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Biomedical subjects

E Wimmer

Publications and source records attributed to E Wimmer.

At least 55 records · Page 3Linked to original sources
PubMed1998

A protein linkage map of the P2 nonstructural proteins of poliovirus.

The yeast two-hybrid system was used to catalog all detectable interactions among the P2 nonstructural cleavage products of poliovirus type 1 (Mahoney). Evidence has been obtained for specific associations among 2A(pro), 2BC, 2C, and 2B. Specifically, 2A(pro) can interact with itself and 2BC and its cleavage products (2B and 2C) interact in all possible combinations, with the exception of 2C/2C. Detected interactions were confirmed in vitro by a glutathione S-transferase pulldown assay, which allowed us to detect 2C/2C association. transdominant-negative mutants of 2B (K. Johnson and P. J. Sarnow, J. Virol. 65:4341-4349, 1991) were examined and were found to retain interaction with wild-type 2B, perhaps reflecting a need for 2B multimerization in viral RNA replication. The multimerization of 2B was examined further by screening a mutagenized library for 2B variants that have lost the ability to bind wild-type 2B. The screen identified two nonconservative missense mutations within a central hydrophobic region, as well as truncations and frameshifts that implicate the C terminus in homointeraction. Introduction of the missense mutations into the genome of the virus conferred a quasi-infectious phenotype, an observation strongly suggesting that the 2B/2B interaction is required for replication of the viral genome.

Carrier Proteins↗
PubMed1998

Mechanism of injury-provoked poliomyelitis.

Skeletal muscle injury is known to predispose its sufferers to neurological complications of concurrent poliovirus infections. This phenomenon, labeled "provocation poliomyelitis," continues to cause numerous cases of childhood paralysis due to the administration of unnecessary injections to children in areas where poliovirus is endemic. Recently, it has been reported that intramuscular injections may also increase the likelihood of vaccine-associated paralytic poliomyelitis in recipients of live attenuated poliovirus vaccines. We have studied this important risk factor for paralytic polio in an animal system for poliomyelitis and have determined the pathogenic mechanism linking intramuscular injections and provocation poliomyelitis. Skeletal muscle injury induces retrograde axonal transport of poliovirus and thereby facilitates viral invasion of the central nervous system and the progression of spinal cord damage. The pathogenic mechanism of provocation poliomyelitis may differ from that of polio acquired in the absence of predisposing factors.

Animals↗
PubMed1998

Brefeldin A inhibits cell-free, de novo synthesis of poliovirus.

Brefeldin A (BFA), an inhibitor of intracellular vesicle-dependent secretory transport, is a potent inhibitor of poliovirus RNA replication in infected cells. We have determined that the unknown mechanism of BFA inhibition of replication is reproduced in the cell-free poliovirus translation, replication, and encapsidation system. Furthermore, we provide evidence suggesting that the cellular mechanism targeted by BFA, the GTP-dependent synthesis of secretory transport vesicles, may be involved in viral RNA replication in the system via a soluble cellular GTP-binding and -hydrolyzing activity. This activity is related to the ARF (ADP-ribosylation factor) family of GTP-binding proteins. ARFs are required for the formation of several classes of secretory vesicles, and some family members are indirectly inactivated by BFA. Peptides that function as competitive inhibitors of ARF activity in cell-free transport systems also inhibit poliovirus RNA replication, and this inhibitory effect can be countered by the addition of exogenous ARF. We suggest that BFA inhibition of replication is diagnostic of a requirement for ARF activity in the cell-free system.

ADP-Ribosylation Factors↗
PubMed1998

Complete protein linkage map of poliovirus P3 proteins: interaction of polymerase 3Dpol with VPg and with genetic variants of 3AB.

