RESUMEN
Rotaviruses have a genome composed of 11 segments of double-stranded RNA (dsRNA) surrounded by three protein layers. The virus contains an RNA-dependent RNA polymerase that synthesizes RNA transcripts corresponding to all segments of the viral genome. These transcripts direct the synthesis of the viral proteins and also serve as templates for the synthesis of the complementary strand to form the dsRNA genome. In this work, we analyzed the kinetics of transcription and replication of the viral genome throughout the replication cycle of the virus using quantitative reverse transcription-PCR. The role of the proteins that form double-layered particles ([DLPs] VP1, VP2, VP3, and VP6) in replication and transcription of the viral genome was analyzed by silencing their expression in rotavirus-infected cells. All of them were shown to be essential for the replication of the dsRNA genome since in their absence there was little synthesis of viral mRNA and dsRNA. The characterization of the kinetics of RNA transcription and replication of the viral genome under conditions where these proteins were silenced provided direct evidence for a second round of transcription during the replication of the virus. Interestingly, despite the decrease in mRNA accumulation when any of the four proteins was silenced, the synthesis of viral proteins decreased when VP2 and VP6 were knocked down, whereas the absence of VP1 and VP3 did not have a severe impact on viral protein synthesis. Characterization of viral particle assembly in the absence of VP1 and VP3 showed that while the formation of triple-layered particles and DLPs was decreased, the amount of assembled lower-density particles, often referred to as empty particles, was not different from the amount in control-infected cells, suggesting that viral particles can assemble in the absence of either VP1 or VP3.
Asunto(s)
ARN Viral/biosíntesis , Rotavirus/fisiología , Transcripción Genética , Replicación Viral , Técnicas de Silenciamiento del Gen/métodos , Cinética , Interferencia de ARN , ARN Bicatenario/biosíntesis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Proteínas Estructurales Virales/antagonistas & inhibidores , Proteínas Estructurales Virales/metabolismo , Ensamble de VirusRESUMEN
The phenotype of a rotavirus SA-11 mutant, ts C, which carries a mutation in the gene coding for the viral RNA polymerase was studied in vitro. ts C viral transcription proved to be sensitive to temperature in a different way to that previously described. Like the wild type, the ts C mutant has an optimum for in vitro transcription at 45 degrees, but mRNA synthesis was inhibited at temperatures over 50 degrees. This mutant also showed a higher resistance to transcriptional inhibition by nucleotide analogues than the wild-type strain. The in vitro minus-strand RNA synthesis catalysed by ts C particles indicates that the mutant does not exhibit the expected increased sensitivity to temperatures over 31 degrees shown by the in vivo phenotype. As with plus-strand synthesis, the optimal temperature for the minus-strand synthesis assay was 45 degrees, but for temperatures over 55 degrees, the number of double-stranded RNA products was altered. Our results suggest that when in vitro plus- and minus-strand RNA synthesis in ts C and wild type are compared, the mutated VP1 motif affects both transcription and minus-strand synthesis, but in different ways. In infected cell cultures, the results also show that the phenotype associated with ts C seems to mainly affect the function of plus-strand RNA synthesis.
Asunto(s)
ARN Polimerasas Dirigidas por ADN/genética , ARN Bicatenario/biosíntesis , ARN Viral/biosíntesis , ARN/biosíntesis , Rotavirus/genética , Animales , Nucleótidos de Desoxiadenina/farmacología , Electroforesis en Gel de Poliacrilamida , Calor , Macaca mulatta , Mutación , Rotavirus/enzimología , Transcripción Genética/efectos de los fármacos , Fosfato de Vidarabina/análogos & derivados , Fosfato de Vidarabina/farmacologíaRESUMEN
Four cDNA clones were generated from the genomic dsRNA of an Australian isolate of pangola stunt Fijivirus (PaSV). Each clone hybridized with nucleic acid extracts from PaSV infected plants but not healthy plants. Further, each clone hybridized with more than one segment of the PaSV dsRNA genome. One clone was used to demonstrate that homology existed between the Australian isolate of PaSV and a South American isolate of PaSV although the isolates differed in the sizes of the genomic dsRNAs and in the vector species. The clone also hybridized with some segments of the maize rough dwarf Fijivirus genome.
Asunto(s)
Poaceae/virología , Reoviridae/aislamiento & purificación , Australia , Autorradiografía , Clonación Molecular , Sondas de ADN , ADN Complementario , Nucleótidos de Desoxicitosina/metabolismo , Genoma Viral , Radioisótopos de Fósforo , ARN Bicatenario/análisis , ARN Bicatenario/biosíntesis , ARN Bicatenario/genética , Reoviridae/genética , Homología de Secuencia de Ácido Nucleico , América del SurRESUMEN
Viral particles purified from species of the protozoan parasite Leishmania braziliensis subsp. guyanensis by centrifugation in CsCl gradients were examined for the presence of viral polymerase. We demonstrated that RNA-dependent RNA polymerase is associated with viral particles. Viral transcription was studied in vitro with pulse-chase experiments and by assaying the RNase sensitivity of the viral transcripts. Viral polymerase synthesized full-length transcripts within 1 h. Double-strained, genome-length, and single-stranded RNAs were produced in this system. The nature of the RNA extracted from virions was also tested by RNase protection assays; both single-stranded and double-stranded RNAs were found.