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At least 433 records · Page 24Linked to original sources
PubMed1994

The 5'-untranslated regions of picornavirus RNAs contain independent functional domains essential for RNA replication and translation.

The role of the 5'-untranslated region (5'UTR) in the replication of enteroviruses has been studied by using a series of poliovirus type 3 (PV3) replicons containing the chloramphenicol acetyltransferase reporter gene in which the 5'UTR was replaced by the 5'UTR of either coxsackievirus B4 or human rhinovirus 14 or composite 5'UTRs derived from sequences of PV3, human rhinovirus 14, coxsackievirus B4, or encephalomyocarditis virus. The results indicate that efficient replication of an enterovirus genome requires a compatible interaction between the 5'-terminal cloverleaf structure and the coding and/or 3'-noncoding regions of the genome. A crucial determinant of this interaction is the stem-loop formed by nucleotides 46 to 81 (stem-loop d). The independence of the cloverleaf structure formed by the 5'-terminal 88 nucleotides and the ribosome landing pad or internal ribosome entry site (IRES) was investigated by constructing a 5'UTR composed of the PV3 cloverleaf and the IRES from encephalomyocarditis virus. Chloramphenicol acetyltransferase gene-containing replicons and viruses containing this recombinant 5'UTR showed levels of replication similar to those of the corresponding genomes containing the complete PV3 5'UTR, indicating that the cloverleaf and the IRES may be regarded as functionally independent and nonoverlapping elements.

Base Sequence↗
PubMed2004

Regulation of Semliki Forest virus RNA replication: a model for the control of alphavirus pathogenesis in invertebrate hosts.

Alphavirus nonstructural proteins are translated as a polyprotein that is ultimately cleaved into four mature proteins called nsP1, nsP2, nsP3, and nsP4 from their order in the polyprotein. The role of this nonstructural polyprotein, of cleavage intermediates, and of mature proteins in synthesis of Semliki Forest virus (SFV) RNA has been studied using mutants unable to cleave one or more of the sites in the nonstructural polyprotein or that had the arginine sense codon between nsP3 and nsP4 changed to an opal termination codon. The results were compared with those obtained for Sindbis virus (SINV), which has a naturally occurring opal codon between nsP2 and nsP3. We found that (1) an active nonstructural protease in nsP2 is required for RNA synthesis. This protease is responsible for all three cleavages in the nonstructural polyprotein. (2) Cleavage between nsP3 and nsP4 (the viral RNA polymerase) is required for RNA synthesis by SFV. (3) SFV mutants that are able to produce only polyprotein P123 and nsP4 synthesize minus-strand RNA early after infection as efficiently as SF wild type but are defective in the synthesis of plus-strand RNA. The presence of sense or opal following nsP3 did not affect this result. At 30 degrees C, they give rise to low yields of virus after a delay, but at 39 degrees C, they are nonviable. (4) SFV mutants that produce nsP1, P23, nsP4, as well as the precursor P123 are viable but produce an order of magnitude less virus than wild type at 30 degrees C and two orders of magnitude less virus at 39 degrees C. The ratio of subgenomic mRNA to genomic RNA is much reduced in these mutants relative to the parental viruses. (5) At 30 degrees C, the variants containing an opal codon grow as well as or slightly better than the corresponding virus with a sense codon. At 39 degrees C, however, the opal variants produce significantly more virus. These results support the conclusion that SFV and SINV, and by extension all alphaviruses, regulate their RNA synthesis in the same fashion after infection. P123 and nsP4 form a minus-strand replicase that synthesizes plus-strand RNA only inefficiently, especially at the higher temperatures found in mammals and birds. A replicase containing nsP1, P23, and nsP4 can make both plus and minus strands, but prefers the promoter for genomic plus sense RNA to that for subgenomic mRNA. The fully cleaved replicase can make only plus-strand RNA, and prefers the promoter for subgenomic mRNA to that for genomic RNA. Alphaviruses alternate between infection of hematophagous arthropods and higher vertebrates. Although the infection of higher vertebrates is acute and often accompanied by disease, continuing transmission of the virus in nature requires that infection of arthropods be persistent and relatively asymptomatic. We propose that this mechanism for control of RNA synthesis evolved to moderate the pathogenicity of the viruses in their arthropod hosts.

