Cell recognition and entry by rhino- and enteroviruses.
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Animals↗
Biomedical subjects
E Wimmer
Publications and source records attributed to E Wimmer.
Interaction of the poliovirus receptor with poliovirus.
The structure of the extracellular, three-domain poliovirus receptor (CD155) complexed with poliovirus (serotype 1) has been determined to 22-A resolution by means of cryo-electron microscopy and three-dimensional image-reconstruction techniques. Density corresponding to the receptor was isolated in a difference electron density map and fitted with known structures, homologous to those of the three individual CD155 Ig-like domains. The fit was confirmed by the location of carbohydrate moieties in the CD155 glycoprotein, the conserved properties of elbow angles in the structures of cell surface molecules with Ig-like folds, and the concordance with prior results of CD155 and poliovirus mutagenesis. CD155 binds in the poliovirus "canyon" and has a footprint similar to that of the intercellular adhesion molecule-1 receptor on human rhinoviruses. However, the orientation of the long, slender CD155 molecule relative to the poliovirus surface is quite different from the orientation of intercellular adhesion molecule-1 on rhinoviruses. In addition, the residues that provide specificity of recognition differ for the two receptors. The principal feature of receptor binding common to these two picornaviruses is the site in the canyon at which binding occurs. This site may be a trigger for initiation of the subsequent uncoating step required for viral infection.
Amino Acid Sequence↗
The antiviral compound 5-(3,4-dichlorophenyl) methylhydantoin inhibits the post-synthetic cleavages and the assembly of poliovirus in a cell-free system.
The mode of action of the antiviral drug 5-(3,4-dichlorophenyl) methylhydantoin (hydantoin) was studied in a cell-free system allowing de novo synthesis of poliovirus. This cell-free system, which is programmed with viral RNA, is suitable for the study of the late stages of poliovirus replication and, thus, for a study of antiviral compounds acting on these late stages. It was shown that, apart from the known inhibition of the assembly of poliovirus, hydantoin also blocks post-synthetic cleavages of poliovirus proteins. Our data demonstrate that the cell-free system is a sensitive tool to study the mode of action of antiviral compounds.
Antiviral Agents↗
A cysteine-rich motif in poliovirus protein 2C(ATPase) is involved in RNA replication and binds zinc in vitro.
Protein 2C(ATPase) of picornaviruses is involved in the rearrangement of host cell organelles, viral RNA replication, and encapsidation. However, the biochemical and molecular mechanisms by which 2C(ATPase) engages in these processes are not known. To characterize functional domains of 2C(ATPase), we have focused on a cysteine-rich motif near the carboxy terminus of poliovirus 2C(ATPase). This region, which is well conserved among enteroviruses and rhinoviruses displaying an amino acid arrangement resembling zinc finger motifs, was studied by genetic and biochemical analyses. A mutation that replaced the first cysteine residue of the motif with a serine was lethal. A mutant virus which lacked the second of four potential coordination sites for zinc was temperature sensitive. At the restrictive temperature, RNA replication was inhibited whereas translation and polyprotein processing, assayed in vitro and in vivo, appeared to be normal. An intragenomic second-site revertant which reinserted the missing coordination site for zinc and recovered RNA replication at the restrictive temperature was isolated. The cysteine-rich motif was sufficient to bind zinc in vitro, as assessed in the presence of 4-(2-pyridylazo)resorcinol by a colorimetric assay. Zinc binding, however, was not required for hydrolysis of ATP. 2C(ATPase) as well as its precursors 2BC and P2 were found to exist in a reduced form in poliovirus-infected cells.
Adenosine Triphosphatases↗
Genetic analysis of a poliovirus/hepatitis C virus (HCV) chimera: interaction between the poliovirus cloverleaf and a sequence in the HCV 5' nontranslated region results in a replication phenotype.
