specific protein synthesis in mutant virus-infected cells was not temperature sensitive but occurred at a slower rate than that of wild type virus at permissive and nonpermissive temperatures. Replication of the mutant virus was sensitive to actinomycin D, in contrast to the wild type parent virus, which was resistant to the drug. Mutant virus stocks contained a small percentage of ts+ viruses that were able to form plaques at the nonpermissive temperature. Nucleotide sequence analysis of genomic RNA from these ts+ viruses revealed a single base change at position 34 from a G to U. In the positive RNA strand, the effect of this mutation is to restore to the hairpin structure the single base pair whose formation was prevented by the original deletion. The ts+ pseudorevertants replicated to similar titers as wild type virus at 33 and 38.5 degrees and were partially sensitive to actinomycin D.
Proc Natl Acad Sci U S A 1986 Oct;83(20):7845-9 Transformation of a human poliovirus receptor gene into mouse cells. The first step in poliovirus replication is binding of virus to a cellular receptor. Mouse L cells, which are resistant to poliovirus infection because they do not bear a poliovirus receptor, were transformed with HeLa cell (human) DNA to poliovirus sensitivity at a frequency of approximately 1 in 50,000 transformants. Monoclonal antibody directed against the HeLa cell poliovirus receptor site was used in rosette assays to identify poliovirus-sensitive L-cell transformants in a background of L-cell tk+ transformants. A cloned cell line, CM-1, was isolated that displayed a surface component recognized by the anti-poliovirus receptor antibody. CM-1 cells were susceptible to infection with all three poliovirus serotypes, and infection could be blocked by the antireceptor antibody. Poliovirus formed plaques in CM-1 and HeLa cells with equal efficiency. CM-1 and HeLa cells produced infectious poliovirus at a similar rate, although yield of virus in CM-1 cells was about 33% less than the yield in HeLa cells. These results suggest that DNA encoding the HeLa cell poliovirus receptor has been introduced into mouse cells, resulting in the expression of the receptor and susceptibility to poliovirus infection.
Mechanism of in vitro synthesis of covalently linked dimeric RNA molecules by the poliovirus replicase. Four RNA fragments of approximately 1,000 to 1,200 nucleotides, representing both the 5' and 3' termini of poliovirus plus- and minus-strand RNAs, were generated by transcription of poliovirus cDNA by using bacteriophage SP6 RNA polymerase. The copying of these templates by the poliovirus replicase invariably produced RNA products approximately twice the size of the templates. In experiments with templates uniformly labeled with 32P it was shown that some of the apparently double-length products were generated by extension from an internal site of the template. Filter hybridization of the labeled in vitro-synthesized products with various unlabeled templates suggested a second mechanism by which double-length molecules could be synthesized; the results can be best explained by de novo synthesis of the first strand by copying of the template RNA, followed by snap-back of the newly synthesized RNA, generating a template-primer structure for the synthesis of the second strand. Highly purified poliovirus replicase was able to support the synthesis of double-length RNA products in response to these templates. These reactions did not require host factor. In contrast, synthesis of genome-length copies of poliovirion RNA by the same replicase was absolutely dependent on added host factor. The synthesis of double-length RNA products did not require either the 3'-terminal poly(A) of plus RNA or sequences within the 3' termini of both plus- and minus-strand RNAs.
Mapping of sequences required for mouse neurovirulence of poliovirus type 2 Lansing. Intracerebral inoculation of mice with poliovirus type 2 Lansing induces a fatal paralysis, while most other poliovirus strains are unable to cause disease in the mouse. To determine the molecular basis for Lansing virus neurovirulence, we determined the complete nucleotide sequence of the Lansing viral genome from cloned cDNA. The deduced amino acid sequence was compared with that of two mouse-avirulent strains. There are 83 amino acid differences between the Lansing and Sabin type 2 strain and 179 differences between the Lansing and Mahoney type 1 strain scattered throughout the genome. To further localize Lansing sequences important for mouse neurovirulence, four intertypic recombinants were isolated by exchanging DNA restriction fragments between the Lansing 2 and Mahoney 1 infectious poliovirus cDNA clones. Plasmids were transfected into HeLa cells, and infectious recombinant viruses were recovered. All four recombinant viruses, which contained the Lansing capsid region and different amounts of the Mahoney genome, were neurovirulent for 18- to 21-day-old Swiss-Webster mice by the intracerebral route. The genome of neurovirulent recombinant PRV5.1 contained only nucleotides 631 to 3413 from Lansing, encoding primarily the viral capsid proteins. Therefore, the ability of Lansing virus to cause paralysis in mice is due to the viral capsid. The Lansing capsid sequence differs from that of the mouse avirulent Sabin 2 strain at 32 of 879 amino acid positions: 1 in VP4, 5 in VP2, 4 in VP3, and 22 in VP1.
Proc Natl Acad Sci U S A 1985 Dec;82(24):8424-8 In vitro synthesis of infectious poliovirus RNA. Replication of the infectious RNA genome of poliovirus is accomplished in cells by the viral RNA polymerase through negative-strand RNA intermediates. Full-length negative-strand poliovirus RNA was synthesized in vitro by transcription of infectious poliovirus cDNA with bacteriophage SP6 DNA-dependent RNA polymerase. When provided with this negative-strand RNA as template, the poliovirus RNA-dependent RNA polymerase synthesized full-length positive-strand molecules. The positive-strand RNAs synthesized in vitro were infectious when transfected into HeLa cells. In contrast, positive-strand copies of poliovirus RNA synthesized in vitro by SP6 polymerase, using a poliovirus cDNA template, were not infectious. Production of infectious positive-strand RNA by the poliovirus polymerase was not observed when magnesium or negative-strand RNA template was omitted from the reaction mixture. Infectivity of the product RNA was not destroyed by DNase treatment. The specific infectivity in HeLa cells of in vitro-synthesized positive-strand RNA was 4 X 10(4) plaque-forming units/micrograms of RNA.