Animals↗
PubMed2004

Defective RNA replication and late gene expression in temperature-sensitive influenza viruses expressing deleted forms of the NS1 protein.

Influenza A virus mutants expressing C-terminally deleted forms of the NS1 protein (NS1-81 and NS1-110) were generated by plasmid rescue. These viruses were temperature sensitive and showed a small plaque size at the permissive temperature. The accumulation of virion RNA in mutant virus-infected cells was reduced at the restrictive temperature, while the accumulation of cRNA or mRNA was not affected, indicating that the NS1 protein is involved in the control of transcription versus replication processes in the infection. The synthesis and accumulation of late virus proteins were reduced in NS1-81 mutant-infected cells at the permissive temperature and were essentially abolished for both viruses at the restrictive temperature, while synthesis and accumulation of nucleoprotein (NP) were unaffected. Probably as a consequence, the nucleocytoplasmic export of virus NP was strongly inhibited at the restrictive temperature. These results indicate that the NS1 protein is essential for nuclear and cytoplasmic steps during the virus cycle.

Active Transport, Cell Nucleus↗
PubMed1994

Novel infectious particles generated by expression of the vesicular stomatitis virus glycoprotein from a self-replicating RNA.

Self-propagating infectious particles were produced in animal cells transfected with an RNA replicon encoding a single viral structural protein, the vesicular stomatitis virus glycoprotein (VSV-G). The replicon is derived from an alphavirus, Semliki Forest virus (SFV), and encodes the SFV RNA replicase, but none of the SFV structural proteins. After transfection of the replicon into tissue culture cells, expression of G protein spread from small foci throughout the culture. Supernatants from the cells contained infectious, virus-like particles that could be passaged and were neutralized by anti-VSV serum. The majority of the infectious particles were smaller and less dense than either VSV or SFV. Characterization by electron microscopy showed membrane-enveloped vesicles that contained the VSV-G protein. Infectious particles were apparently generated by budding of vesicles containing VSV-G protein and the RNA replicon. These experiments reveal that an enveloped infectious agent can be much simpler than previously thought.

Animals↗
PubMed1991

Studies on the regulation of influenza virus RNA replication: a differential inhibition of the synthesis of vRNA segments in shift-up experiments with ts mutants.

The regulation of influenza virus vRNA synthesis in the course of the reproduction cycle was studied with the use of a series of ts mutants in shift-up experiments. The synthesis of vRNA segments was registered by means of polyacrylamide gel electrophoresis of nucleocapsid-associated RNA isolated from the infected cells labelled with [3H]uridine after the shift-up to a semi-permissive temperature. Each mutant exhibited a specific differential pattern of vRNA synthesis inhibition after the shift-up. The most affected segments were either vRNA 4, vRNAs 4 and 7, or vRNAs 4, 6, and 7 in cells infected, respectively, with ts mutants C15 (ts lesion in PB1 gene), C45 (ts lesion in PA gene) and CmN3 (ts lesion in NS gene). The synthesis of vRNAs 1, 2, and 3 was relatively resistant to the shift-up in the cells infected with C15 or C45 and more sensitive in the cells infected with C44 (ts lesion in PB2 gene) or CmN3. The replication of the "early" genes (vRNAs 5 and 8) was generally least affected by the shift-up. The results are discussed in connection with the "early-late" transition of vRNA synthesis pattern in the course of infection.

Animals↗
PubMed2003

Threonine 157 of influenza virus PA polymerase subunit modulates RNA replication in infectious viruses.

