RESUMO
Trichomonas vaginalis viruses (TVV), which may regulate P270 gene expression in the protozoan pathogen T. vaginalis, are a group of divergent double-stranded (ds) RNA viruses. In the present study, the complete 4674-bp cDNA sequence of a 4.6-kb ds RNA from a newly identified TVV2-1 isolate was determined. The sequence of the plus-strand mRNA contains four open reading frames, which encode overlapping cap and pol genes in the reading frame 2 and reading frame 1, respectively, and two putative serine-threonine-rich basic proteins VP3 and VP4 in the third reading frame. An 85-kDa capsid protein and a 160-kDa CAP-POL fusion protein were identified in crude viruses by Western blotting experiments using antisera raised against gene-specific oligopeptides. In conjunction with the presence of a potential ribosomal slippery heptanucleotide G GGC CCC within the overlap of the cap and pol genes, these observations suggest that the pol gene of TVV2-1 is translated via a -1 ribosomal frameshifting event during translation of the cap gene. Our results also provide insight into the conservation among divergent dsRNA species from TVV and suggest that the genome of TVV2-1 may encode two extra genes in addition to the cap and pol genes.
Assuntos
DNA Complementar/genética , Vírus de RNA/genética , Trichomonas vaginalis/virologia , Sequência de Aminoácidos , Animais , Capsídeo/genética , Clonagem Molecular , DNA Complementar/química , RNA Polimerases Dirigidas por DNA/genética , Genoma Viral , Modelos Moleculares , Dados de Sequência Molecular , Fases de Leitura Aberta , Vírus de RNA/isolamento & purificação , Vírus de RNA/ultraestrutura , RNA Viral/química , RNA Viral/genética , Alinhamento de Sequência , Análise de Sequência de DNARESUMO
Co-infection by a 0.5-kb small double-stranded (ds) RNA together with Trichomonas vaginalis virus (TVV) genomic 4.6-kb dsRNA is commonly observed in a number of T. vaginalis isolates. By molecular cloning and primer extension experiments, the 497-bp cDNA sequence of a 0.5-kb dsRNA co-infecting with TVV-T1 in T vagina/is T1 isolate was elucidated. Consistent with the replication cycle of a typical dsRNA virus, a plus-strand viral RNA beginning at +1 of the 0.5-kb dsRNA was identified in infected T. vaginalis T1 cells by primer extension and Northern hybridization studies. The 0.5-kb dsRNA was separately encased in TVV capsids from the viral genomic dsRNA, as shown by protein analysis and electron microscopic examination of viral particles purified by multiple rounds of CsCl gradient centrifugation. The riboprobes transcribed from a cloned cDNA of the 0.5-kb dsRNA exhibited strong hybridization to a small dsRNA in a T vaginalis T9 isolate, which harbors a TVV-T9 distantly related to TVV-T1, but the same probes showed very little hybridization to the viral genomic dsRNA of both TVV-T1 and TVV-T9. Very little sequence homology between the 0.5-kb dsRNA and the 4.6-kb dsRNA in TVV-T1 was found by computer-assisted analysis, suggesting that the small dsRNA in T. vaginalis T1 is not derived from the genome of TVV-T1 or other distantly related T. vaginalis viruses. These results suggest that the small dsRNAs in T vaginalis are satellite RNAs of T. vaginalis virus.
Assuntos
Vírus de RNA/genética , RNA de Cadeia Dupla/genética , RNA Satélite/genética , Trichomonas vaginalis/virologia , Animais , Sequência de Bases , DNA Complementar/análise , DNA Complementar/genética , Dados de Sequência Molecular , Vírus de RNA/fisiologia , Alinhamento de Sequência , Replicação Viral/genéticaRESUMO
Trichomonas vaginalis virus (TVV) is a nonsegmented double-stranded (ds) RNA virus that infects the pathogenic protozoan T. vaginalis. To study the virus, we cloned the genomic ds RNA of a TVV-T1 isolate and obtained a contiguous 4647-bp cDNA sequence. Two overlapping genes separated by a + 1 reading frame shift were identified on the plus strand but none on the complementary strand RNA in this sequence. The upstream gene probably encodes a 75-kDa capsid protein, and the downstream gene encodes an 86-kDa polypeptide which is probably the viral RNA-dependent RNA polymerase (RDRP). A potential ribosomal slippage heptamer (C CUU UUU) was identified within the 14-nt overlap of the two genes. The genomic organization and RDRP sequence in TVV-T1 exhibit similarity to those of Saccharomyces cerevisiae virus and Leishmania RNA virus, suggesting that these viruses originate from common ancestry, but are only distantly related to Giardia lamblia virus.