Enterovirus 70 receptor utilization Is controlled by capsid residues that also regulate host range and cytopathogenicity.
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Age-dependent poliovirus replication in the mouse central nervous system is determined by internal ribosome entry site-mediated translation.
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Hepatitis C virus internal ribosome entry site-dependent translation in Saccharomyces
cerevisiae is independent of polypyrimidine tract-binding protein, poly(rC)-binding
protein 2, and La protein.
J. Virol. 2005 79:10126-10137.
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Amino Acid Changes in Proteins 2B and 3A Mediate Rhinovirus Type 39 Growth
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Poliovirus tropism
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Transgenic mouse
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Kinetic Analysis of the Effect of Poliovirus Receptor on Viral Uncoating: the Receptor as a Catalyst
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It is too early to stop polio vaccination.
Bull World Health Organ. 2000;78(3):359-60.
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Two distinct binding affinities of poliovirus for its cellular receptor.
J Biol Chem. 2000 Jul 28;275(30):23089-96.
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Defects in nuclear and cytoskeletal morphology and mitochondrial localization in spermatozoa of mice lacking nectin-2, a component of cell-cell adherens junctions.
Mol Cell Biol. 2000 Apr;20(8):2865-73.
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An antiviral compound that blocks structural transitions of poliovirus prevents receptor binding at low temperatures.
J Virol. 2000 Apr;74(8):3929-31.
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Three-dimensional structure of poliovirus receptor bound to poliovirus.
Proc Natl Acad Sci U S A. 2000 Jan 4;97(1):73-8.
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ASM Press, Washington, DC.1999.
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The murine homolog (Mph) of human herpesvirus entry protein B (HveB) mediates entry of pseudorabies virus but not herpes simplex virus types 1 and 2.
J Virol. 1999 May;73(5):4493-7.
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Characterization of mouse lines transgenic with the human poliovirus receptor gene.
Microb Pathog. 1998 Jul;25(1):43-54.
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Persistent echovirus infection of mouse cells expressing the viral receptor VLA-2.
Virology. 1997 Sep 1;235(2):293-301.
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The polio eradication effort: should vaccine eradication be next?
Science. 1997 Aug 8;277(5327):779-80.
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Expression of the poliovirus receptor in intestinal epithelial cells is not sufficient to permit poliovirus replication in the mouse gut.
J Virol. 1997 Jul;71(7):4915-20.
pdf file
Cold-adapted poliovirus mutants bypass a postentry replication block.
J Virol. 1997 Jun;71(6):4728-35.
pdf file
Attenuating mutations in the poliovirus 5' untranslated region alter its interaction with polypyrimidine tract-binding protein.
J Virol. 1997 May;71(5):3826-33.
pdf file
CD44 is not required for poliovirus replication.
J Virol. 1997 Apr;71(4):2793-8.
pdf file
Differences in the UV-crosslinking patterns of the poliovirus 5'-untranslated region with cell proteins from poliovirus-susceptible and -resistant tissues.
Virology. 1997 Jan 20;227(2):505-8.
pdf file
Early events in poliovirus infection: virus-receptor interactions.
Proc Natl Acad Sci U S A. 1996 Oct 15;93(21):11378-81. Review.
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The poliovirus receptor: a hook, or an unzipper?
Structure. 1996 Jul 15;4(7):769-73. Review.
pdf file
Poliovirus biology and pathogenesis.
Curr Top Microbiol Immunol. 1996;206:305-25. Review. No abstract available.
Determinants of attenuation and temperature sensitivity in the type 1 poliovirus Sabin vaccine.
J Virol. 1995 Aug;69(8):4972-8.
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Poliovirus variants selected on mutant receptor-expressing cells identify capsid residues that expand receptor recognition.
J Virol. 1995 Aug;69(8):4823-9.
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Transgenic strategies for studying poliovirus pathogenesis and immunology.
In: "Strategies in Transgenic Animal Science: (G.M. Monastersky, J.M.
Robl, eds.).
ASM Press, Washington, DC. 1995 pp. 271-286.
Early events in infection: Receptor binding and cell entry.
In: "Human Enterovirus Infections" (H. Rotbart, ed.).
ASM Press, Washington, DC. 1995 pp. 73-94.
Soluble receptor-resistant poliovirus mutants identify surface and internal capsid residues that control interaction with the cell receptor.
EMBO J. 1994 Dec 15;13(24):5855-62.
Homolog-scanning mutagenesis reveals poliovirus receptor residues important for virus binding and replication.
J Virol. 1994 Apr;68(4):2578-88.
A monoclonal antibody that blocks poliovirus attachment recognizes the lymphocyte homing receptor CD44.
J Virol. 1994 Mar;68(3):1301-8.
Transgenic mice and the pathogenesis of poliomyelitis.
Arch Virol Suppl. 1994;9:79-86. Review.
Infectious cDNA, cell receptors and transgenic mice in the study of Sabin's poliovirus vaccines.
Biologicals. 1993 Dec;21(4):365-9.
Characterization of poliovirus conformational alteration mediated by soluble cell receptors.
Virology. 1993 Nov;197(1):501-5.
Characterisation of L cells expressing the human poliovirus receptor for the specific detection of polioviruses in vitro.
J Virol Methods. 1993 Mar;41(3):333-40. No abstract available.
Poliovirus attenuation and pathogenesis in a transgenic mouse model for poliomyelitis.
Dev Biol Stand. 1993;78:109-16. Review.
Virus-receptor interaction in poliovirus entry and pathogenesis.
The Harvey Lectures 1993 87:1-16.
Poliovirus spreads from muscle to the central nervous system by neural pathways.
J Infect Dis. 1992 Oct;166(4):747-52.
Molecular cloning and expression of a murine homolog of the human poliovirus receptor gene.
