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PubMed1984

Defective replication of porcine transmissible gastroenteritis virus in a continuous cell line.

During a search for established cell lines to produce large quantities of porcine transmissible gastroenteritis virus (TGEV), we observed bright immunofluorescent staining 6- 12h after infection of pig kidney derived LLC-PK1 line. Infectious virus yield was, however, 2 log10 lower than that from secondary adult pig thyroid (APT/2) cell cultures, although small plaques were visible by three days in cultures maintained under agarose, suggesting limited replication. Attempts to adapt TGEV to the LLC-PK1 cell line by 10 serial 20h passes were unsuccessful. Procedures to purify virions from infected LLC-PK1 cells produced less than 1% of the particles isolated from parallel APT/2 cultures. Examination of intracellular viral RNA in actinomycin-D treated cells revealed similar amounts of genomic RNA and the 4 major subgenomic species in both cell types, suggesting that there was no defect in viral RNA replication. In vitro translation of polyadenylated RNA from infected APT/2 and LLC-PK1 cells, followed by immune precipitation of the products, showed similar profiles of precursors to structural polypeptides, confirming the functional integrity of the viral messengers in the restrictive cell. Comparison of the viral polypeptides synthesised following infection of the two cell types showed that similar species were synthesised in both, corresponding to a group of 28-30,000 mol. wt. envelope glycopolypeptides, a 47,000 mol. wt. nucleoprotein and peplomer glycopolypeptides of about 200,000 mol. wt. The rate of viral polypeptide synthesis in LLC-PK1 cells was reproducibly higher than in APT/2, resulting in the earlier detection of bands and greater incorporation of isotope. Tunicamycin at 1 microgram/ml had a similar effect in both cells, preventing glycosylation of the 26,000 mol. wt. precursor of the envelope glycopolypeptides and synthesis of the 200,000 peplomer glycoprotein. Degradation of the nucleoprotein from 47,000 to 42,000 mol. wt. although detectable in both cells was more marked in the LLC-PK1 cultures. Phosphorylation of these proteins was readily demonstrated in both cells, although phosphorylation of host proteins and, to some extent, viral envelope proteins was considerably greater in the LLC-PK1. The significance of this finding with respect to virus maturation is being investigated.

Animals↗

PubMed1997

Interaction of hepatitis A virus (HAV) precursor proteins 3AB and 3ABC with the 5' and 3' termini of the HAV RNA.

RNA secondary structures within the terminal nontranslated regions of entero- and rhinoviral genomes interact specifically with viral nonstructural proteins and are required in cis for viral RNA replication. Here we show that recombinant hepatitis A virus (HAV) polypeptide 3ABC specifically interacts in vitro with secondary RNA structures formed at both the 5' and 3' terminus of the viral genome. Similar to protein 3AB, HAV 3ABC bound to the 3' terminal RNA structure which did not interact with the mature proteinase 3C. In contrast to 3AB, 3ABC interacted with RNA stem-loop IIa and combinations of individual secondary structure elements of the 5' noncoding region. RNA binding of the precursor polypeptide 3ABC was 50 times stronger than that of 3AB and 3C, implicating a specific role of this stable processing intermediate in viral genome replication.

3C Viral Proteases↗

PubMed1992

Selection of a ribozyme that functions as a superior template in a self-copying reaction.

The sunY ribozyme is derived from a self-splicing RNA group I intron. This ribozyme was chosen as a starting point for the design of a self-replicating RNA because of its small size. As a means of facilitating the self-replication process, the size of this ribozyme was decreased by the deletion of nonconserved structural domains; however, when such deletions were made, there were severe losses of enzymatic activity. In vitro genetic selection was used to identify mutations that reactivate a virtually inactive sunY deletion mutant. A selected mutant with five substitution mutations scattered throughout the primary sequence showed greater catalytic activity than the original ribozyme under the selection conditions. The sunY ribozyme and its small selected variant can both catalyze template-directed oligonucleotide assembly. The small size and reduced secondary structure of the selected variant results in an enhancement, relative to that of the original ribozyme, of its rate of self-copying. This engineered ribozyme is able to function effectively both as a catalyst and as a template in self-copying reactions.

