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V I Agol

Publications and source records attributed to V I Agol.

At least 19 recordsLinked to original sources
PubMed2001

Cell-specific proteins regulate viral RNA translation and virus-induced disease.

Translation initiation of the picornavirus genome is regulated by an internal ribosome entry site (IRES). The IRES of a neurovirulent picornavirus, the GDVII strain of Theiler's murine encephalomyelitis virus, requires polypyrimidine tract-binding protein (PTB) for its function. Although neural cells are deficient in PTB, they express a neural-specific homologue of PTB (nPTB). We now show that nPTB and PTB bind similarly to multiple sites in the GDVII IRES, rendering it competent for efficient translation initiation. Mutation of a PTB or nPTB site results in a more prominent decrease in nPTB than PTB binding, a decrease in activity of nPTB compared with PTB in promoting translation initiation, and attenuation of the neurovirulence of the virus without a marked effect on virus growth in non-neural cells. The addition of a second-site mutation in the mutant IRES generates a new PTB (nPTB) binding site, and restores nPTB binding, translation initiation and neurovirulence. We conclude that the tissue-specific expression and differential RNA-binding properties of PTB and nPTB are important determinants of cell-specific translational control and viral neurovirulence.

Animals↗
PubMed2001

Molecular mechanisms of translation initiation in eukaryotes.

Translation initiation is a complex process in which initiator tRNA, 40S, and 60S ribosomal subunits are assembled by eukaryotic initiation factors (eIFs) into an 80S ribosome at the initiation codon of mRNA. The cap-binding complex eIF4F and the factors eIF4A and eIF4B are required for binding of 43S complexes (comprising a 40S subunit, eIF2/GTP/Met-tRNAi and eIF3) to the 5' end of capped mRNA but are not sufficient to promote ribosomal scanning to the initiation codon. eIF1A enhances the ability of eIF1 to dissociate aberrantly assembled complexes from mRNA, and these factors synergistically mediate 48S complex assembly at the initiation codon. Joining of 48S complexes to 60S subunits to form 80S ribosomes requires eIF5B, which has an essential ribosome-dependent GTPase activity and hydrolysis of eIF2-bound GTP induced by eIF5. Initiation on a few mRNAs is cap-independent and occurs instead by internal ribosomal entry. Encephalomyocarditis virus (EMCV) and hepatitis C virus epitomize distinct mechanisms of internal ribosomal entry site (IRES)-mediated initiation. The eIF4A and eIF4G subunits of eIF4F bind immediately upstream of the EMCV initiation codon and promote binding of 43S complexes. EMCV initiation does not involve scanning and does not require eIF1, eIF1A, and the eIF4E subunit of eIF4F. Initiation on some EMCV-like IRESs requires additional noncanonical initiation factors, which alter IRES conformation and promote binding of eIF4A/4G. Initiation on the hepatitis C virus IRES is even simpler: 43S complexes containing only eIF2 and eIF3 bind directly to the initiation codon as a result of specific interaction of the IRES and the 40S subunit.

Amino Acid Sequence↗
PubMed2001

Poliovirus protein 3A inhibits tumor necrosis factor (TNF)-induced apoptosis by eliminating the TNF receptor from the cell surface.

Viral infections often trigger host defensive reactions by activating intrinsic (intracellular) and extrinsic (receptor-mediated) apoptotic pathways. Poliovirus is known to encode an antiapoptotic function(s) suppressing the intrinsic pathway. Here, the effect of poliovirus nonstructural proteins on cell sensitivity to tumor necrosis factor (TNF)-induced (i.e., receptor-mediated) apoptosis was studied. This sensitivity is dramatically enhanced by the viral proteinase 2A, due, most likely, to inhibition of cellular translation. On the other hand, cells expressing poliovirus noncapsid proteins 3A and 2B exhibit strong TNF resistance. Expression of 3A neutralizes the proapoptotic activity of 2A and results in a specific suppression of TNF signaling, including the lack of activation of NF-kappaB, due to elimination of the TNF receptor from the cell surface. In agreement with this, poliovirus infection results in a dramatic decrease in TNF receptor abundance on the surfaces of infected cells as early as 4 h postinfection. Poliovirus proteins that confer resistance to TNF interfere with endoplasmic reticulum-Golgi protein trafficking, and their effect on TNF signaling can be imitated by brefeldin A, suggesting that the mechanism of poliovirus-mediated resistance to TNF is a result of aberrant TNF receptor trafficking.