J Virol. 1992 May;66(5):2807-13.
A mutation present in the amino terminus of Sabin 3 poliovirus VP1 protein is attenuating.
J Virol. 1992 May;66(5):3194-7.
Construction of an infectious cDNA clone of echovirus 6.
Virus Res. 1992 Jan;22(1):71-8.
Human poliovirus receptor gene expression and poliovirus tissue tropism in transgenic mice.
J Virol. 1992 Jan;66(1):296-304.
Interaction of poliovirus with its cell receptor.
Sem. in Virol. 1992 3:473-482.
Poliovirus vaccines.
Biotechnology. 1992;20:205-22. Review. No abstract available.
Mutational analysis of the cellular receptor for poliovirus.
J Virol. 1991 Jul;65(7):3873-6.
Viruses: not the simplest form of life after all.
Molecular Biology of Human Pathogenic Viruses: a Keystone Symposium, Lake Tahoe, CA, USA, March 8-15, 1991.
New Biol. 1991 Jun;3(6):575-9. No abstract available.
Host range determinants located on the interior of the poliovirus capsid.
EMBO J. 1991 May;10(5):1067-74.
Down regulation of poliovirus receptor RNA in HeLa cells resistant to poliovirus infection.
J Virol. 1991 Apr;65(4):1829-35.
Identification of two determinants that attenuate vaccine-related type 2 poliovirus.
J Virol. 1991 Mar;65(3):1377-82.
Virus-receptor interaction in poliovirus entry and pathogenesis.
Harvey Lect. 1991-92;87:1-16. Review.
Picornaviruses.
Current Topics in Microbiol. and Immunol. vol. 161. Review.
Poliovirus mutants resistant to neutralization with soluble cell receptors.
Science. 1990 Dec 14;250(4987):1596-9.
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Heterogeneous expression of poliovirus receptor-related proteins in human cells and tissues.
Mol Cell Biol. 1990 Nov;10(11):5700-6.
Transgenic mice expressing a human poliovirus receptor: a new model for poliomyelitis.
Cell. 1990 Oct 19;63(2):353-62.
Neutralization of poliovirus by cell receptors expressed in insect cells.
J Virol. 1990 Oct;64(10):4697-702.
Cell receptors for picornaviruses.
Curr Top Microbiol Immunol. 1990;161:1-22. Review. No abstract available.
Inhibition of translation in cells infected with a poliovirus 2Apro mutant correlates with phosphorylation of the alpha subunit of eucaryotic initiation factor 2.
J Virol. 1989 Dec;63(12):5069-75.
Cell proteins bind to multiple sites within the 5' untranslated region of poliovirus RNA.
Proc Natl Acad Sci U S A. 1989 Nov;86(21):8299-303.
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New model for the secondary structure of the 5' non-coding RNA of poliovirus is supported by biochemical and genetic data that also show that RNA secondary structure is important in neurovirulence.
J Mol Biol. 1989 May 20;207(2):379-92.
Differences in replication of attenuated and neurovirulent polioviruses in human neuroblastoma cell line SH-SY5Y.
J Virol. 1989 May;63(5):2357-60.
Mapping of attenuating sequences of an avirulent poliovirus type 2 strain.
J Virol. 1989 May;63(5):1884-90.
Cellular receptor for poliovirus: molecular cloning, nucleotide sequence, and expression of a new member of the immunoglobulin superfamily.
Cell. 1989 Mar 10;56(5):855-65.
Isolation and characterization of HeLa cell lines blocked at different steps in the poliovirus life cycle.
J Virol. 1989 Jan;63(1):43-51.
Poliovirus host range is determined by a short amino acid sequence in neutralization antigenic site I.
Science. 1988 Jul 8;241(4862):213-5.
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Translational efficiency of poliovirus mRNA: mapping inhibitory cis-acting elements within the 5' noncoding region.
J Virol. 1988 Jul;62(7):2219-27.
Construction and characterization of poliovirus subgenomic replicons.
J Virol. 1988 May;62(5):1687-96.
Cap-independent translation of poliovirus mRNA is conferred by sequence elements within the 5' noncoding region.
Mol Cell Biol. 1988 Mar;8(3):1103-12.
Poliovirus neurovirulence.
Adv Virus Res. 1988;34:217-46. Review. No abstract available.
Reduced mouse neurovirulence of poliovirus type 2 Lansing antigenic variants selected with monoclonal antibodies.
Virology. 1987 Dec;161(2):429-37.
A mouse model for poliovirus neurovirulence identifies mutations that attenuate the virus for humans.
J Virol. 1987 Sep;61(9):2917-20.
Viral sequences required for neurovirulence of poliovirus.
Bioessays. 1986 Dec;5(6):266-70. Review. No abstract available.
Poliovirus temperature-sensitive mutant containing a single nucleotide deletion in the 5'-noncoding region of the viral RNA.
Virology. 1986 Dec;155(2):498-507.
Transformation of a human poliovirus receptor gene into mouse cells.
Proc Natl Acad Sci U S A. 1986 Oct;83(20):7845-9.
pdf file
Mechanism of in vitro synthesis of covalently linked dimeric RNA molecules by the poliovirus replicase.
J Virol. 1986 May;58(2):459-67.
Mapping of sequences required for mouse neurovirulence of poliovirus type 2 Lansing.
J Virol. 1986 Feb;57(2):515-25.
In vitro synthesis of infectious poliovirus RNA.
Proc Natl Acad Sci U S A. 1985 Dec;82(24):8424-8.
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Molecular cloning of the mouse ouabain resistance gene.
Proc Natl Acad Sci U S A 1984 81:1489-1493.
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Studying poliovirus with infectious cloned cDNA.
Rev Infect Dis. 1984 May-Jun;6 Suppl 2:S514-5.
Molecular cloning and characterization of hepatitis A virus cDNA.