Internal ribosomal entry sites (IRESs) can function in foreign viral genomes or in artificial dicistronic mRNAs. We describe an interaction between the wild-type hepatitis C virus (HCV)-specific sequence and the poliovirus (PV) 5'-terminal cloverleaf in a PV/HCV chimeric virus (containing the HCV IRES), resulting in a replication phenotype. Either a point mutation at nucleotide (nt) 29 or a deletion up to nt 40 in the HCV 5' nontranslated region relieved the replication block, yielding PV/HCV variants replicating to high titers. Fortuitous yet crippling interactions between an IRES and surrounding heterologous RNA must be considered when IRES-based dicistronic expression vectors are being constructed.
5' Untranslated Regions↗
Identification of an RNA hairpin in poliovirus RNA that serves as the primary template in the in vitro uridylylation of VPg.
The first step in the replication of the plus-stranded poliovirus RNA is the synthesis of a complementary minus strand. This process is initiated by the covalent attachment of UMP to the terminal protein VPg, yielding VPgpU and VPgpUpU. We have previously shown that these products can be made in vitro in a reaction that requires only synthetic VPg, UTP, poly(A), purified poliovirus RNA polymerase 3D(pol), and Mg(2+) (A. V. Paul, J. H. van Boom, D. Filippov, and E. Wimmer, Nature 393:280-284, 1998). Since such a poly(A)-dependent process cannot confer sufficient specificity to poliovirus RNA replication, we have developed a new assay to search for a viral RNA template in conjunction with viral or cellular factors that could provide this function. We have now discovered a small RNA hairpin in the coding region of protein 2C as the site in PV1(M) RNA that is used as the primary template for the in vitro uridylylation of VPg. This hairpin has recently been described in poliovirus RNA as being an essential structure for the initiation of minus strand RNA synthesis (I. Goodfellow, Y. Chaudhry, A. Richardson, J. Meredith, J. W. Almond, W. Barclay, and D. J. Evans, J. Virol. 74:4590-4600, 2000). The uridylylation reaction either with transcripts of cre(2C) RNA or with full-length PV1(M) RNA as the template is strongly stimulated by the addition of purified viral protein 3CD(pro). Deletion of the cre(2C) RNA sequences from minigenomes eliminates their ability to serve as template in the reaction. A similar signal in the coding region of VP1 in HRV14 RNA (K. L. McKnight and S. M. Lemon, RNA 4:1569-1584, 1998) and the poliovirus cre(2C) can be functionally exchanged in the assay. The mechanism by which the VPgpUpU precursor, made specifically on the cre(2C) template, might be transferred to the site where it serves as primer for poliovirus RNA synthesis, remains to be determined.
Base Sequence↗
Genetic and biochemical studies of poliovirus cis-acting replication element cre in relation to VPg uridylylation.
In addition to highly conserved stem-loop structures located in the 5'- and 3'-nontranslated regions, genome replication of picornaviruses requires cis-acting RNA elements located in the coding region (termed cre) (K. L. McKnight and S. M. Lemon, J. Virol. 70:1941-1952, 1996; P. E. Lobert, N. Escriou, J. Ruelle, and T. Michiels, Proc. Natl. Acad. Sci. USA 96:11560-11565, 1999; I. Goodfellow, Y. Chaudhry, A. Richardson, J. Meredith, J. W. Almond, W. Barclay, and D. J. Evans, J. Virol. 74:4590-4600, 2000). cre elements appear to be essential for minus-strand RNA synthesis by an as-yet-unknown mechanism. We have discovered that the cre element of poliovirus (mapping to the 2C coding region of poliovirus type 1; nucleotides 4444 to 4505 in 2C), which is homologous to the cre element of poliovirus type 3, is preferentially used as a template for the in vitro uridylylation of VPg catalyzed by 3D(pol) in a reaction that is greatly stimulated by 3CD(pro) (A. V. Paul, E. Rieder, D. W. Kim, J. H. van Boom, and E. Wimmer, J. Virol. 74:10359-10370, 2000). Here we report a direct correlation between mutations that eliminate, or severely reduce, the in vitro VPg-uridylylation reaction and produce replication phenotypes in vivo. None of the genetic changes significantly influenced translation or polyprotein processing. A substitution mapping to the first A (A4472C) of a conserved AAACA sequence in the loop of PV-cre(2C) eliminated the ability of the cre RNA to serve as template for VPg uridylylation and abolished RNA infectivity. Mutagenesis of the second A (A4473C; AAACA) severely reduced the yield of VPgpUpU and RNA infectivity was restored only after reversion to the wild-type sequence. The effect of substitution of the third A (A4474G; AAACA) was less severe but reduced both VPg uridylylation and virus yield. Disruption of base pairing within the upper stem region of PV-cre(2C) also affected uridylylation of VPg. Virus derived from transcripts containing mutations in the stem was either viable or quasi-infectious.
