Biology of hepatitis delta virus.
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Hepatitis Delta Virus↗
Biomedical subjects
R A Bhat
Publications and source records attributed to R A Bhat.
Suppression of the translation defect phenotype specific for a virus-associated RNA-deficient adenovirus mutant in monkey cells by simian virus 40.
Human cells infected with adenovirus type 2 (Ad2) or Ad5 require VAI RNA for efficient translation of viral mRNAs at late times after infection. The Ad5 mutant dl-sub720 synthesized neither virus-associated I (VAI) nor VAII RNAs, and infection of human cells with this mutant resulted in reduced virion polypeptide synthesis. Infection of monkey cells with this mutant also resulted in drastic reduction of polypeptide synthesis compared with wild-type (WT) adenovirus infections. Steady-state levels of hexon-specific mRNA were found to be comparable in WT- and mutant-infected monkey cells. The in vitro translation experiments showed that double-mutant- and WT-infected cells contained comparable levels of translatable hexon mRNA (and other adenovirus late mRNAs), suggesting that the severe inhibition of hexon protein synthesis in the VA mutant involves a translation block. Preinfection of monkey cells with simian virus 40 fully restored the efficient translation of this mRNA in the VA mutant infections to the level observed in WT-infected cultures. These results raise the possibility that simian virus 40 may encode or induce factors that suppress the translation block that occurs during adenovirus infections in the absence of the VA RNAs.
Adenoviruses, Human↗
Construction and analysis of additional adenovirus substitution mutants confirm the complementation of VAI RNA function by two small RNAs encoded by Epstein-Barr virus.
Adenovirus VAI RNA is essential for the efficient initiation of translation of viral mRNAs at late times after infection. Recently, by constructing an adenovirus type 5 substitution mutant, we showed that the Epstein-Barr virus encoded two small RNAs complemented for the VAI RNA function in the adenovirus type 5 lytic growth (Bhat and Thimmappaya, Proc. Natl. Acad. Sci. USA 80:4789-4793, 1983). This observation was based on our inability to propagate an adenovirus type 5 mutant lacking functional VAI and VAII genes. Subsequently, it was found that this mutant was viable and able to grow to a low titer. Therefore, we examined the complementation of the VAI RNA function by the Epstein-Barr virus-encoded RNAs by constructing additional adenovirus type 5 substitution mutants containing multiple copies of the Epstein-Barr virus-encoded RNA genes in nonessential early transcriptional region III. The new substitution mutants synthesized viral polypeptides at late times at levels comparable to those observed in wild type-infected cells. Our results convincingly demonstrated that the two Epstein-Barr virus-encoded RNAs can efficiently complement for the VAI RNA-mediated translational defect in adenovirus-infected cells.
Adenoviruses, Human↗
Structural requirements of adenovirus VAI RNA for its translation enhancement function.
Recently, by genetic and biochemical approaches, it has been shown that adenovirus VAI RNA is required for efficient translation of viral mRNAs at late times after infection. To understand the nucleotide sequences and the domains of the VAI RNA that are responsible for the role of VAI RNA in enhancement of translation, a mutational analysis of the VAI gene was undertaken. Deletion, substitution, and insertion mutations covering most of the nucleotide sequences of VAI RNA were introduced into the VAI gene at the plasmid level. These mutant genes were then reintroduced into the virus, and growth properties of the mutant viruses were studied. The majority of the mutants retained normal or nearly normal levels of biological function. Mutations in the region between +43 and +53 and between +107 and the 3' end of the gene resulted in a considerable loss of activity. These mutants, however, grew significantly better than did an adenovirus type 5 mutant lacking both functional VAI and VAII genes, indicating that they retain a portion of their activity. Because no one mutation was able to completely abolish the function, we suggest that the VAI RNA may have multiple functional sites for its translation modulation function. These multiple sites may be short oligonucleotide sequences that may interact with cellular or viral components or both during translation.
Adenoviruses, Human↗
Adenovirus mutants with DNA sequence perturbations in the intragenic promoter of VAI RNA gene allow the enhanced transcription of VAII RNA gene in HeLa cells.