Animals↗
PubMed2001

Some problems of molecular biology of poliovirus infection relevant to pathogenesis, viral spread and evolution.

Molecular mechanisms of poliovirus reproduction in the human gut remain largely unexplored. Nevertheless, there are grounds to believe that the virus spreads from cell to cell, like that from person to person during natural circulation, and involves a relatively small proportion of the highly heterogeneous viral population generated by the previous host. This mechanism of random sampling is responsible for the majority of fixed mutations, and contributes to the maintenance of a certain level of viral fitness (virulence). In the long term, random sampling may lead to the decrease in fitness and even to extinction of some viral evolutionary branches, explaining cases of self-limiting poliovirus infection in immunodeficient patients. A low propensity of the Sabin viruses for natural circulation may also be a related phenomenon. The trend to decrease in fitness may be interrupted by the appearance of rare, fitter (more virulent) variants, which may be responsible for poliomyelitis outbreaks caused by wild type virus, and for the development of paralytic disease in chronic carriers of the Sabin vaccine. All these evolutionary events are largely stochastic and hence are unpredictable in principle.

Digestive System↗
PubMed2000

Early alteration of nucleocytoplasmic traffic induced by some RNA viruses.

A HeLa cell line expressing the green fluorescent protein fused to the SV40 T-antigen nuclear localization signal (EGFP-NLS) was established. Fluorescence in these cells was confined to the nuclei. After poliovirus infection, cytoplasmic fluorescence in a proportion of cells could be detected by 1 h postinfection (p.i.) and in virtually all of the fluorescent cells by 2 h p.i. The relocation could be prevented by cycloheximide but not by inhibition of poliovirus replication by guanidine. HCl. Nuclear exit of a protein composed of three copies of GFP fused to the NLS also occurred upon poliovirus infection. A similar redistribution of EGFP-NLS took place upon infection with coxsakievirus B3 and, to a lesser extent, with vesicular stomatitis virus. The EGFP-NLS efflux was not due to the loss of NLS. Thus, some positive-strand and negative-strand RNA viruses trigger a rapid nonspecific relocation of nuclear proteins.

Antigens, Polyomavirus Transforming↗
PubMed2000

A cell cycle-dependent protein serves as a template-specific translation initiation factor.

Cap-independent translation initiation on picornavirus mRNAs is mediated by an internal ribosomal entry site (IRES) in the 5' untranslated region (5' UTR) and requires both eukaryotic initiation factors (eIFs) and IRES-specific cellular trans-acting factors (ITAFs). We show here that the requirements for trans-acting factors differ between related picornavirus IRESs and can account for cell type-specific differences in IRES function. The neurovirulence of Theiler's murine encephalomyelitis virus (TMEV; GDVII strain) was completely attenuated by substituting its IRES by that of foot-and-mouth disease virus (FMDV). Reconstitution of initiation using fully fractionated translation components indicated that 48S complex formation on both IRESs requires eIF2, eIF3, eIF4A, eIF4B, eIF4F, and the pyrimidine tract-binding protein (PTB) but that the FMDV IRES additionally requires ITAF(45), also known as murine proliferation-associated protein (Mpp1), a proliferation-dependent protein that is not expressed in murine brain cells. ITAF(45) did not influence assembly of 48S complexes on the TMEV IRES. Specific binding sites for ITAF(45), PTB, and a complex of the eIF4G and eIF4A subunits of eIF4F were mapped onto the FMDV IRES, and the cooperative function of PTB and ITAF(45) in promoting stable binding of eIF4G/4A to the IRES was characterized by chemical and enzymatic footprinting. Our data indicate that PTB and ITAF(45) act as RNA chaperones that control the functional state of a particular IRES and that their cell-specific distribution may constitute a basis for cell-specific translational control of certain mRNAs.