Bacteriophage T4↗

PubMed2003

Clinical isolates of GB virus type C vary in their ability to persist and replicate in peripheral blood mononuclear cell cultures.

GB virus C/hepatitis G virus (GBV-C) replication in vitro is inefficient and inconsistent. In this study, clinical isolates of GBV-C were evaluated using peripheral blood mononuclear cell (PBMC) based culture methods. Isolates varied consistently in their ability to persistently replicate, and yield increased in cells grown without PHA/IL-2 stimulation. The deduced polyprotein sequence of an isolate that replicated well was determined (GenBank AY196904) and compared to 20 full-length GBV-C sequences. Fourteen of the 16 unique amino acid polymorphisms identified were in the coding regions for nonstructural proteins associated with interferon resistance and RNA replication. These data indicate that clinical GBV-C isolates vary in their ability to persist in culture, do not require PHA/IL-2 stimulation, and that sequence variability in key regulatory regions may affect growth in PBMC cultures. Since GBV-C appears to inhibit HIV replication in a coinfection model, these studies should facilitate determination of the mechanism of this interaction.

Amino Acid Sequence↗

PubMed1994

Secretory pathway function, but not cytoskeletal integrity, is required in poliovirus infection.

We examined the importance of two interactions between poliovirus and its host cell: the putative association between viral proteins and a rearranged intermediate filament (IF) network and the apparent requirement for functional vesicle budding machinery within the host-cell secretory pathway. Poliovirus capsid proteins appeared to associate with reorganized IF proteins during infection. Treatment of cells with cytochalasin D and nocodazole in combination disrupted normal cytoskeletal organization and prevented the poliovirus-induced redistribution of IF proteins to a juxtanuclear location. However, this treatment had no effect on viral yields from single-cycle infections, indicating that neither cytoskeletal integrity nor a specific poliovirus-induced rearrangement of IF proteins is required in the poliovirus life cycle. In contrast, we report that the inhibition of poliovirus replication by brefeldin A (BFA), an inhibitor of secretory membrane traffic, is specific to the host cell. Polioviral yields were not affected by BFA in two BFA-resistant cell lines, demonstrating that BFA targets a host protein or process required by poliovirus. No BFA-resistant virus was detected in these experiments, further supporting the hypothesis that poliovirus replication requires secretory pathway function, perhaps for the generation of vesicles on which viral RNA replication complexes are assembled.

Animals↗

PubMed2004

In vitro replication of hepatitis E virus (HEV) genomes and of an HEV replicon expressing green fluorescent protein.

Hepatitis E virus (HEV) RNA replication occurred in seven of nine primate cell cultures transfected with in vitro transcripts of an infectious cDNA clone. Cell-to-cell spread did not occur in cell cultures, but rhesus monkeys inoculated with lysates of HEV-transfected PLC/PRF/5 and Huh-7 cells became infected with HEV. A replicon with the ORF2 and ORF3 genes deleted and replaced with the green fluorescent protein gene also replicated in the same primate cells that supported the replication of the full-length genome. Fluorescence-activated cell sorter analysis confirmed that the 7mG cap structure was critical for efficient infectivity, although replication could be initiated at a very low level in its absence. HEV virions were also able to infect a limited number of cells of certain lines.

Animals↗

PubMed2003

The effect of ribavirin and IMPDH inhibitors on hepatitis C virus subgenomic replicon RNA.

