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

Bhat, Ramesh A.; Thimmappaya, Bayar

doi:10.1093/nar/12.19.7377

Cited by 60 publications

(64 citation statements)

References 20 publications

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“…In contrast, VA RNAII levels are reduced to only about 5% at the higher multiplicity (100 FFU/cell). This finding is in agreement with previous results showing that the VA RNAII promoter is much weaker than the VA RNAI promoter and loses activity under limiting conditions (8). It should be noted that all our experi- The VA RNA genes were separated near the apical loop into two halves and cloned into a reporter mRNA, generating target regions complementary to the 5Ј and 3Ј halves of both VA RNAs (see reference 3).…”

Section: Resultssupporting

confidence: 93%

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Adenovirus Virus-Associated RNAII-Derived Small RNAs Are Efficiently Incorporated into the RNA-Induced Silencing Complex and Associate with Polyribosomes

Xu¹,

Segerman²,

Zhou³

et al. 2007

J Virol

111

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Adenovirus type 5 encodes two highly structured short RNAs, the virus-associated (VA) RNAI and RNAII. Both are processed by Dicer into small RNAs that are incorporated into the RNA-induced silencing complex (RISC). We show here, by cloning of small RNAs, that approximately 80% of Ago2-containing RISC immunopurified from late-infected cells is associated with VA RNA-derived small RNAs (mivaRNAs). Most surprisingly, VA RNAII, which is expressed at 20-fold lower levels compared to that of VA RNAI, appears to be the preferred substrate for Dicer and accounts for approximately 60% of all small RNAs in RISC. The mivaRNAs are derived from the 3 strand of the terminal stems of the VA RNAs, with the major fraction of VA RNAII starting at position 138. The small RNAs derived from VA RNAI were more heterogeneous in size, with the two predominant small RNAs starting at positions 137 and 138. Collectively, our results suggest that the mivaRNAs are efficiently used for RISC assembly in late-infected cells. Potentially, they function as miRNAs, regulating translation of cellular mRNAs. In support of this hypothesis, we detected a fraction of the VA RNAII-derived mivaRNAs on polyribosomes.

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Section: Resultssupporting

confidence: 93%

“…4). This result was completely unexpected, since VA RNAI has been shown to be the major VA RNA expressed during a lytic adenovirus infection (8). To verify that the VA RNAs were expressed at the expected ratio in the 293-Ago2 cell line, under our experimental conditions, we quantitated VA RNA expression by Northern blotting.…”

Section: Resultsmentioning

confidence: 89%

Adenovirus Virus-Associated RNAII-Derived Small RNAs Are Efficiently Incorporated into the RNA-Induced Silencing Complex and Associate with Polyribosomes

Xu¹,

Segerman²,

Zhou³

et al. 2007

J Virol

111

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“…The transcriptional control underlying the differential expression of the two VA genes is not fully understood. Evidence points toward competition between the two VA promoters for a transcription factor present in limiting quantities (4).…”

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confidence: 99%

Multiple proteins bind to VA RNA genes of adenovirus type 2.

Dyke¹,

Roeder²

1987

Mol. Cell. Biol.

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Using fractionated HeLa cell nuclear extracts and both nuclease (DNase I) cleavage and chemical cleavage (methidiumpropyl-EDTA-Fe(II) protection methodologies, we demonstrated the presence of three proteins which interacted specifically, yet differentially, with the two VA genes of adenovirus type 2. One, previously identified as transcription initiation factor TFIIIC, bound to a site centered on the transcriptionally essential B-block concensus element of the VAI gene and, with a lower affinity, to the analogous site in the VAII gene. Another, identified as the cellular protein involved in adenovirus replication, nuclear factor I, bound to sites immediately downstream from the two VAI terminators (at approximately +160 and +200 (3,13,18). Spacing between the A and B blocks is important for maintaining transcriptional activity (3, 7). At least three cellular proteins are necessary to reconstitute transcription from the VA genes in vitro (37). These include the well-characterized RNA polymerase III and two subcellular fractions which have been functionally designated TFIIIB and TFIIIC. A component in TFIIIC has been shown by physical methods to interact directly with DNA sequences around the B-block consensus element (8,15,22 stoichiometric interaction of TFIIIB with the TFIIIC-VA gene complex, followed by rapid association of RNA polymerase III to form a complete preinitiation complex (8,20).During the course of adenovirus infection, the relative rates of synthesis of the two VA RNAs differ markedly (38). Transcription from both genes commences shortly after infection, with both RNAs being produced at roughly equivalent rates. This pattern continues until the onset of viral DNA replication, approximately 8 to 10 h postinfection, at which time VAII synthesis levels off while VAI synthesis accelerates. Thus, at late times the ratio of VAI to VAII RNA present may be as high as 40:1 (40). The transcriptional control underlying the differential expression of the two VA genes is not fully understood. Evidence points toward competition between the two VA promoters for a transcription factor present in limiting quantities (4).To understand the mechanistic basis for differential regulation of the VA genes, as well as the roles of sequence and factors in the mechanism of RNA polymerase ITT-mediated specific transcription, we undertook a study of those proteins which interact with the VA genes and their effects on transcription. With fractionated HeLa cell nuclear extracts and both DNase I (16) and methidiumpropyl-EDTA-Fe(II) (MPE) (35, 41) cleavage protection methodologies, three proteins were shown to interact specifically, yet differentially, with the two VA genes. Two of these proteins were identified as the polymerase III transcription factor TFIIIC and the DNA replication protein nuclear factor I (NFI), whereas the third, a VA gene binding protein designated VBP, was previously unrecognized. The possible functions of these proteins in VA transcription were analyzed in reconstituted systems. Whereas NFI exhibited no...

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“…The short VA RNA gene region was detected by the PCR using several strains of Ad40 and Ad41 having proven genome differences (van der Avoort et al, 1989) (data not shown). As with Adl2, the existence of one gene precludes the possibility of provision of a backup VA RNA as seen in Ad2 (Bhat & Thimmappaya, 1984). Nevertheless, it is not clear how the Ad40 and Ad41 VA RNAs might affect interferon tolerance, if indeed they do, except that the VA RNA of Ad41 may adopt more than one conformation, which could conceivably affect its interaction with the dsRNA-activated inhibitor of translation (DAI).…”

confidence: 99%

“…synthesis (S6derlund et al, 1976), but in VA RNA I gene mutants, the VA RNA n may partially substitute for it (Thimmappaya et al, 1982;Bhat & Thimmappaya, 1984). In contrast to Ad2, Adl2 of subgroup A has only one VA RNA species (F6hring et al, 1979;Shu et al, 1986).…”

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confidence: 99%