Base Sequence↗
PubMed2000

Prothymosin alpha fragmentation in apoptosis.

We observed fragmentation of an essential proliferation-related human nuclear protein prothymosin alpha in the course of apoptosis induced by various stimuli. Prothymosin alpha cleavage occurred at the DDVD(99) motif. In vitro, prothymosin alpha could be cleaved at D(99) by caspase-3 and -7. Caspase hydrolysis disrupted the nuclear localization signal of prothymosin alpha and abrogated the ability of the truncated protein to accumulate inside the nucleus. Prothymosin alpha fragmentation may therefore be proposed to disable intranuclear proliferation-related function of prothymosin alpha in two ways: by cleaving off a short peptide containing important determinants, and by preventing active nuclear uptake of the truncated protein.

Apoptosis↗
PubMed2000

Cross-talk between orientation-dependent recognition determinants of a complex control RNA element, the enterovirus oriR.

The coxsackie B3 virus oriR is an element of viral RNA thought to promote the assembly of a ribonucleoprotein complex involved in the initiation of genome replication. The mutual orientation of its two helical domains X and Y is determined by a kissing interaction between the loops of these domains. Here, a genetic approach was worked out to identify spatial orientation-dependent recognition signals in these helices. Spatial orientation changes (due to linear and rotational shifts) were introduced by appropriate insertions/deletions of a single base pair into one or both of the domains, and phenotypic consequences caused by these mutations were studied. The insertion of a base pair into domain Y caused a defect in viral reproduction that could be suppressed by a base-pair insertion into domain X. Similarly, a defect in viral replication caused by a base-pair deletion from domain X could be suppressed by a base-pair deletion from domain Y. Thus, certain areas of the two domains should cross-talk to one another in the sense that a change of space position of one of them required an adequate reply (change of space position) from the other. Phenotypic effects of the local rotation of one or more base pairs (and of some other mutations) in either domain X or domain Y suggested that the two most distal base pairs of these domains served as orientation-dependent recognizable signals. The results were also consistent with the notion that the recognition of the distal base pair of domain Y involved a mechanism similar to the intercalation of an amino acid residue.

Animals↗
PubMed2000

Competing death programs in poliovirus-infected cells: commitment switch in the middle of the infectious cycle.

Productive poliovirus infection of HeLa cells leads to the canonical cytopathic effect (CPE), whereas certain types of abortive infection result in apoptosis. To define the time course of commitment to the different types of poliovirus-induced death, inhibitors of viral replication (guanidine HCl) or translation (cycloheximide) were added at different times postinfection (p.i.). Early in the infection (during the first approximately 2 h p.i.), predominantly proapoptotic viral function was expressed, rendering the cells committed to apoptosis, which developed several hours after viral expression was arrested. In the middle of infection, concomitantly with the onset of fast generation of viral progeny, the implementation of the viral apoptotic program was abruptly interrupted. In particular, activation of an Asp-Glu-Val-Asp (DEVD)-specific caspase(s) occurring in the apoptosis-committed cells was prevented by the ongoing productive infection. Simultaneously, the cells retaining normal or nearly normal morphology became committed to CPE, which eventually developed regardless of whether or not further viral expression was allowed to proceed. The implementation of the poliovirus-induced apoptotic program was suppressed in HeLa cells overexpressing the Bcl-2 protein, indicating that the fate of poliovirus-infected cells depends on the balance of host and viral pro- and antiapoptotic factors.

Apoptosis↗
PubMed2000

Evolution of circulating wild poliovirus and of vaccine-derived poliovirus in an immunodeficient patient: a unifying model.