Poliovirus has evolved to maximize its genomic information by producing multifunctional viral proteins. The P3 nonstructural proteins harbor various activities when paired with different binding partners. These viral polypeptides regulate host cell macromolecular synthesis and function as proteinases, as RNA binding proteins, or as RNA-dependent RNA polymerase. A cleavage product of the P3 region is the genome-linked protein VPg that is essential in the initiation of RNA synthesis. We have used an inducible yeast two-hybrid system to analyze directly protein-protein interactions among P3 proteins. Sixteen signals of homo- or heterodimer interactions have been observed and have been divided into three groups. Of interest is the newly discovered affinity of VPg to 3Dpol that suggests direct interaction between these molecules in genome replication. A battery of 3AB variants (eight clustered-charge-to-alanine changes and five single-amino-acid mutations) has been used to map the binding determinants of 3AB-3AB interaction which were found to differ from the amino acids critical for the 3AB-3Dpol interaction. The viral proteinase 3Cpro was not found to interact with other 3Cpro molecules or with any other P3 polypeptide in yeast cells, a result confirmed by glutaraldehyde cross-linking. The weak apparent interaction between 3AB and 3CDpro scored in the yeast two-hybrid system was in contrast to a strong signal by far-Western blotting. The results elucidate, in part, previous results of biochemical and genetic analyses. The role of the interactions in RNA replication is addressed.

Amino Acid Sequence↗
PubMed1997

Genetic recombination of poliovirus in a cell-free system.

Genetic recombination of plus-strand RNA viruses is an important process for promoting genetic variation. By using genetically marked poliovirus RNAs, we have demonstrated that genetic recombination can occur in a cell-free system that generates infective virus from added poliovirus RNA. Recombinant polioviruses were isolated, and the region of crossing over was roughly mapped. Recombinants could be isolated even under conditions where the yield of viruses from one of the parental RNAs was depressed to levels comparable to or less than the yield of recombinant viruses, an observation suggesting that only one of the recombining RNAs needs to be replication-competent. The generation of poliovirus recombinants in a cell-free system offers new possibilities for studying recombination and evolution of RNA viruses.

Biological Evolution↗
PubMed1997

The promoters for human and monkey poliovirus receptors. Requirements for basic and cell type-specific activity.

The cellular receptors for poliovirus (PVR) are glycoproteins belonging to the immunoglobulin superfamily. Functional receptors for poliovirus are only expressed by primates; known rodent homologues lack the ability to bind virus due to amino acid differences. Human poliovirus infections are targeted to the gastrointestinal tract and, rarely, to motor neurons in the central nervous system. Available evidence suggests that poliovirus uses only one cellular receptor, implying that the tissue tropism of poliovirus is likely to be related to the expression of the human PVR (hPVR). However, low levels of expression of hPVR-specific mRNAs can be detected in many human tissues other than the apparent target cells. The nonpathogenic function of hPVR is unknown. For a study of the transcriptional control of hPVR expression, we have isolated and characterized the promoter of the hPVR gene. Deletion analysis defined an approximately 280 base pair minimal promoter fragment that: 1) lacks TATA- and CAAT-like elements, 2) is distinguished by a high GC content, and 3) promotes transcription at multiple start sites. The pattern of activity caused by transfection of serial 5'- and 3'-promoter deletions is almost identical in HEp2, HeLa, COS-1, and mouse L929 cells, indicating a similar transcriptional regulation of the hPVR promoter in these cell lines. However, on transfection of Raji cells, a Burkitt's lymphoma cell line harboring a transcriptionally inactive hPVR gene, all promoter reporter constructs tested exerted only residual activity. These results suggest that the cis-element(s) governing cell type-specific hPVR expression resides in the minimal promoter region. We also report the sequences of the promoters of two monkey homologues to hPVR (AGMalpha1 and AGMalpha2). Transcripts encoding the monkey poliovirus receptors originate from a region analogous to that identified for hPVR transcripts.

Animals↗
PubMed1996

Generation of a novel poliovirus with a requirement of hepatitis C virus protease NS3 activity.

Hepatitis C virus (HCV) is the major etiologic agent of non-A, non-B hepatitis. One of the difficulties in developing anti-HCV drugs is the lack of an efficient HCV cultivation system. We have generated an artificial surrogate virus suitable for testing the antiviral effects of drugs affecting HCV protease NS3, an enzyme believed to be essential for HCV proliferation. The surrogate virus genome is composed of most of the poliovirus genome and HCV protease NS3 and an NS3-specific cleavage site. The activity of HCV protease NS3 is required for proliferation of this chimeric virus. The antiviral efficacy of HCV protease inhibitors can, therefore, be evaluated by examining the effects of the drugs on the surrogate virus proliferation.