Proc Natl Acad Sci U S A. 1983 Oct;80(19):5885-9.
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Cloned poliovirus complementary DNA is infectious in mammalian cells.
Science. 1981 Nov 20;214(4523):916-9.
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Nucleotide sequence and evolution of a mammalian alpha-tubulin messenger RNA.
J Mol Biol. 1981 Sep 5;151(1):101-20. No abstract available.
Molecular cloning of poliovirus cDNA and determination of the complete nucleotide sequence of the viral genome.
Proc Natl Acad Sci U S A. 1981 Aug;78(8):4887-91.
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Genetic structure and genetic variation of influenza viruses.
Philos Trans R Soc Lond B Biol Sci. 1980 Feb 25;288(1029):299-305.
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The 3' and 5'-terminal sequences of influenza A, B and C virus RNA segments are highly conserved and show partial inverted complementarity.
Gene. 1980 Feb;8(3):315-28.
Isolation of influenza C virus recombinants.
J Virol. 1979 Dec;32(3):1006-14.
Influenza B virus genome: assignment of viral polypeptides to RNA segments.
J Virol. 1979 Jan;29(1):361-73.
The genes of influenza virus: Analysis of influenza B virus strains.
In: Negative Strand Viruses and the Host Cell (R.D. Barry and B.W.J. Mahy, eds.) 1978, pp. 27-36.
Academic Press, London.
Hepatitis C virus internal ribosome entry site-dependent translation in
Saccharomyces cerevisiae is independent of polypyrimidine tract-binding
protein, poly(rC)-binding protein 2, and La protein.
Rosenfeld AB, Racaniello VR
Translation initiation of some viral and cellular mRNAs occurs by ribosome
binding to an internal ribosome entry site (IRES). Internal initiation
mediated by the hepatitis C virus (HCV) IRES in Saccharomyces cerevisiae
was shown by translation of the second open reading frame in a bicistronic
mRNA. Introduction of a single base change in the HCV IRES, known to abrogate
internal initiation in mammalian cells, abolished translation of the second
open reading frame. Internal initiation mediated by the HCV IRES was independent
of the nonsense-mediated decay pathway and the cap binding protein eIF4E,
indicating that translation is not a result of mRNA degradation or 5'-end-dependent
initiation. Human La protein binds the HCV IRES and is required for efficient
internal initiation. Disruption of the S. cerevisiae genes that encode
La protein orthologs and synthesis of wild-type human La protein in yeast
had no effect on HCV IRES-dependent translation. Polypyrimidine tract-binding
protein (Ptb) and poly-(rC)-binding protein 2 (Pcbp2), which may be required
for HCV IRES-dependent initiation in mammalian cells, are not encoded
within the S. cerevisiae genome. HCV IRES-dependent translation in S.
cerevisiae was independent of human Pcbp2 protein and stimulated by the
presence of human Ptb protein. These findings demonstrate that the genome
of S. cerevisiae encodes all proteins necessary for internal initiation
of translation mediated by the HCV IRES.
Harris JR, Racaniello VR
Many steps of viral replication are dependent on the interaction
of viral proteins with host cell components. To identify rhinovirus
proteins involved in such interactions, human rhinovirus 39 (HRV39),
a virus unable to replicate in mouse cells, was adapted to efficient
growth in mouse cells producing the viral receptor ICAM-1 (ICAM-L
cells). Amino acid changes were identified in the 2B and 3A proteins
of the adapted virus, RV39/L. Changes in 2B were sufficient to
permit viral growth in mouse cells; however, changes in both 2B
and 3A were required for maximal viral RNA synthesis in mouse cells.
Examination of infected HeLa cells by electron microscopy demonstrated
that human rhinoviruses induced the formation of cytoplasmic membranous
vesicles, similar to those observed in cells infected with other
picornaviruses. Vesicles were also observed in the cytoplasm of
HRV39-infected mouse cells despite the absence of viral RNA replication.
Synthesis of picornaviral nonstructural proteins 2C, 2BC, and 3A
is known to be required for formation of membranous vesicles. We
suggest that productive HRV39 infection is blocked in ICAM-L cells
at a step posttranslation and prior to the formation of a functional
replication complex. The observation that changes in HRV39 2B and
3A proteins lead to viral growth in mouse cells suggests that one
or both of these proteins interact with host cell proteins to promote
viral replication.
Kauder, S.E. and Racaniello, V.R.
Poliovirus replication is limited to a few organs, including the brain
and spinal cord. This restricted tropism may be a consequence of organ-specific
differences in translation initiation by the poliovirus internal ribosome
entry site (IRES). A C-to-U mutation at base 472 in the IRES of the
Sabin type 3 poliovirus vaccine strain, known to attenuate neurovirulence,
may further restrict tropism by eliminating viral replication in the
CNS. To determine the relationship between IRES-mediated translation
and poliovirus tropism, recombinant human adenoviruses were used to
express bicistronic mRNAs in murine organs. The IRESs of poliovirus,
the cardiotropic coxsackievirus B3 (CVB3), and the hepatotropic hepatitis
C virus (HCV) mediate translation in many organs, including those
that do not support viral replication. A translation defect associated
with the Sabin type 3 IRES was observed in all organs examined. Poliovirus
type 1 and recombinant polioviruses dependent on the IRES of CVB3
or HCV replicate in the CNS of mice and cause paralysis. Although
the type 3 Sabin strain is an effective vaccine, polioviruses with
a U at base 472 of the IRES cause paralysis in newborn mice. Tropism
of wild-type and vaccine strains of poliovirus is therefore determined
after internal ribosome entry.
Brown DM, Kauder SE, Cornell CT, Jang GM, Racaniello VR, Semler BL.