Ad2 VAI gene strongly competes for transcription with VAII gene in vitro. It has been suggested that this competition may be a basis for the large excess of VAI gene transcription in virus infected cells at late times. We have studied the effect of the DNA sequence perturbations of the intragenic promoter of the VAI gene on transcription of VAII gene at the level of viral chromosome. Several Ad5 mutants with mutations in the promoter of VAI gene were constructed and transcription of their VAI and VAII genes were analyzed in the infected cells. It was found that transcription of VAII gene increased dramatically when either Box A or Box B promoter sequences of VAI gene were mutated or when the entire VAI gene was replaced by a DNA segment with an unrelated DNA sequence. Thus, at late times, active transcription of VAI gene appears to partially repress transcription of VAII gene. Those mutants which synthesized large quantities of VAII RNA only grew more slowly yielding a titer which was 1/10 of that of their parent but 5 to 6 fold higher than that of an Ad5 mutant lacking both VAI and VAII genes.
Adenoviruses, Human↗
Two small RNAs encoded by Epstein-Barr virus can functionally substitute for the virus-associated RNAs in the lytic growth of adenovirus 5.
Adenovirus (Ad) serotypes 2 and 5 synthesize large amounts of two low molecular weight RNAs designated virus-associated (VA) 1 and 2. Recently, genetic and biochemical approaches have been used to show that Ad2 VA1 RNA is required for efficient translation of viral mRNAs produced at late times after infection. Primate cells harboring the Epstein-Barr virus genome (EBV) synthesize large amounts of two low molecular weight RNAs: EBER1 and EBER2. Striking similarities of gene organization have been noted between the genes coding for Ad5 VA RNAs and the EBV EBERs [Rosa, M. D., Gottlieb, E., Lerner, M. R. & Steitz, J. A. (1981) Mol. Cell. Biol. 1, 785-796]. To examine whether the EBRs can functionally substitute for the VA RNAs for the lytic growth of Ad5, we have constructed an Ad5 substitution mutant in which the two VA RNA genes have been deleted and replaced by an EBV DNA segment coding for the two EBERs. The resulting Ad5 mutant synthesizes large amounts of the EBERs and is viable. Thus, two small RNAs of EBV origin, having primary and secondary structures different from those of the VA RNAs, can functionally substitute for the VA RNAs in the lytic growth of Ad5. These results are discussed in the context of mechanism of function of the VA RNAs.
Adenoviruses, Human↗
Organization of the noncontiguous promoter components of adenovirus VAI RNA gene is strikingly similar to that of eucaryotic tRNA genes.
The intragenic transcriptional control region (internal promoter) of the adenovirus type 2 VAI RNA gene was mutated by deletion, insertion, and substitution of DNA sequences at the plasmid level. The mutant plasmids were assayed for in vitro transcriptional activity by using HeLa cell extracts. The mutant clones with substitution or insertion of DNA sequences or both between nucleotides +18 and +53 of the VAI RNA gene were all transcriptionally active, although to various extents. Substitution of unrelated DNA sequences up to +26 or between +54 and +61 abolished the transcriptional activity completely. Based on these results, the intragenic promoter sequences of the VAI RNA gene can be subdivided into two components: element A, +10 to +18; and element B, +54 to +69. The distance between the A and B components could be enlarged from its normal 35 base pairs to 75 base pairs without destroying the transcriptional activity. However, a deletion of 4 or 6 base pairs in the DNA segment separating the A and B components (segment C) reduced the transcriptional activity of the genes to less than 2% of that of the wild type. When the VAI RNA gene with its element A or B was substituted for the corresponding element A or B of the Xenopus laevis tRNAMet gene, the hybrid genes transcribed close to the level of the wild-type VAI RNA gene and about 10- to 20-fold more efficiently than the tRNAMet gene. Thus, the organization of DNA sequences in the internal promoter of the VAI RNA gene appears to be very similar to that of eucaryotic tRNA genes. This similarity suggests an evolutionary relationship of the VAI RNA gene to tRNA genes.
Adenoviridae↗