Base Sequence↗
Genetic recombination of poliovirus in vitro and in vivo: temperature-dependent alteration of crossover sites.
Genetic recombination that occurs with high frequency during poliovirus genome replication is a process whose molecular mechanism is poorly understood. Studies of genetic recombination in a cell-free system in vitro and in infected tissue culture cells in vivo have led to the unexpected observation that temperature strongly influences the loci at which cross-over between the two recombining RNA strands occurs. Specifically, cross-over between two genetically marked RNA strands in vitro and in vivo at 34 degrees C occurred over a wide range of the genome. In contrast, recombination in vivo at 37 and 40 degrees C yielded cross-over patterns that had shifted dramatically to a region encoding nonstructural proteins. Preferential selection of recombinants at 37 and 40 degrees C was ruled out by analyses of the growth kinetics of the recombinants. During the studies of recombination in the cell-free system we found that there is a direct correlation between the ability of a poliovirus RNA molecule to replicate in the cell-free system and its capacity to complement de novo virus synthesis programmed by another viral RNA.
Binding Sites↗
Characterization of the nucleoside triphosphatase activity of poliovirus protein 2C reveals a mechanism by which guanidine inhibits poliovirus replication.
The highly conserved non-structural protein 2C of picornaviruses is involved in viral genome replication and encapsidation and in the rearrangement of intracellular structures. 2C binds RNA, has nucleoside triphosphatase activity, and shares three motifs with superfamily III helicases. Motifs "A" and "B" are involved in nucleotide triphosphate (NTP) binding and hydrolysis, whereas a function for motif "C" has not yet been demonstrated. Poliovirus RNA replication is inhibited by millimolar concentrations of guanidine hydrochloride (GdnHCl). Resistance and dependence to GdnHCl map to 2C. To characterize the nucleoside triphosphatase activity of 2C, we purified poliovirus recombinant 2C fused to glutathione S-transferase (GST-2C) from Escherichia coli. GST-2C hydrolyzed ATP with a Km of 0.7 mM. Other NTPs, including GTP, competed with ATP for binding to 2C but were poor substrates for hydrolysis. Mutation of conserved residues in motif A and B abolished ATPase activity, as did mutation of the conserved asparagine residue in motif C, an observation indicating the involvement of this motif in ATP hydrolysis. GdnHCl at millimolar concentrations inhibited ATP hydrolysis. Mutations in 2C that confer poliovirus resistant to or dependent on GdnHCl increased the tolerance to GdnHCl up to 100-fold.
Acid Anhydride Hydrolases↗
Identification and characterization of the cis-acting elements of the human CD155 gene core promoter.
The CD155 protein is the founding member of a new group of related molecules within the immunoglobulin superfamily sharing a V-C2-C2 domain structure and significant amino acid identity. We have recently isolated the promoter of the CD155 gene so that we may determine the transcription factors that regulate its expression and possibly gain insight into the cell biology of this gene. Here we report the mapping of three cis-elements within the CD155 core promoter, designated FPI, II, and III. The results of linker scanning mutagenesis suggest that all three of these cis-elements are required in varying degrees for the promoter activity of the core promoter fragment. The relative contribution of each region ranked in the following order: III > II > I. Interestingly, footprint and electrophoretic mobility shift assays show that FPIII binding activity is much reduced in a human cell line that does not express CD155. Additionally, protein binding to FPI and FPII was also investigated. DNase I footprinting using recombinant hAP-2alpha indicated that this transcription factor bound to both the FPI and FPII regions of the CD155 core promoter fragment. Electrophoretic mobility shift assays and supershift analysis confirmed the binding of AP-2 from crude nuclear extracts to FPI and to FPII. Lastly, cotransfection of the CD155 promoter with an AP-2alpha expression vector indicates that overexpression of AP-2alpha modulated the promoter activity of a CD155 promoter construct.