The recent development of in vitro hepatitis C virus (HCV) RNA replication systems has provided useful tools for studying the intracellular anti-HCV activity of ribavirin. Ribavirin has been shown to: (1) induce "error catastrophe" in poliovirus, Proc. Natl. Acad. Sci. USA 98, 6895-6900), (2) be a pseudo-substrate of the HCV RNA-dependent RNA polymerase (RdRp) in vitro, J. Biol. Chem. 276, 46094-46098), and (3) increase mutations in HCV RNA in the binary T7 polymerase/HCV cDNA replication system, J. Virol. 76, 8505-8517). These findings have led to the hypothesis that ribavirin may also induce error catastrophe in HCV. However, the functional relevance of ribavirin-induced HCV RNA mutagenesis is unclear. By use of a colony formation assay, in which RNA is isolated from the HCV subgenomic replicon system following treatment, the impact of ribavirin, inosine-5'-monophosphate dehydrogenase (IMPDH) inhibitors, and the combination was assessed. Ribavirin reduced HCV replicon colony-forming efficiency (CFE) in a dose-dependent fashion, suggesting that ribavirin may be misincorporated into replicon RNA and result in an anti-replicon effect analogous to error catastrophe. This effect was markedly suppressed by addition of exogenous guanosine. Combination treatment with ribavirin and mycophenolic acid (MPA) or VX-497, both potent, nonnucleoside IMPDH inhibitors, led to a greatly enhanced anti-replicon effect. This enhancement was reversed by inclusion of guanosine with the treatment. In contrast, MPA or VX-497 alone had only marginal effects on both the quantity and quality (CFE) of replicon RNA, suggesting that although IMPDH inhibition is an important contributing factor to the overall ribavirin anti-HCV replicon activity, IMPDH inhibition by itself is not sufficient to exert an anti-HCV effect. Sequencing data targeting the neo gene segment of the HCV replicon indicated that ribavirin together with MPA or VX-497 increased the replicon error rate by about two-fold. Taken together these results further suggest that lethal mutagenesis may be an effective anti-HCV strategy. The colony formation assay provides a useful tool for evaluating mutagenic nucleoside analogs for HCV therapy. Finally, the data from combination treatment indicate potential therapeutic value for an enhanced anti-HCV effect when using ribavirin in combination with IMPDH inhibition.

Antimetabolites↗

PubMed2005

Enhancement of internal ribosome entry site-mediated translation and replication of hepatitis C virus by PD98059.

Translation initiation of hepatitis C virus (HCV) occurs in an internal ribosome entry site (IRES)-dependent manner. We found that HCV IRES-dependent protein synthesis is enhanced by PD98059, an inhibitor of the extracellular signal-regulated kinase (ERK) signaling pathway, while cellular cap-dependent translation was relatively unaffected by the compound. Treatment of cells with PD98059 allowed for robust HCV replication following cellular incubation with HCV-positive serum. Though the molecular mechanism underlying IRES enhancement remains elusive, PD98059 is a potent accelerator of HCV RNA replication.

Base Sequence↗

PubMed2005

Virus evolution: fitting lifestyles to a T.

The structure of a double-stranded RNA virus outer shell has revealed unexpected similarities with virions of positive-strand RNA viruses. These similarities intersect with emerging parallels in RNA replication to create intriguing evolutionary possibilities.

Biological Evolution↗

PubMed2004

Regulation of picornavirus gene expression.

Members of the Picornaviridae are positive- strand RNA viruses that cause a variety of human diseases such as poliomyelitis, the common cold, myocarditis, and hepatitis. Although the diseases caused by picornaviruses are diverse, the genome organization and mechanisms of gene expression are highly conserved among family members. This review will discuss the mechanisms of viral gene expression including cap-independent translation initiation, host cell translation shut off, viral polyprotein processing, and RNA replication.

Gene Expression Regulation, Viral↗

PubMed2003

Promising candidates for the treatment of chronic hepatitis C.

Chronic hepatitis C virus (HCV) infection is the cause of an emerging global pandemic of chronic liver disease. Current pegylated IFN-alpha/ribavirin combination therapies are merely 54 - 56% efficacious and are often poorly tolerated. Popular strategies to improve upon existing therapies include efforts to decrease the dosing regime, improve the safety profile and specifically target the liver, the site of HCV replication. A clear goal of novel therapies is to significantly improve the therapeutic response for HCV-infected patients. One popular scheme to accomplish this is to directly target the viral enzymes involved in HCV RNA replication. While peptidomimetics have been pursued as potent and specific inhibitors of the serine protease, nucleoside analogues and non-nucleoside small molecules have been explored as RNA-dependent RNA polymerase inhibitors with promising potential. Advances in the understanding of HCV replication at the molecular level that stem from the use of the subgenomic replicon system, in vitro enzyme assays and from co-crystallographic structure solutions of the replication enzymes with novel inhibitors have propelled these compounds into clinical development. As these candidates are developed further, there is great hope for a cure for all those chronically infected with HCV.