Amino Acid Sequence↗
PubMed1996

Internal ribosomal entry site substitution eliminates neurovirulence in intergeneric poliovirus recombinants.

Neuropathogenicity of poliovirus can be attenuated by mutations in the internal ribosomal entry site (IRES) within the 5' nontranslated region of its genome. The Sabin vaccine strains used in prevention of poliomyelitis carry such mutations in their IRES elements. In addition, mutations within the structural and nonstructural proteins of Sabin strains may equally contribute to the attenuation phenotype. Despite their effectiveness as vaccines, the Sabin strains retain a neuropathogenic potential in animal models for poliomyelitis and, at a very low rate, they can cause poliomyelitis in vaccine recipients. The elimination of the neurocytopathic phenotype was achieved through the exchange of the entire poliovirus IRES with its counterpart from human rhinovirus type 2 without affecting growth properties in nonneuronal cells. The attenuating effect of the human rhinovirus type 2 IRES within the context of a poliovirus genome has been mapped to the 3' portion of this genetic element.

Animals↗
PubMed1996

Poliovirus chimeras replicating under the translational control of genetic elements of hepatitis C virus reveal unusual properties of the internal ribosomal entry site of hepatitis C virus.

Chimeric genomes of poliovirus (PV) have been constructed in which the cognate internal ribosomal entry site (IRES) element was replaced by genetic elements of hepatitis C virus (HCV). Replacement of PV IRES with nt 9-332 of the genotype Ib HCV genome, a sequence comprising all but the first eight residues of the 5' nontranslated region (5'NTR) of HCV, resulted in a lethal phenotype. Addition of 366 nt of the HCV core-encoding sequence downstream of the HCV 5'NTR yielded a viable PV/HCV chimera, which expressed a stable, small-plaque phenotype. This chimeric genome encoded a truncated HCV core protein that was fused to the N terminus of the PV polyprotein via an engineered cleavage site for PV proteinase 3CPpro. Manipulation of the HCV core-encoding sequence of this viable chimera by deletion and frameshift yielded results suggesting that the 5'-proximal sequences of the HCV open reading frame were essential for viability of the chimera and that the N-terminal basic region of the HCV core protein is required for efficient replication of the chimeric virus. These data suggest that the bona fide HCV IRES includes genetic information mapping to the 5'NTR and sequences of the HCV open reading frame. PV chimeras replicating under translational control of genetic elements of HCV can serve to study HCV IRES function in vivo and to search for anti-HCV chemotherapeutic agents.

Base Sequence↗
PubMed1996

Genetic variation of the poliovirus genome with two VPg coding units.

Amongst the picornaviruses, poliovirus encodes a single copy of the genome-linked protein, VPg wheras foot-and-mouth disease virus uniquely encodes three copies of VPg. We have previously shown that a genetically engineered poliovirus genome containing two tandemly arranged VPgs is quasi-infectious (qi) that, upon genome replication, inadvertently deleted one complete VPg sequence. Using two genetically marked viral genomes with two VPg sequences, we now provide evidence that this deletion occurs via homologous recombination. The mechanism was abrogated when the second VPg was engineered such that its nucleotide sequence differed from that of the first VPg sequence by 36%. Such genomes also expressed a qi phenotype, but progeny viruses resulted from (i) random deletions yielding single VPg coding sequences of varying length lacking the Q*G cleavage site between the VPgs and (ii) mutations in the AKVQ*G cleavage sites between the VPgs at either the P4, P1 or P1' position. These variants present a unique genetic system defining the cleavage signals recognized in 3Cpro-catalyzed proteolysis. We propose a recognition event in the cis cleavages of the polyprotein P2-P3 region, and we present a hypothesis why the poliovirus genome does not tolerate two tandemly arranged VPg sequences.

Amino Acid Sequence↗
PubMed1996

Inhibition of basal transcription by poliovirus: a virus- encoded protease (3Cpro) inhibits formation of TBP-TATA box complex in vitro.