Base Sequence↗
Paradoxes of the replication of picornaviral genomes.
A wealth of experimental data on the mechanism of the picornavirus genome replication has accumulated. Not infrequently, however, conclusions derived from these data appear to contradict each other. On the one hand, initiation of a complementary RNA strand can be demonstrated to occur in a solution containing only the poliovirus RNA polymerase, VPg, uridine triphosphate, poly(A) template and appropriate ions. On the other hand, convincing experiments suggest that efficient initiation of a viral complementary RNA strand requires complex cis-acting signals on the viral RNA template, additional viral and possibly cellular proteins as well as a membrane-containing environment. On the one hand, there is evidence that the viral RNA, in order to be replicated, should first be translated, but on the other hand, the viral RNA polymerase appears to be unable to overcome the ribosome barrier. Possible solutions for these and several other similar paradoxes are discussed, along with less contradictory results on the properties of the picornaviral replicative proteins. Recent results suggesting that recombination and other rearrangements of the viral RNA genomes may be accomplished not only by the replicative template switching but also by nonreplicative mechanisms are also briefly reviewed.
Genome, Viral↗
Poliovirus/Hepatitis C virus (internal ribosomal entry site-core) chimeric viruses: improved growth properties through modification of a proteolytic cleavage site and requirement for core RNA sequences but not for core-related polypeptides.
H.-H. Lu and E. Wimmer (Proc. Natl. Acad. Sci. USA 93:1412-1417, 1996) have demonstrated that the internal ribosomal entry site (IRES) of poliovirus (PV) can be functionally replaced by the related genetic element from hepatitis C virus (HCV). One important finding of this study was that open reading frame sequences 3' of the initiating AUG, corresponding to the open reading frame of the HCV core polypeptide, are required to create a viable chimeric virus. This made necessary the inclusion of a PV 3C protease (3Cpro) cleavage site for proper polyprotein processing to create the authentic N terminus of the PV capsid precursor. Chimeric PV/HCV (P/H) viruses, however, grew poorly relative to PV. The goal of this study was to determine the molecular basis of impaired replication and enhance the growth properties of this chimeric virus. Genetic modifications leading to a different proteinase (PV 2Apro) cleavage site between the HCV core sequence and the PV polyprotein (P/H701-2A) proved far superior with respect to viral protein expression, core-PV fusion polyprotein processing, plaque phenotype, and viral titer than the original prototype PV/HCV chimera containing the PV 3Cpro-specific cleavage site (P/H701). We have used this new virus model to answer two questions concerning the role of the HCV core protein in P/H chimeric viral proliferation. First, a derivative of P/H701-2A with frameshifts in the core-encoding sequence was used to demonstrate that production of the core protein was not necessary for the translation and replication of the P/H chimera. Second, a viral construct with a C-terminal truncation of 23 amino acids of the core gene was used to show that a signal sequence for signal peptidase processing, when present in the viral construct, is detrimental to P/H virus growth. The novel P/H chimera described here are suitable models for analyzing the function(s) of the HCV elements by genetic analyses in vivo and for antiviral drug discovery.
3C Viral Proteases↗
Dual stem loops within the poliovirus internal ribosomal entry site control neurovirulence.