We previously reported the isolation of a mutant poliovirus lacking the
entire genomic RNA 3' noncoding region. Infection of HeLa cell monolayers
with this deletion mutant revealed only a minor defect in the levels of
viral RNA replication. To further analyze the consequences of the genomic
3' noncoding region deletion, we examined viral RNA replication in a neuroblastoma
cell line, SK-N-SH cells. The minor genomic RNA replication defect in HeLa
cells was significantly exacerbated in the SK-N-SH cells, resulting in a
decreased capacity for mutant virus growth. Analysis of the nature of the
RNA replication deficiency revealed that deleting the poliovirus genomic
3' noncoding region resulted in a positive-strand RNA synthesis defect.
The RNA replication deficiency in SK-N-SH cells was not due to a major defect
in viral translation or viral protein processing. Neurovirulence of the
mutant virus was determined in a transgenic mouse line expressing the human
poliovirus receptor. Greater than 1,000 times more mutant virus was required
to paralyze 50% of inoculated mice, compared to that with wild-type virus.
These data suggest that, together with a cellular factor(s) that is limiting
in neuronal cells, the poliovirus 3' noncoding region is involved in positive-strand
synthesis during genome replication.
Transgenic mouse model for echovirus myocarditis and paralysis.
Hughes, S.A., Thaker, H.M., and Racaniello, V.R.
Echoviruses have been
implicated in multiple human disease syndromes, including aseptic
meningitis, paralysis, and heart disease, but no animal model
is available for studying the pathogenesis of infection. Production
of human integrin very
late antigen 2, a receptor for echovirus type 1, in transgenic mice conferred
susceptibility to viral infection. Intracerebral inoculation of newborn transgenic
mice with echovirus leads to paralysis and wasting. No disease was observed
in
infected nontransgenic mice. In paralyzed mice significant damage was observed
in the outer layers of the cerebrum, and numerous condensed neuronal nuclei
were present. In contrast, intracerebral inoculation of adolescent
(3- to 4-week-old)
transgenic mice with echovirus type 1 did not lead to paralysis but an acute
wasting phenotype and myocarditis. These findings establish human very late
antigen 2 transgenic mice as a model for echovirus pathogenesis.
Béatrice Baury, B., D. Masson, B. M. McDermott Jr., A. Jarry, H. M.
Blottière, C L. Laboisse, P. Lustenberger, V. R. Racaniello, and M.G.
Denis
Institut National de la Santé et de la
Recherche Médicale U539, Faculté de Médecine, 1 rue Gaston
Veil, 44035 Nantes, France
Department of Microbiology, Columbia University College of Physicians & Surgeons,
New York, NY 10032, USA
The CD155 gene is a member of the immunoglobulin superfamily. We first demonstrate
the existence of soluble CD155 (sCD155) isoforms in culture medium conditioned
by CD155-expressing cells, in human serum and in cerebrospinal fluid. sCD155
concentration was measured in human serum and cerebrospinal fluid using a specific
ELISA. Analysis of conditioned media indicated that sCD155 release does not
require protease activity. In order to determine which tissues are responsible
for
sCD155 expression, we have quantified CD155 mRNAs in human normal tissues.
The highest expression was observed in liver. The CD155 transcript is the most
abundant
and the proportion of the CD155 and CD155 variants was similar between the
tissues. Finally, serum purified sCD155 reduces poliovirus entry mediated by
membrane-bound CD155. The high level of CD155 synthesis in many tissues and
the presence of sCD155 in biological fluids suggest the existence of an important
role for the protein in cellular function.
Harris, JR, Racaniello, VR
Department of Microbiology, Columbia University
College of Physicians and Surgeons, New York, New York 10032
Rhinovirus type 16 was found to replicate in mouse L cells that express
the viral receptor, human intercellular adhesion molecule 1 (ICAM-1). However,
infection of these cells at low multiplicity of infection leads to no discernible
cytopathic effect, and low virus titers are produced. A variant virus, 16/L,
was isolated after alternate passage of rhinovirus 16 between HeLa and ICAM-1
L cells. Infection of mouse cells with 16/L leads to higher virus titers,
increased production of RNA, and total cytopathic effect. Three amino acid
changes were identified in the P2 region of virus 16/L, and the adaptation
phenotype mapped to two changes in protein 2C. The characterization of a
rhinovirus host range mutant will facilitate the investigation of cellular
proteins required for efficient viral growth and the development of a murine
model for rhinovirus infection.
Tsang, SK, McDermott, BM, Racaniello, VR, Hogle, JM
Committee on Higher Degrees in Biophysics, Harvard University, Cambridge, Massachusetts 02138; Department of Microbiology, Columbia University College of Physicians and Surgeons, New York, New York 10032; and Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115.
McDermott BM Jr, Rux AH, Eisenberg RJ, Cohen GH, Racaniello VR
Department of Microbiology, Columbia University College of Physicians & Surgeons,
New York, New York 10032, USA.
Bouchard MJ, Dong Y, McDermott BM Jr, Lam DH, Brown KR, Shelanski M, Bellve AR, Racaniello VR
Departments of Microbiology, Columbia University College of Physicians and Surgeons, New York, New York 10032, USA.
Dove AW, Racaniello VR
Department of Microbiology, Columbia University College of Physicians and Surgeons, New York, New York 10032, USA.
Belnap DM, McDermott BM Jr, Filman DJ, Cheng N, Trus BL, Zuccola HJ, Racaniello VR, Hogle JM, Steven AC
Laboratory of Structural Biology, National Institute of Arthritis, Musculoskeletal and Skin Diseases, Bethesda, MD 20892, USA.