Host cell RNA polymerase II (pol II)-mediated transcription is inhibited by poliovirus infection. We demonstrate here that both TATA- and initiator-mediated basal transcription is inhibited in extracts prepared from poliovirus-infected HeLa cells. This inhibition can be reproduced by incubation of uninfected HeLa cell extracts with purified, recombinant poliovirus protease, 3Cpro. Transient-transfection assays demonstrate that 3Cpro, in the absence of other viral proteins, is able to inhibit cellular pol II-mediated transcription in vivo. Three lines of evidence suggest that inactivation of TATA-binding protein (TBP) is the major cause of inhibition of basal transcription by poliovirus. First, RNA pol II transcription in poliovirus-infected cell extract is fully restored by bacterially expressed TBP. Second, addition of purified TBP restores transcription in heat-treated nuclear extracts from mock- and virus-infected cells to identical levels. Finally, using a gel mobility shift assay, we demonstrate that incubation of TBP with the viral protease (3Cpro) inhibits its ability to bind TATA sequence in vitro. These results suggest that inhibition of pol II transcription in mammalian cells infected with poliovirus is, at least in part, due to the inability of modified TBP to bind pol II promoter sequences.

3C Viral Proteases↗
PubMed1995

Canyon rim residues, including antigenic determinants, modulate serotype-specific binding of polioviruses to mutants of the poliovirus receptor.

Several mouse cell lines expressing hybrid human poliovirus receptors (hPVRs) bearing mutations in the first immunoglobulin-like domain were previously characterized for their defective binding and replication of poliovirus type 1 Mahoney (G. Bernhardt, J. Harber, A. Zibert, M. DeCrombrugghe, and E. Wimmer, Virology, 203, 344-356, 1994). Here we report that these mutant hPVRs were utilized to explore differences in the binding behavior of the three serotypes of poliovirus. Type 3 polioviruses (both Sabin and the neurovirulent Leon strain) clearly bound to the hPVR mutant Q130G/GD, but were incapable of initiating infection. Also, binding at 25 degrees of poliovirus types 2 and 3 to cell lines expressing the hPVR mutants P84SYS/HPGA, L99GAE/AAAA, and D117F was greater than type 1 poliovirus. Further study of the serotype-specific interaction with mutant hPVRs was accomplished with antigenic hybrid viruses. Improved binding by antigenic hybrid viruses demonstrated that serotype-specific binding to mutant hPVRs is, in part, determined by the amino acid sequence of neutralization antigenic sites (NAgs) and the probable conformational rearrangement of amino acids adjacent to the NAg sites. Finally, site-directed mutants of poliovirus were utilized to determine the relative contributions, to hPVR interactions, of individual amino acids with solvent accessible side chains in the viral canyon. Of the 18 viable virus mutants produced, 3 (D1226A, I1089A, and VPEK1166HPGA) expressed impaired replication phenotypes on the mutant hPVR cell lines P84SYS/HYSA and D117F. A location at the rim of the poliovirus canyon was implicated for the interaction of the amino terminal domain of the poliovirus receptor with conserved and serotype-specific viral surface amino acids. The possible involvement of elements of neutralization antigenic sites in receptor binding may explain, in part, why poliovirus exists in only three serotypes.

Animals↗
PubMed1995

Cis-acting elements of the encephalomyocarditis virus internal ribosomal entry site.

Translation initiation of encephalomyocarditis virus (EMCV) mRNA occurs by ribosomal entry into the 5' untranslated region. Internal ribosome binding to EMCV mRNA requires a viral cis-acting element, termed the internal ribosomal entry site (IRES), and cellular trans-acting factors. The polypyrimidine tract binding protein (PTB) has been identified as one such trans-acting factor required for EMCV IRES-dependent translation. Using a dicistronic mRNA and an in vitro translation system, we have identified cis-acting elements of the EMCV IRES required for IRES-dependent translation. The results identify several regions of the IRES that are required for efficient IRES-dependent translation, including the PTB binding site. Other regions of the IRES may act only as spacer sequences, analogous to the spacer sequences found in rhinovirus and enterovirus IRES elements, to link essential regions of the IRES together. The flexibility of one region of the EMCV IRES was demonstrated by an insertion of 125 nucleotides that had little effect on IRES function while the constraint imposed on another region was demonstrated by a 3-nucleotide deletion that nearly abolished IRES-dependent translation.