Antiviral Agents↗

PubMed1995

Foot and mouth disease virus replication in bovine skin Langerhans cells under in vitro conditions detected by RT-PCR.

The replication of foot and mouth disease virus (FMDV) was studied in isolated bovine skin Langerhans cells (LC), in keratinocytes from epidermal cell suspension, and in migrating LC obtained from cultured bovine epidermal sheets in vitro. Viral RNA replication in infected cells was determined by the reverse transcriptase-polymerase chain reaction (RT-PCR) of the negative FMDV RNA strand and by the plaque forming assay of FMDV. It was established that bovine skin LC, keratinocytes, and migratory bovine LC infected with FMDV strain 01 Geshur supported virus replication. This RT-PCR method to detect the negative strand of FMDV RNA in migratory bovine skin LC may be useful for determining FMD virus replication in tissue cells.

Amino Acid Sequence↗

PubMed2004

A phosphorothioate antisense oligodeoxynucleotide specifically inhibits coxsackievirus B3 replication in cardiomyocytes and mouse hearts.

Antisense oligodeoxynucleotides (AS-ODNs) are promising therapeutic agents for the treatment of virus-induced diseases. We previously reported that coxsackievirus B3 (CVB3) infectivity could be inhibited effectively in HeLa cells by phosphorothioate AS-ODNs complementary to different regions of the 5' and 3' untranslated regions of CVB3 RNA. The most effective target is the proximal terminus of the 3' untranslated region. To further investigate the potential antiviral role of the AS-ODN targeting this site in cardiomyocytes (HL-1 cell line), corresponding AS-ODN (AS-7) was transfected into the HL-1 cells and followed by CVB3 infection. Analyses by RT-PCR, Western blotting and plaque assay demonstrated that AS-7 strongly inhibits viral RNA and viral protein synthesis as compared to scrambled AS-ODNs. The percent inhibitions of viral RNA transcription and capsid protein VP1 synthesis were 87.6 and 40.1, respectively. Moreover, AS-7 could inhibit ongoing CVB3 infection when it was given after virus infection. The antiviral activity was further evaluated in a CVB3 myocarditis mouse model. Adolescent A/J mice were intravenously administrated with AS-7 or scrambled AS-ODNs prior to and after CVB3 infection. Following a 4-day therapy, the myocardium CVB3 RNA replication decreased by 68% and the viral titers decreased by 0.5 log(10) in the AS-7-treated group as compared to the group treated with the scrambled AS-ODNs as determined by RT-PCR, in situ hybridization and viral plaque assay. Taken together, our results demonstrated a great potential for AS-7 to be further developed into an effective treatment towards viral myocarditis as well as other diseases caused by CVB3 infection.

3' Untranslated Regions↗

PubMed1987

Host factor-induced template modification during synthesis of poliovirus RNA in vitro.

Poliovirus RNA polymerase, 3Dpol, transcribes poliovirus RNA in vitro in the presence of a host factor (HF) preparation from uninfected HeLa cells to yield heterogeneous-size product RNAs. The products include some molecules larger than the template that represent self-primed elongations of the template from a 3'-terminal hairpin. We showed that transcription proceeded through the formation of a modified RNA intermediate that was generated by nucleolytic cleavage of the template by HF in the absence of nucleoside triphosphates. Cleavage resulted in the loss of the original poly(A) 3' end and the generation of new, heterogeneous 3' ends that formed self-priming structures that could then be elongated by 3Dpol or reverse transcriptase. The two stages of the reaction, (i) cleavage to yield self-priming templates and (ii) subsequent chain elongation to yield heterogeneous-size products up to nearly dimer length, could be separated. RNAs whose original 3' ends were chemically oxidized so as to prevent chain elongation showed no reduction in template activity after preincubation with HF. We conclude that an HF preparation that contains a low level of nuclease activity is sufficient to activate RNA templates for transcription by 3Dpol to generate up to apparent dimer-length products. This reaction likely has little relevance to the mechanism of poliovirus RNA replication in vivo. It is likely that numerous other factors or activities, in addition to 3Dpol, would also result in transcription of poliovirus RNA in vitro.