Shukla D, Rowe CL, Dong Y, Racaniello VR, Spear PG
A mouse member of the immunoglobulin superfamily, originally designated the murine poliovirus receptor homolog (Mph), was found to be a receptor for the porcine alphaherpesvirus pseudorabies virus (PRV). This mouse protein, designated here murine herpesvirus entry protein B (mHveB), is most similar to one of three related human alphaherpesvirus receptors, the one designated HveB and also known as poliovirus receptor-related protein 2. Hamster cells resistant to PRV entry became susceptible upon expression of a cDNA encoding mHveB. Anti-mHveB antibody and a soluble protein composed of the mHveB ectodomain inhibited mHveB-dependent PRV entry. Expression of mHveB mRNA was detected in a variety of mouse cell lines, but anti-mHveB antibody inhibited PRV infection in only a subset of these cell lines, indicating that mHveB is the principal mediator of PRV entry into some mouse cell types but not others. Coexpression of mHveB with PRV gD, but not herpes simplex virus type 1 (HSV-1) gD, inhibited entry activity, suggesting that PRV gD may interact directly with mHveB as a ligand that can cause interference. By analogy with HSV-1, envelope-associated PRV gD probably also interacts directly with mHveB during viral entry.
Deatly AM, Taffs RE, McAuliffe JM, Nawoschik SP, Coleman JW, McMullen G, Weeks-Levy C, Johnson AJ, Racaniello VR
Viral Vaccine Research, Wyeth-Lederle Vaccines and Pediatrics, Pearl River, New York 10965, USA.
Zhang S, Racaniello VR
Department of Microbiology, Columbia University College of Physicians and Surgeons, 701 West 168th Street, New York, New York, 10032, USA.
Alan W. Dove, Vincent R. Racaniello *
Department of Microbiology, Columbia University College of Physicians and Surgeons, New York, NY 10032, USA.
Zhang S, Racaniello VR
Department of Microbiology, Columbia University College of Physicians & Surgeons,
New York, New York 10032, USA.
J Virol 1997 Jun;71(6):4728-35
Dove AW, Racaniello VR
Department of Microbiology, Columbia University College of Physicians & Surgeons,
New York, New York 10032, USA.
J Virol 1997 May;71(5):3826-33
Gutierrez AL, Denova-Ocampo M, Racaniello VR, del Angel RM
Departamento de Patologia Experimental, Centro de Investigacion y de Estudios y de Estudios Avanzados del IPN, Mexico City, Mexico.
Bouchard MJ, Racaniello VR
Department of Microbiology, Columbia University College of Physicians and Surgeons, New York, New York 10032, USA.
Gutierrez-Escolano AL, Medina F, Racaniello VR, Del Angel RM
Departamento de Patologia Experimental, Centro de Investigacion y de Estudios Avanzados del Instituto Politecnico Nacional, Mexico City, Mexico.
Racaniello VR
Department of Microbiology, Columbia University College of Physicians and Surgeons, New York, NY 10032, USA. racaniello@cuccfa.ccc.columbia.edu
Racaniello VR
Department of Microbiology, Columbia University College of Physicians & Surgeons,
New York, NY 10032, USA. racaniello@cuccfa.ccc.columbia.edu
Bouchard MJ, Lam DH, Racaniello VR
Department of Microbiology, Columbia University College of Physicians & Surgeons,
New York, NY 10032, USA.To identify determinants of attenuation in
the poliovirus type 1 Sabin vaccine strain, a series of recombinant viruses
were constructed
by using infectious cDNA clones of the virulent type 1 poliovirus
P1/Mahoney and the attenuated type 1 vaccine strain P1/Sabin. Intracerebral
inoculation
of these viruses into transgenic mice which express the human receptor
for poliovirus identified regions of the genome that conferred reduced neurovirulence.
Exchange of smaller restriction fragments and site-directed mutagenesis
were
used to identify the nucleotide changes responsible for attenuation.
P1/Sabin mutations at nucleotides 935 of VP4, 2438 of VP3, and 2795 and 2879
of VP1
were all shown to be determinants of attenuation. The recombinant
viruses and site-directed mutants were also used to identify the nucleotide
changes
which are involved in the temperature sensitivity of P1/Sabin. Determinants
of this phenotype in HeLa cells were mapped to changes at nucleotides
935 of VP4, 2438 of VP3, and 2741 of VP1. The 3Dpol gene of P1/Sabin, which
contains
three amino acid differences from its parent P1/Mahoney, also contributes
to the temperature sensitivity of P1/Sabin; however, mutants containing
individual amino acid changes grew as well as P1/Mahoney at elevated temperatures,
suggesting
that either some combination or all three changes are required for
temperature sensitivity. In addition, the 3'-noncoding region of P1/Sabin
augments the
temperature-sensitive phenotype conferred by 3Dpol. Although nucleotide
2741, 3Dpol, and the 3'-noncoding region of P1/Sabin contribute to the temperature
sensitivity of P1/Sabin, they do not contribute to attenuation in
transgenic
mice expressing the poliovirus receptor, demonstrating that determinants
of attenuation and temperature sensitivity can be genetically separated.
Colston EM, Racaniello VR
Department of Microbiology, Columbia University College of Physicians & Surgeons,
New York, NY 10032, USA.
Colston E, Racaniello VR
Department of Microbiology, Columbia University College of Physicians & Surgeons,
New York, NY 10032.
Morrison ME, He YJ, Wien MW, Hogle JM, Racaniello VR
Department of Microbiology, Columbia University College of Physicians & Surgeons,
New York, New York 10032.
Shepley MP, Racaniello VR
Department of Microbiology, Columbia University College of Physicians and Surgeons, New York, New York 10032.
Racaniello VR, Ren R
Department of Microbiology, Columbia University College of Physicians and Surgeons, New York, New York.
Racaniello VR
Department of Microbiology, Columbia University College of Physicians & Surgeons,
New York, NY 10032.
Gomez Yafal A, Kaplan G, Racaniello VR, Hogle JM
Department of Biological Chemistry and Molecular Pharmacology, Harvard University Medical School, Boston, Massachusetts 02115.
Racaniello VR, Ren R, Bouchard M
Department of Microbiology, College of Physicians and Surgeons, Columbia University, New York, NY 10032.