We determined nucleotide sequences of the VP1 and 2AB genes and portions of the 2C and 3D genes of two evolving poliovirus lineages: circulating wild viruses of T geotype and Sabin vaccine-derived isolates from an immunodeficient patient. Different regions of the viral RNA were found to evolve nonsynchronously, and the rate of evolution of the 2AB region in the vaccine-derived population was not constant throughout its history. Synonymous replacements occurred not completely randomly, suggesting the need for conservation of certain rare codons (possibly to control translation elongation) and the existence of unidentified constraints in the viral RNA structure. Nevertheless the major contribution to the evolution of the two lineages came from linear accumulation of synonymous substitutions. Therefore, in agreement with current theories of viral evolution, we suggest that the majority of the mutations in both lineages were fixed as a result of successive sampling, from the heterogeneous populations, of random portions containing predominantly neutral and possibly adverse mutations. As a result of such a mode of evolution, the virus fitness may be maintained at a more or less constant level or may decrease unless more-fit variants are stochastically generated. The proposed unifying model of natural poliovirus evolution has important implications for the epidemiology of poliomyelitis.

Adolescent↗
PubMed1999

Structural requirements of the higher order RNA kissing element in the enteroviral 3'UTR.

The origin of replication ( oriR ) involved in the initiation of (-) strand enterovirus RNA synthesis is a quasi-globular multi-domain RNA structure which is maintained by a tertiary kissing interaction. The kissing interaction is formed by base pairing of complementary sequences within the predominant hairpin-loop structures of the enteroviral 3' untranslated region. In this report, we have fully characterised the kissing interaction. Site-directed mutations which affected the different base pairs involved in the kissing interaction were generated in an infectious coxsackie B3 virus cDNA clone. The kissing interaction appeared to consist of 6 bp. Distortion of the interaction by mispairing of each of the base pairs involved in this higher order RNA structure resulted in either temperature sensitive or lethal phenotypes. The nucleotide constitution of the base which gaps the major groove of the kissing domain was not relevant for virus growth. The reciprocal exchange of the complete sequence involved in the kissing resulted in a mutant virus with wild type virus growth characteristics arguing that the base pair constitution is of less importance for the initiation of (-) strand RNA synthesis than the existence of the tertiary structure itself.

3' Untranslated Regions↗
PubMed1999

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↗
PubMed1999

An attenuated variant of the GDVII strain of Theiler's virus does not persist and does not infect the white matter of the central nervous system.

The DA strain of Theiler's virus causes a persistent and demyelinating infection of the white matter of spinal cord, whereas the GDVII strain causes a fatal gray-matter encephalomyelitis. Studies with recombinant viruses showed that this difference in phenotype is controlled mainly by the capsid. However, conflicting results regarding the existence of determinants of persistence in the capsid of the GDVII strain have been published. Here we show that a GDVII virus whose neurovirulence has been attenuated by an insertion in the 5' noncoding region does not persist in the central nervous systems of mice. Furthermore, this virus infects the gray matter efficiently, but not the white matter. These results confirm the absence of determinants of persistence in the GDVII capsid. They suggest that the DA capsid controls persistence by allowing the virus to infect cells in the white matter of the spinal cord.

Animals↗
PubMed1999

Nonreplicative RNA recombination in poliovirus.

Current models of recombination between viral RNAs are based on replicative template-switch mechanisms. The existence of nonreplicative RNA recombination in poliovirus is demonstrated in the present study by the rescue of viable viruses after cotransfections with different pairs of genomic RNA fragments with suppressed translatable and replicating capacities. Approximately 100 distinct recombinant genomes have been identified. The majority of crossovers occurred between nonhomologous segments of the partners and might have resulted from transesterification reactions, not necessarily involving an enzymatic activity. Some of the crossover loci are clustered. The origin of some of these "hot spots" could be explained by invoking structures similar to known ribozymes. A significant proportion of recombinant RNAs contained the entire 5' partner, if its 3' end was oxidized or phosphorylated prior to being mixed with the 3' partner. All of these observations are consistent with a mechanism that involves intermediary formation of the 2',3'-cyclic phosphate and 5'-hydroxyl termini. It is proposed that nonreplicative RNA recombination may contribute to evolutionarily significant RNA rearrangements.

3' Untranslated Regions↗
PubMed1999

Distinct attenuation phenotypes caused by mutations in the translational starting window of Theiler's murine encephalomyelitis virus.