Carrier Proteins↗

PubMed1986

Nucleotide sequence and newly formed phosphodiester bond of spontaneously ligated satellite tobacco ringspot virus RNA.

The satellite RNA of tobacco ringspot virus (STobRV RNA) replicates and becomes encapsidated in association with tobacco ringspot virus. Previous results show that the infected tissue produces multimeric STobRV RNAs of both polarities. RNA that is complementary to encapsidated STobRV RNA, designated as having the (-) polarity, cleaves autolytically at a specific ApG bond. Purified autolysis products spontaneously join in a non-enzymic reaction. We report characteristics of this RNA ligation reaction: the terminal groups that react, the type of bond in the newly formed junction and the nucleotide sequence of the joined RNA. The nucleotide sequence of the ligated RNA shows that joining of the reacting RNAs restored an ApG bond. The junction ApG has a 3'-to-5' phosphodiester bond. Thus the net ligation reaction of STobRV (-)RNA is the precise reversal of autolysis. We discuss this new type of RNA ligation reaction and its implications for the formation of multimeric STobRV RNAs during replication.

Base Sequence↗

PubMed2005

Testing the modularity of the N-terminal amphipathic helix conserved in picornavirus 2C proteins and hepatitis C NS5A protein.

The N-terminal region of the picornaviral 2C protein is predicted to fold into an amphipathic alpha-helix that is responsible for the protein's association with membranes in the viral RNA replication complex. We have identified a similar sequence in the N-terminal region of NS5A of hepaciviruses that was recently shown to form an amphipathic alpha-helix. The conservation of the N-terminal region in two apparently unrelated proteins of two different RNA virus families suggested that this helix might represent an independent module. To test this hypothesis, we constructed chimeric poliovirus (PV) genomes in which the sequence encoding the N-terminal 2C amphipathic helix was replaced by orthologous sequences from other picornaviral genomes or a similar sequence from NS5A of HCV. Effects of the mutations were assessed by measuring the accumulation of viable virus and viral RNA in HeLa cells after transfection, examining membrane morphology in cells expressing chimeric proteins and by in vitro analysis of RNA translation, protein processing and negative strand RNA synthesis in HeLa cell extracts. The chimeras manifested a wide range of growth and RNA synthesis phenotypes. The results are compatible with our hypothesis, although they demonstrate that helix exchangeability may be restricted due to requirements for interactions with other viral components involved in virus replication.

Amino Acid Sequence↗

PubMed2004

Structure of foot-and-mouth disease virus RNA-dependent RNA polymerase and its complex with a template-primer RNA.

Genome replication in picornaviruses is catalyzed by a virally encoded RNA-dependent RNA polymerase, termed 3D. The enzyme performs this operation, together with other viral and probably host proteins, in the cytoplasm of their host cells. The crystal structure of the 3D polymerase of foot-and-mouth disease virus, one of the most important animal pathogens, has been determined unliganded and bound to a template-primer RNA decanucleotide. The enzyme folds in the characteristic fingers, palm and thumb subdomains, with the presence of an NH2-terminal segment that encircles the active site. In the complex, several conserved amino acid side chains bind to the template-primer, likely mediating the initiation of RNA synthesis. The structure provides essential information for studies on RNA replication and the design of antiviral compounds.

Amino Acid Motifs↗

PubMed1981

Prolonged intracerebral infection with poliovirus in asymptomatic mice.

A heretofore unrecognized prolonged asymptomatic infection has been identified in mice intracerebrally inoculated with Lansing strain type II poliovirus. Virus was detected by infectivity assay and nucleic acid hybridization and was found in brains as long as 77 days after injection. Viral RNA replication occurred, indicating that persisting virus was not an inactive residuum of the infecting dose. Virus was sometimes found by infectivity even when viral RNA could not be demonstrated by hybridization, but detection by nucleic acid hybridization alone never occurred. Among 56 animals--including mice that died of infection, mice with prolonged infection, and survivors from which virus was not recovered--no animal had a serum neutralization titer above 1:2 in a plaque reduction assay Moreover, brain homogenates of 30 survivors without virus did not have neutralization titers above 1:4 in the plaque reduction assay. These findings were the same as in mock-infected mice.

Animals↗