Ren R, Racaniello VR
Department of Microbiology, Columbia University College of Physicians & Surgeons,
New York, New York 10032.
Morrison ME, Racaniello VR
Department of Microbiology, Columbia University College of Physicians & Surgeons,
New York, New York 10032.
Tatem JM, Weeks-Levy C, Georgiu A, DiMichele SJ, Gorgacz EJ, Racaniello VR, Cano FR, Mento SJ
Lederle Laboratories, Pearl River, New York 10965.
Blackburn RV, Racaniello VR, Righthand VF
Department of Immunology and Microbiology, Wayne State University, Detroit, MI 48201.
Ren R, Racaniello VR
Department of Microbiology, Columbia University College of Physicians & Surgeons,
New York, New York 10032.
Freistadt MS, Racaniello VR
Department of Microbiology, Columbia University College of Physicians and Surgeons, New York, New York 10032.
Moss EG, Racaniello VR
Department of Microbiology, Columbia University College of Physicians and Surgeons, New York, NY 10032.
Kaplan G, Racaniello VR
Department of Microbiology, College of Physicians and Surgeons, Columbia University, New York, New York 10032.
Ren RB, Moss EG, Racaniello VR
Department of Microbiology, College of Physicians & Surgeons, Columbia
University, New York, New York 10032.
Racaniello VR
Department of Microbiology, Columbia University College of Physicians and Surgeons, New York, New York.
Kaplan G, Peters D, Racaniello VR
Department of Microbiology, College of Physicians and Surgeons of Columbia University, New York, NY 10032.
Freistadt MS, Kaplan G, Racaniello VR
Department of Microbiology, Columbia University College of Physicians & Surgeons,
New York, New York 10032.
Ren RB, Costantini F, Gorgacz EJ, Lee JJ, Racaniello VR
Department of Microbiology, College of Physicians and Surgeons Columbia University, New York, New York 10032.
Kaplan G, Freistadt MS, Racaniello VR
Department of Microbiology, College of Physicians and Surgeons, Columbia University, New York, New York 10032.
O'Neill RE, Racaniello VR
Department of Microbiology, Columbia University College of Physicians and Surgeons, New York, New York 10032.
del Angel RM, Papavassiliou AG, Fernandez-Tomas C, Silverstein SJ, Racaniello VR
Departmento de Genetica y Biologia Molecular, Centro de Investigacion y de Estudios Avanzados, Mexico City, Distrito Federal.
Skinner MA, Racaniello VR, Dunn G, Cooper J, Minor PD, Almond JW
Department of Microbiology, University of Reading, England.
La Monica N, Racaniello VR
Department of Microbiology, Columbia University College of Physicians and Surgeons, New York, New York 10032.
Moss EG, O'Neill RE, Racaniello VR
Department of Microbiology, Columbia University College of Physicians and Surgeons, New York, New York 10032.
Mendelsohn CL, Wimmer E, Racaniello VR
Department of Microbiology, Columbia University College of Physicians and Surgeons, New York, New York 10032.
Kaplan G, Levy A, Racaniello VR
Department of Microbiology, Columbia University College of Physicians & Surgeons,
New York, New York 10032.
Murray MG, Bradley J, Yang XF, Wimmer E, Moss EG, Racaniello VR
Department of Microbiology, School of Medicine, State University of New York, Stony Brook 11794.
Pelletier J, Kaplan G, Racaniello VR, Sonenberg N
Department of Biochemistry, McGill University, Montreal, Quebec, Canada.
Kaplan G, Racaniello VR
Department of Microbiology, Columbia University College of Physicians & Surgeons,
New York, New York 10032.
Pelletier J, Kaplan G, Racaniello VR, Sonenberg N
Department of Biochemistry, McGill University, Montreal, Quebec, Canada.
La Monica N, Kupsky WJ, Racaniello VR
Department of Microbiology, Columbia University of Physicians & Surgeons,
New York, New York 10032.
La Monica N, Almond JW, Racaniello VR
Department of Microbiology, Columbia University of Physicians & Surgeons,
New York, New York 10032.
Racaniello VR, Meriam C
Department of Microbiology, Columbia University of Physicians & Surgeons,
New York, New York 10032.
Mendelsohn C, Johnson B, Lionetti KA, Nobis P, Wimmer E, Racaniello VR
Department of Microbiology, Columbia University of Physicians & Surgeons,
New York, New York 10032.
Lubinski JM, Kaplan G, Racaniello VR, Dasgupta A
La Monica N, Meriam C, Racaniello VR
Kaplan G, Lubinski J, Dasgupta A, Racaniello VR
Proc Natl Acad Sci U S A 1984 Mar;81(5):1489-93
Molecular cloning of the mouse ouabain-resistance gene.
Levenson R, Racaniello V, Albritton L, Housman D.
DNA prepared from ouabain-resistant mouse cells was able to transform ouabain-sensitive CV-1 cells to ouabain resistance after DNA-mediated gene transfer. The murine DNA fragment responsible for ouabain resistance was detected on the background of CV-1 DNA by virtue of a repetitive DNA sequence element that reacts positively with a mouse repeat DNA clone. CV-1 DNA is nonreactive with this probe. Southern analysis of several independently derived ouabain-resistant transformants indicates that the mouse ouaR gene is located on a 6.5-kilobase EcoRI restriction fragment. The 6.5-kilobase DNA fragment was initially isolated from a lambda phage library made from a ouabain-resistant secondary transformant and subsequently was subcloned in the plasmid vector pAT153. This plasmid was able to transform wild-type CV-1 cells to ouabain resistance at a frequency of about 10 cells per ng of DNA.
Rev Infect Dis 1984 May-Jun;6 Suppl 2:S514-5
Studying poliovirus with infectious cloned cDNA.