Binding Sites↗
PubMed1995

Intragenomic complementation of a 3AB mutant in dicistronic polioviruses.

We report the construction of a poliovirus genome [pPVM-VPg(3F4A)] harboring a double mutation in VPg. This mutant, in which the tyrosine and the threonine at residues 3 and 4 of the VPg region were replaced by phenylalanine and alanine, respectively, is lethal, that is, all RNA synthesis was abolished and no revertants could be isolated. Using the properties of dicistronic polioviruses (with the general genotype PV 5'NTR-3AB-EMCV IRES-PV ORF-3'NTR), we have observed that the defect in RNA synthesis of the VPg(3F4A) mutant could be rescued by providing wild-type protein 3AB from the first open reading frame in trans. We conclude that the 3AB provided by the first cistron of the dicistronic construct was capable of "intragenomic complementation." Intragenomic complementation, however, was inefficient. Thus, the dicistronic RNAs were only quasi-infectious, and even first-passage viruses were found to have reverted to a functioning VPg in the polyprotein. This phenomenon underlines the role of polypeptide 3AB in multiple functions of viral proliferation. First-passage viruses, all of which expressed a small-plaque phenotype, had retained the original dicistronic genotype. At the fourth passage, however, all isolates were monocistronic, and they displayed complex genetic rearrangements revealing interesting information regarding IRES function.

Amino Acid Sequence↗
PubMed1995

The human poliovirus receptor. Receptor-virus interaction and parameters of disease specificity.

The host range of poliovirus is determined by the expression of the hPVR, a member of the immunoglobulin superfamily. We characterized hPVR proteins biochemically and found them to be complex-type glycoproteins. The outermost V-like domain of three extracellular domains harbors the PVR function. A panel of single or multiple amino acid exchanges were introduced throughout this domain in order to localize regions involved in virus-receptor interactions. Putative contact amino acids were found to reside in the C'C"D and DE regions. Binding and uptake of poliovirus paralleled virus replication in all mutants tested suggesting that virus binding was affected without abrogating the ability to mediate subsequent events in the infection. Although the primate PVR is essential in conferring susceptibility to poliovirus infection, certain strains can induce neurological disease in rodents. Mouse neurovirulent PV isolates of divergent serotypical origin each provoked a distinctive, characteristic neurological syndrome upon intracerebral infection of wild-type mice. We analyzed clinical and histopathological features of diffuse encephalomyelitis caused by these PV strains and compared the condition with poliomyelitis in mice transgenic for the hPVR. Diffuse PV encephalomyelitis in wild-type mice could be distinguished clinically and histopathologically from hPVR-mediated poliomyelitis in trangenic mice. We localized the determinants of mouse neurovirulence of PV1(LS-a), a derivative of PV1 (Mahoney), in a portion of the viral genome encompassing parts of the capsid protein VP1 as well as the nonstructural protein 2A. Mouse neuropathogenicity could possibly be conferred by reduced particle stability of PV1(LS-a) inasmuch as we found particles to be thermolabile.

Amino Acid Sequence↗
PubMed1995

Translation of encephalomyocarditis virus RNA by internal ribosomal entry.

Picornavirus 5' NCRs contain IRES elements that have been divided into two groups, exemplified by PV (type 1) and EMCV (type 2). These elements are functionally related and have an intriguing level of structural and sequence similarity. Some conserved RNA sequences and/or structures may correspond to cis-acting elements involved in IRES function, so that there may also be similarities in the mechanism by which the two types or IRES promote initiation. The function of both types of IRES element appears to depend on a cellular 57 kDa polypeptide, which has been identified as the predominantly nuclear hnRNP protein PTB. However, a specific function for p57/PTB in translation has not yet been established. These two groups can be differentiated on the basis of their requirements for trans-acting factors. The EMCV IRES functions efficiently in a broader range of eukaryotic cell types than type 1 IRES elements, probably because the latter require additional factor(s). A second distinction between these IRES element is that initiation occurs directly at the 3' border of type 2 IRES elements, whereas a nonessential spacer of between 30 nt and 154 nt separates type 1 IRES elements from the downstream initiation codon.

Base Sequence↗