VAI Gene Research Articles

Complementation of adenovirus virus-associated RNA I gene deletion by expression of a mutant eukaryotic translation initiation factor.

Adenovirus VA RNAs (virus-associated RNAs) are small polymerase III transcripts that are required for efficient initiation of mRNA translation late in adenovirus infection. VAI RNA prevents double-stranded RNA (dsRNA) activation of the interferon-induced protein kinase (DAI kinase). Activation of this kinase results in phosphorylation of the alpha subunit of eukaryotic translation initiation factor 2 (eIF-2 alpha) and correlates with inhibition of translation initiation. In this report we show growth complementation of adenoviruses harboring deletions in the VAI gene in cell lines expressing a serine-to-alanine mutant of eIF-2 alpha. This serine-to-alanine mutant is resistant to phosphorylation by DAI kinase. These results directly show that the primary function of VAI RNA in the lytic adenovirus infection is the inhibition of eIF-2 alpha phosphorylation by DAI kinase and identify eIF-2 alpha as the target that mediates the effects of DAI kinase activation. Cells that express a mutant eIF-2 alpha will enable the isolation of specific host-range mutants for other types of viruses that are defective in the ability to inhibit DAI kinase.

Human transcription factor IIIC (TFIIIC): Purification, polypeptide structure, and the involvement of thiol groups in specific DNA binding

Human transcription factor IIIC (TFIIIC) is an initiation factor required for the in vitro transcription of 5 S RNA, tRNA, and adenovirus viral-associated (VA) RNA genes by RNA polymerase III. A TFIIIC activity which complemented purified TFIIIB and RNA polymerase III fractions for VA transcription was highly purified from cultured HeLa cells. This activity copurified through all chromatographic procedures, including B-block oligodeoxynucleotide affinity chromatography, with the two forms of TFIIIC detected by gel mobility shift assays with the VA gene (Hoeffler, W.K., Kovelman, R., and Roeder, R.G. (1988) Cell 53, 907–920). Both specific binding activity to the VAI gene and TFIIIC transcription activity were inhibited by the alkylating agents diisopropyl fluorophosphate, N-tosyl-L-phenylalanine chloromethyl ketone (TPCK), and N-ethylmaleimide, and to a lesser extent by N α-p-tosyl-L-lysine chloromethyl ketone, whereas neither activity was inhibited by phenylmethylsulfonyl fluoride. These data suggest further that the DNA binding and transcription assays scored the same protein(s). TPCK and N-ethylmaleimide inactivated TFIIIC solely through thiol group modification, since prior modification with the reversible thiol reagent 2,2′-dithiopyridine prevented permanent inactivation. The involvement of reduced thiol groups in the specific binding of TFIIIC to the VAI gene was further indicated by an increase in TFIIIC binding activity upon addition of dithiothreitol. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that a Mr = 126,000 polypeptide both eluted from a B-block oligodeoxynucleotide affinity column with the DNA binding and transcription activities of TFIIIC and was specifically cross-linked by UV to a 5-bromo-2-deoxynucleotide-substituted B-block oligodeoxynucleotide. The near identity of the TFIIIC molecular weight determined by gel filtration on SOTA Phase GF 200 (Mr = 140,000) suggests that TFIIIC in solution (in the presence of 0.3 M NaCl at pH 7.0) consists of a single polypeptide which is fairly globular in nature.

Ordering promoter binding of class III transcription factors TFIIIC1 and TFIIIC2.

The separation of the mammalian class III transcription factor TFIIIC into two functional components, termed TFIIIC1 and TFIIIC2, enabled an analysis of their functions in transcription initiation. Template competition assays were used to define the order with which these factors interact in vitro to form stable preinitiation complexes on the adenovirus VAI and Drosophila melanogaster tRNA(Arg) genes. The interaction between these genes and TFIIIC2, the factor that binds with high affinity to the B block, was both necessary and sufficient for template commitment. When either the VAI or tRNA(Arg) gene was preincubated with TFIIIC2 alone, transcription of a second gene added subsequently was excluded, indicating that TFIIIC2 bound stably to the first template. Furthermore, the interaction between TFIIIC2 and these genes must occur prior to that of TFIIIC1 or TFIIIB. Once TFIIIC2 was bound, TFIIIC1 could bind to the tRNA(Arg) and VAI genes, although its interaction with the VAI gene was less stable than that with the tRNA(Arg) gene. TFIIIB activity bound stably to the complex of both genes with TFIIIC2. These results demonstrate that TFIIIC2 is the first transcription factor to bind to these genes and that TFIIIB and TFIIIC1 can then interact in either order to form a preinitiation complex.

Ordering promoter binding of class III transcription factors TFIIIC1 and TFIIIC2.

The separation of the mammalian class III transcription factor TFIIIC into two functional components, termed TFIIIC1 and TFIIIC2, enabled an analysis of their functions in transcription initiation. Template competition assays were used to define the order with which these factors interact in vitro to form stable preinitiation complexes on the adenovirus VAI and Drosophila melanogaster tRNA(Arg) genes. The interaction between these genes and TFIIIC2, the factor that binds with high affinity to the B block, was both necessary and sufficient for template commitment. When either the VAI or tRNA(Arg) gene was preincubated with TFIIIC2 alone, transcription of a second gene added subsequently was excluded, indicating that TFIIIC2 bound stably to the first template. Furthermore, the interaction between TFIIIC2 and these genes must occur prior to that of TFIIIC1 or TFIIIB. Once TFIIIC2 was bound, TFIIIC1 could bind to the tRNA(Arg) and VAI genes, although its interaction with the VAI gene was less stable than that with the tRNA(Arg) gene. TFIIIB activity bound stably to the complex of both genes with TFIIIC2. These results demonstrate that TFIIIC2 is the first transcription factor to bind to these genes and that TFIIIB and TFIIIC1 can then interact in either order to form a preinitiation complex.