In the human central nervous system, susceptibility to poliovirus (PV) infection is largely confined to a specific subpopulation of neuronal cells. PV tropism is likely to be determined by cell-external components such as the PV receptor CD155, as well as cell-internal constraints such as the availability of a suitable microenvironment for virus propagation. We reported previously that the exchange of the cognate internal ribosomal entry site (IRES) within the 5' nontranslated region of PV with its counterpart from human rhinovirus type 2 (HRV2) can eliminate the neuropathogenic phenotype in a transgenic mouse model for poliomyelitis without diminishing the growth properties in HeLa cells. We now show that attenuation of neurovirulence of PV/HRV2 chimeras is not confined to CD155 transgenic mice but is evident also after intraspinal inoculation into Cynomolgus monkeys. We have dissected the PV and HRV2 IRES elements to determine those structures responsible for neurovirulence (or attenuation) of these chimeric viruses. We report that two adjacent stem loop structures within the IRES cooperatively determine neuropathogenicity.
5' Untranslated Regions↗
Internal ribosomal entry site scanning of the poliovirus polyprotein: implications for proteolytic processing.
Based on previous studies of dicistronic polioviruses carrying two internal ribosomal entry sites (IRESes), we performed a novel experiment of IRES scanning through a polypeptide by inserting sequentially the IRES of encephalomyocarditis virus into the open reading frame (ORF) of the poliovirus polyprotein at selected 3Cpro-specific Q*G cleavage sites. No cytopathic effects were observed after transfection of HeLa cells with any of the dicistronic constructs, and no virus was recovered. In vitro translation of the dicistronic RNA transcripts in HeLa cell-free extracts revealed that multiple defects in the processing of the P2-P3 domain of the polyprotein is the primary reason for the lethal phenotypes. Surprisingly, the interruption of 3Cpro-catalyzed cleavages downstream of 2C interfered with the 2Apro-catalyzed, primary cleavage between P1 and P2. In contrast, insertion of a foreign coding sequence (V3 loop of human immunodeficiency virus type 1 gp120) into the ORF of the polyprotein at the 2C-3A junction yielded a viable virus that appeared to be genetically stable over several passages. The results of these experiments, which are generally applicable to analyses of viral polyproteins or multidomain polypeptides, suggest that processing of the P2-P3 domain by 3C-3CDpro is rapid and accurate only in the context of the unperturbed P2-P3 precursor; this is consistent with cleavages occurring in cis. Moreover, an intact 2C-3A precursor is not required for viral proliferation.
3C Viral Proteases↗
Protein-primed RNA synthesis by purified poliovirus RNA polymerase.
A small protein, VPg, is covalently linked to the 5' end of the plus-stranded poliovirus genomic RNA. Poliovirus messenger RNA, identical in nucleotide sequence to genomic RNA, is not capped at its 5' end by the methylated structure that is common to most eukaryotic mRNAs. These discoveries presented two problems. First, as cap structures are usually required for translation of mRNA into protein, how does this uncapped viral RNA act as a template for translation? Second, what is the function of VPg? The identification of the internal ribosomal-entry site, which allows the entry of ribosomes into viral mRNA independently of the 5' mRNA end, has solved the first conundrum. Here we describe the resolution of the second problem. VPg is linked to the genomic RNA through the 5'-terminal uridylic acid of the RNA. We show that VPg can be uridylylated by the poliovirus RNA polymerase 3Dpol. Uridylylated VPg can then prime the transcription of polyadenylate RNA by 3Dpol to produce VPg-linked poly(U). Initiation of transcription of the poliovirus genome from the polyadenylated 3' end therefore depends on VPg.
Amino Acid Sequence↗
Poliovirus and its cellular receptor: a molecular genetic dissection of a virus/receptor affinity interaction.
The ability of a virus to attach to a susceptible host cell is of utmost importance for the initiation of viral life cycle. Cell surface proteins called viral receptors mediate the initial steps of virus attachment and uptake. Poliovirus (PV) is one of the most studied animal viruses and its interaction with its cellular receptor, the human poliovirus receptor (hPVR) has been well characterized. This review will present our current understanding of the PV/hPVR interaction at the genetic and biochemical level. In addition, we will also discuss the implications of the PV/hPVR interaction on PV tissue tropism and the evolution of the three PV serotypes.