Racaniello VR
The application of molecular cloning to the study of poliovirus is discussed. Initially cDNA clones representing the entire genome of poliovirus type 1 (Mahoney) were prepared. These molecular clones were used for determining the complete nucleotide sequence of the viral genome. Subsequently a single cDNA clone representing the entire poliovirus genome was constructed. When this cDNA clone was transfected into cultured mammalian cells, infectious poliovirus was produced. The transfection-derived poliovirus was identical to wild-type virus by several criteria, including RNase T1-oligonucleotide fingerprint and 5' end nucleotide sequence. The availability of infectious cloned cDNA should make possible new approaches to the study of poliovirus.
Proc Natl Acad Sci U S A 1983 Oct;80(19):5885-9
Molecular cloning and characterization of hepatitis A virus cDNA.
Ticehurst JR, Racaniello VR, Baroudy BM, Baltimore D, Purcell RH, Feinstone SM
Double-stranded cDNA was synthesized from hepatitis A virus (HAV) RNA and inserted into the Pst I site of pBR322. Restriction endonuclease digestion and cross-hybridization of fragments yielded a map of overlapping cloned cDNAs that included at least 99% of the viral genome. Molecular clones containing HAV cDNA were identified by hybridizing cloned cDNA to electrophoretically resolved RNA from uninfected and HAV-infected tissue culture cells. Cloned cDNA probes specifically hybridized to RNA from infected cells, and the predominant species identified had the characteristic genomic length of picornaviral RNA (approximately equal to 7,500 nucleotides). A partial sequence from the 3' end of the genome revealed 414 bases in an open reading frame followed by two closely spaced stop codons, a 60-base noncoding region, and a tract of poly(A).
Science 1981 Nov 20;214(4523):916-9
Cloned poliovirus complementary DNA is infectious in mammalian cells.
Racaniello VR, Baltimore D
A complete, cloned complementary DNA copy of the RNA genome of poliovirus was constructed in the Pst I site of the bacterial plasmid pBR322. Cultured mammalian cells transfected with this hybrid plasmid produced infectious poliovirus. Cells transfected with a plasmid which lacked the first 115 bases of the poliovirus genome did not produce virus.
Proc Natl Acad Sci U S A 1981 Aug;78(8):4887-91
Molecular cloning of poliovirus cDNA and determination of the complete nucleotide sequence of the viral genome.
Racaniello VR, Baltimore D
The complete 7410 nucleotide sequence of poliovirus type I genome was obtained from cloned cDNA. Double-stranded poliovirus cDNA was synthesized and inserted into the Pst I site of plasmid pBR322, and three clones were derived that together provided DNA copies of the entire poliovirus genome. Two of the clones contained inserts of 2.5 and 6.5 kilobases and represented all but the 5' 115 bases of poliovirus RNA. A third clone was generated from primer-extended DNA and contained sequences from the 5' end of the viral RNA. An open reading frame that was identified in the nucleotide sequence starting 743 bases from the 5' end of the RNA and extending to a termination codon 71 bases from the 3' end contained known poliovirus polypeptide sequence.
Philos Trans R Soc Lond B Biol Sci 1980 Feb 25;288(1029):299-305
Genetic structure and genetic variation of influenza viruses.
Palese P, Racaniello VR, Desselberger U, Young J, Baez M
The following contribution summarizes our most recent results concerning analysis of the influenza A, B and C virus genomes. In addition, we present data on the extent of genetic variation of H1N1 influenza viruses isolated during the months following the 1977 outbreak of H1N1 influenza in China and Russia. A detailed description of these results is published elsewhere.
The 3' and 5'-terminal sequences of influenza A, B and C virus RNA segments are highly conserved and show partial inverted complementarity.
Desselberger U, Racaniello VR, Zazra JJ, Palese P
The 3'- and 5'-terminal nucleotides of the genome segments of an influenza A, B, and C virus were identified by directly sequencing viral RNA using two different sequencing techniques. A high degree of conservation at the 3' ends as well as at the 5' ends was observed among the genome segments of each virus and among the segments of the three different virus types. A uridine-rich region was observed from positions 17 through 22 at the 5' end of each segment. Moreover, the conserved 3' and 5'-terminal sequences showed partial and inverted complementarity. This feature results in very similar sequences at the 3' ends of the plus and minus strand RNAs and may also enable single-strand RNAs of influenza virus to form "panhandle" structures. Inverted complementary repeats may play an important role in initiation of viral RNA replication.
J Virol 1979 Dec;32(3):1006-14
Isolation of influenza C virus recombinants.
Racaniello VR, Palese P
Recombinants between two different influenza C viruses were isolated. In MDCK (canine kidney) cells, one strain, C/JJ/50, caused lytic plaques, whereas C/JHG/66 virus did not produce clear plaques. From a mixed infection of MDCK cells with C/JHG/66 virus and UV-inactivated C/JJ/50 virus, clones were isolated which possessed the clear-plaque phenotype. Fingerprint analyses indicated that the RNAs of parent viruses had different oligonucleotide patterns and that one of the clones derived from the mixed infection was formed by reassortment of parental genes. This recombinant clone most likely inherited RNAs 1, 2, 3, 6, and 7 from C/JGH/66 virus and RNAs 4 and 5 from C/JJ/50 virus.
Influenza B virus genome: assignment of viral polypeptides to RNA segments.
Racaniello VR, Palese P
It was shown that all eight RNA segments of influenza B viruses are most likely monocistronic and code for eight virus-specific polypeptides. A genetic map of the influenza B virus genome was established, and six polypeptides (P1 protein, nucleoprotein, hemagglutinin, neuraminidase, M protein, and nonstructural protein) were unambiguously assigned to specific RNA segments. Molecular weight estimates of the eight individual genes are obtained by using the glyoxal method. These results suggest that each influenza B virus RNA segment has a greater molecular weight than the influenza A virus RNA segment which codes for the analogous gene product.
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