Upon initiation of translation of picornavirus RNA, the ribosome is believed to bind the internal ribosome entry site of the template and then to form a productive complex with a downstream RNA segment, the starting window. The presence or absence of an AUG triplet within the starting window of the RNA of Theiler's murine encephalomyelitis virus (a picornavirus) is known to modulate its neurovirulence. In this study, mutants of this virus in which the starting windows, lying upstream of the viral polyprotein reading frame, had AUGs with different nonoptimal contexts were engineered. Upon intracerebral inoculation of mice, the mutants proved to be partially attenuated, as judged by a significant increase in the dose causing paralysis in 50% of the animals (PD50). Mutants with similar PD50s might differ from one another by eliciting either a severe, fatal tetraplegy or only mild, recoverable neurologic lesions. Some of the mutants triggered a chronic inflammatory reaction in the white matter of the spinal cord in the absence of detectable viral RNA or antigen. Thus, point mutations changing the context of an AUG within the starting window outside the polyprotein reading frame may differently affect the morbidity and mortality caused by a viral infection and may result in distinct attenuation phenotypes.

Animals↗
PubMed1998

Two types of death of poliovirus-infected cells: caspase involvement in the apoptosis but not cytopathic effect.

The death of poliovirus-infected cells may occur in two forms: canonical cytopathic effect (CPE) (on productive infections) or apoptosis (when the viral reproduction is hindered by certain drugs or some other restrictive conditions). Morphological manifestations of the CPE and apoptosis, being distinct, share some traits (e.g., chromatin condensation and nuclear deformation). It was shown here that a permeable caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp-(OMe) fluoromethyl ketone (zVAD.fmk), prevented the development of the poliovirus-induced apoptosis on abortive infection. The apoptotic pathway could be dissected by an inhibitor of chymotrypsin-like serine proteases, N-tosyl-l-phenylalanine chloromethyl ketone (TPCK), which prevented the cleavage of DNA to oligonucleosome-sized pieces and nuclear fragmentation but did not suppress cellular shrinkage, cytoplasmic blebbing, and partial chromatin condensation. These results demonstrate that caspase activation is involved in the execution phase of the viral apoptosis and suggest that a nuclear subset of the apoptotic program is under a separate control, involving a TPCK-sensitive event. Neither zVAD.fmk nor TPCK, at the concentrations affecting the apoptotic response, exerted appreciable influence on the virus growth or cellular pathological changes on productive infection, indicating that the pathways leading to the poliovirus-evoked CPE and apoptosis are different.

Amino Acid Chloromethyl Ketones↗
PubMed1997

Kissing of the two predominant hairpin loops in the coxsackie B virus 3' untranslated region is the essential structural feature of the origin of replication required for negative-strand RNA synthesis.

Higher-order RNA structures in the 3' untranslated region (3'UTR) of enteroviruses are thought to play a pivotal role in viral negative-strand RNA synthesis. The structure of the 3'UTR was predicted by thermodynamic calculations using the STAR (structural analysis of RNA) computer program and experimentally verified using chemical and enzymatic probing of in vitro-synthesized RNA. A possible pseudoknot interaction between the 3D polymerase coding sequence and domain Y and a "kissing" interaction between domains X and Y was further studied by mutational analysis, using an infectious coxsackie B3 virus cDNA clone (domain designation as proposed by E. V. Pilipenko, S. V. Maslova, A. N. Sinyakov, and V.I. Agol (Nucleic Acids Res. 20:1739-1745, 1992). The higher-order RNA structure of the 3'UTR appeared to be maintained by an intramolecular kissing interaction between the loops of the two predominant hairpin structures (X and Y) within the 3'UTR. Disturbing this interaction had no effect on viral translation and processing of the polyprotein but exerted a primary effect on viral replication, as was demonstrated in a subgenomic coxsackie B3 viral replicon, in which the capsid P1 region was replaced by the luciferase gene. Mutational analysis did not support the existence of the pseudoknot interaction between hairpin loop Y and the 3D polymerase coding sequence. Based on these experiments, we constructed a three-dimensional model of the 3'UTR of coxsackie B virus that shows the kissing interaction as the essential structural feature of the origin of replication required for its functional competence.

Base Sequence↗