Previous results have shown a correlation between the decrease in protease activity of several influenza A virus PA protein mutants and the capacity to replicate of the corresponding mutant ribonucleoproteins (RNPs) reconstituted in vivo. In this work we studied the phenotype of mutant viruses containing these mutations. Viruses with a T162A mutation, which showed a very moderate decrease both in protease and replication activities of reconstituted RNPs, showed a wild-type phenotype. Viruses with a T157A mutation, which presented a severe decrease in protease activity and replication of RNPs, showed a complex phenotype: (i) transport to the nucleus of PAT157A protein was delayed, (ii) virus multiplication was reduced at both low and high multiplicities, (iii) transcriptive synthesis was unaltered while replicative synthesis, especially cRNA, was diminished, and (iv) viral pathogenesis in mice was reduced, as measured by loss of body weight and virus titers in lungs. Finally, recombinant viruses with a T157E mutation in PA protein, which resulted in a drastic reduction of protease and replication activities of RNPs, were not viable. These results indicate that residue T157 in PA protein is important for the capacity of viral polymerase to synthesize cRNA.

Animals↗
PubMed1998

Sequence analysis of a functional polymerase (L) gene of bovine respiratory syncytial virus: determination of minimal trans-acting requirements for RNA replication.

The complete nucleotide sequence of a functional clone of the large polymerase (L) gene of bovine respiratory syncytial virus (BRSV) strain A51908 was determined by analysis of cloned cDNAs obtained from genomic and mRNAs. The BRSV L gene is 6573 nt in length and the derived polypeptide has 2162 aa. Alignment of the sequences of the BRSV L gene, and its encoded protein, with sequences of the L gene and protein of human respiratory syncytial virus strain A2 showed 77% identity at the nucleotide level and 84% identity at the amino acid level. By comparison, the L gene and protein of avian pneumovirus showed only 50% identity at the nucleotide level and 64% identity at the amino acid level. A minigenome was constructed to encode a BRSV vRNA analogue containing the gene for chloramphenicol acetyltransferase (CAT) under the control of putative BRSV transcription motifs and flanked by the BRSV genomic termini. Transfection of plasmids encoding the BRSV minigenome, nucleocapsid protein (N), phosphoprotein (P) and L protein, each under the control of T7 promoter, into cells infected with a vaccinia virus recombinant expressing the T7 RNA polymerase gave rise to CAT activity and progeny with the minigenome. This result indicates that the N, P and L proteins are necessary and sufficient for transcription and replication of the BRSV minigenome and are functional. Further, inclusion of small amounts of the M2 protein along with the N, P and L proteins greatly augmented minigenome transcription.

Amino Acid Sequence↗
PubMed1991

Cerulenin, an inhibitor of lipid synthesis, blocks vesicular stomatitis virus RNA replication.

The replication of genomes of animal viruses in the cytoplasm of susceptible cells is usually coupled to specialized membrane structures. The inhibitor of lipid synthesis cerulenin blocks the formation of vesicular stomatitis virus polypeptides when added to cells soon after virus entry, but has much less effect on viral translation, or the acylation of the glycoprotein G, when cerulenin is added later during infection. By contrast, cerulenin powerfully blocks viral RNA synthesis or the incorporation of glycerol into lipids when present at any time after VSV-infection. These findings suggest that the synthesis of VSV RNA is dependent on continuous synthesis of lipids.

Cerulenin↗
PubMed1995

Interaction between the 5'-terminal cloverleaf and 3AB/3CDpro of poliovirus is essential for RNA replication.

On the basis of sequence alignments and secondary structure comparisons of the first 100 nucleotides of enterovirus and rhinovirus RNAs, chimeric constructs in which this region of poliovirus type 1 Mahoney [PV1(M)] is replaced with that of human rhinovirus type 2 (HRV2) or HRV14 have been engineered. These chimeric constructs contain the internal ribosomal entry site of either poliovirus or encephalomyocarditis virus. Independent of the internal ribosomal entry site elements, only the constructs containing either the PV1(M) or HRV2 cloverleaf sequences yielded viable viruses. The secondary structures of all three cloverleaves are quite similar. However, highly purified polioviral proteins 3CDpro and 3AB together bound to the PV1(M) and HRV2 cloverleaves, albeit with different affinities, whereas the HRV14 homolog did not interact with these proteins to any appreciable extent. These results support a mechanism of poliovirus genomic replication in which the formation of a complex between the cloverleaf structure and the 3CDpro/3AB proteins of poliovirus plays an essential role.

3C Viral Proteases↗