Animals↗
Formation of 4,4-dialkoxycyclohexa-2,5-dienone N-(thiol-S-yl)imine during reaction of 4-alkoxynitrosobenzenes with thiols in alcoholic solvents.
During the interaction of nitrosoarenes with glutathione in aqueous media, intermediate generation of a highly resonance-stabilized sulfenamide cation has been repeatedly suggested. Most intermediates and end products could be explained by reactions of this sulfenamide cation with different nucleophiles such as excess thiol, solvent water, and metabolically produced arylamine. The present paper presents evidence for adduct formation of the sulfenamide cation with solvent alcohol at neutral pH. Sulfenamide cations generated from 4-nitrosophenetole and 4-nitrosoanisole, respectively, are strongly suggested to form the metastable ketals 4-ethoxy-4-methoxycyclohexa-2,5-dienone N-(glutathion-S-yl)imine and 4,4-dimethoxycyclohexa-2,5-dienone N-(glutathion-S-yl)imine, respectively, during reaction with solvent methanol. Reaction of the two sulfenamide cations in ethanol yielded 4,4-diethoxycyclohexa-2, 5-dienone N-(glutathion-S-yl)imine and 4-ethoxy-4-methoxycyclohexa-2, 5-dienone N-(glutathion-S-yl)imine, respectively. Although the metastability of the ketals did not allow isolation of pure solid material, chromatographic and chemical behavior as well as tandem MS fragmentation substantiate a ketal structure of these intermediates. To confirm the proposed structure, new compounds, 2, 6-dimethyl-4-nitrosophenetole, 2,6-dimethyl-4-nitrophenetole, 2, 6-dimethyl-4-phenetidine, and N-(glutathion-S-yl)-N-hydroxy-4-aminoacetophenone, were synthesized and included in supportive experiments. In summary, the detection of ketals corroborates once more the occurrence of a sulfenamide cation which obviously not only reacts with soft nucleophiles such as GSH but, to a limited extent, also reacts with hard nucleophiles. The toxicological significance of this result is discussed.
Alcohols↗
Expression of foreign proteins by poliovirus polyprotein fusion: analysis of genetic stability reveals rapid deletions and formation of cardioviruslike open reading frames.
Using a strategy developed by R. Andino, D. Silvera, S. D. Suggett, P. I. Achacoso, C. J. Miller, D. Baltimore, and M. B. Feinberg (Science 265:1448-1451, 1994), we constructed recombinant polioviruses by fusing the open reading frame (ORF) of the green fluorescent protein gene (gfp) of Aequorea victoria or the gag gene (encoding p17-p24) of human immunodeficiency virus type 1 (HIV-1) to the N terminus of the poliovirus polyprotein. All poliovirus expression vectors constructed by us and those obtained from Andino et al. were found to be severely impaired in viral replication and genetically unstable. Upon replication, inserted sequences were rapidly deleted as early as the first growth cycle in HeLa cells. However, the vector viruses did not readily revert to the wild-type sequence but rather retained some of the insert plus the artificial 3Cpro/3CDpro cleavage site, engineered between the heterologous sequence and the poliovirus polyprotein, to give rise to genotypes reminiscent of cardioviruses. These virus variants that carry a small leader polypeptide were now relatively stable, and they grew better than their progenitor strains. Reverse transcription followed by PCR and sequence analysis of the genomic RNAs reproducibly revealed a few preferred genotypes among the isolated deletion variants. The remaining truncated inserts were retained through subsequent passages. In the immediate vicinity of the deletion borders, we observed short direct sequence repeats that we propose are involved in aligning RNA strands for illegitimate (nonhomologous) RNA recombination during minus-strand synthesis. On the basis of our results, which are at variance with published data, the utility of poliovirus vectors to express proteins > 10 kDa in size through fusion with the polyprotein needs to be reevaluated.
Amino Acid Sequence↗