Surprises in polymerase III transcription

One of the basic truths to have emerged from the short history of eukaryotic molecular biology-and probably the main reason that the field is so excitingis that things frequently turn out very differently than anticipated. There are any number of examples of this, including genomic DNA rearrangements, 5’ capping, splicing and transsplicing, and non-template-encoded nucleotide incorporation. But almost nowhere has the truth,of “expect the unexpected” been more evident than with transcription by RNA polymerase III (pol Ill). Promoters for transcription by pol Ill were the first eukaryotic control regions to be carefully mapped, and based on prior results with prokaryotic RNA polymerases many investigators believed that they would also reside upstream of the initiation site. The first major surprise came when the control region of the Xenopus 5s gene was found to be within the coding region (Sakonju et al., Cell 79, 13-25, 1980); in this classic example, the sequences essential for transcription are actually closer to the 3’ than the 5’ end of the gene. Sequence elements necessary and sufficient for transcription of the adenovirus-encoded VAI gene and several tRNA genes were also shown to reside within the gene body (reviewed in Ciliberto et al., Curr. Topics Dev. Biol. 78, 59-88, 1983). Only after the “fact” of gene-internal pol Ill promoters was well established in textbooks did it become generally recognized that crucial elements of pol III promoters can also reside external to the coding region. First considered almost an oddity of the Bombyx system, the requirement for upstream sequences (in conjunction with geneinternal promoter domains) has now been documented in a variety of organisms and for numerous pol Ill-transcribed genes, yncluding those for several tRNAs, 5s RNA, and 7SL RNA (e.g., Larson et al., PNAS, 80, 3416-3420, 1983; Johnson and Raymond, JBC 259, 5990-5994, 1984; Morton and Sprague, PNAS 87, 5519-5922, 1984; Ullu and Weiner, Nature 378, 371-374, 1985). In at least some instances, the need for these external promoter domains (located within ~25 bp upstream of the initiation site) only becomes apparent under stringent reaction conditions. And now we have come full circle, with the again unexpected revelation that promoter domains essential for pol Ill transcription can also reside entirely upstream of the gene body. Deletion derivatives that lack the entire region downstream of the initiation site of the 7SK gene and the U6 gene can still be actively transcribed (Murphy et al., Cell 57,81-87, 1987; Das et al., EMBO J. 7, 1988, in press). These genes do contain a box A sequence-a geneinternal element that, in conjunction with box B (tRNA) or box C (5s) has been present in all pol Ill promoters (see Minireview

A comprehensive collection of point mutations in the internal promoter of the adenoviral VAI gene.

Point mutations in the adenoviral VAI gene promoter were generated by in vitro mutagenesis with sodium bisulfite and were identified by three phenotypes: the disappearance of certain restriction endonuclease cleavage sites, altered rates of transcription in cell-free extracts, and altered mobility of the RNA product on denaturing acrylamide gels. 25 different point mutations were assayed for their effect on VAI transcription in HeLa S100 extracts. Mutations which change the transcriptional ability of the gene were localized in the internal control regions which are highly conserved among genes transcribed by RNA polymerase III. The magnitude of the change in transcription correlates with the degree of conservation in the consensus sequence at the site of the mutation. Most of the mutations either do not change the transcription rate or reduce transcription less than 6-fold. Two mutations severely reduce the amount of RNA produced, whereas three mutations increase the production of VAI RNA. A second site revertant that partially restores transcription to a mutant gene that produces no detectable VAI has also been isolated.

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

Using fractionated HeLa cell nuclear extracts and both nuclease (DNase I) cleavage and chemical cleavage (methidiumpropyl-EDTA X 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). The third, a previously unrecognized VA gene binding protein termed VBP, bound immediately upstream of the B-block element in the VAI gene but showed no binding to VAII. Possible roles for these proteins in VA gene transcription were investigated in in vitro assay systems reconstituted with partially purified transcription factors (RNA polymerase III, TFIIIB, and TFIIIC). Although TFIIIC activity was present predominantly in fractions containing B-block binding activity, there was not complete correspondence between functional and DNA binding activities. The nuclear factor I-like protein had no effect when added to a complete transcription reaction. The presence of VBP appeared to depress the intrinsic ratio of VAI-VAII synthesis, thereby simulating the relative transcription levels observed early in adenovirus infection of HeLa cells. These observations suggest a model, involving both intragenic binding factors (VBP and TFIIIC) and variable template concentrations, for the differential regulation of VA transcription during the course of adenovirus infection.

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

Using fractionated HeLa cell nuclear extracts and both nuclease (DNase I) cleavage and chemical cleavage (methidiumpropyl-EDTA X 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). The third, a previously unrecognized VA gene binding protein termed VBP, bound immediately upstream of the B-block element in the VAI gene but showed no binding to VAII. Possible roles for these proteins in VA gene transcription were investigated in in vitro assay systems reconstituted with partially purified transcription factors (RNA polymerase III, TFIIIB, and TFIIIC). Although TFIIIC activity was present predominantly in fractions containing B-block binding activity, there was not complete correspondence between functional and DNA binding activities. The nuclear factor I-like protein had no effect when added to a complete transcription reaction. The presence of VBP appeared to depress the intrinsic ratio of VAI-VAII synthesis, thereby simulating the relative transcription levels observed early in adenovirus infection of HeLa cells. These observations suggest a model, involving both intragenic binding factors (VBP and TFIIIC) and variable template concentrations, for the differential regulation of VA transcription during the course of adenovirus infection.

Competitive and cooperative functioning of the anterior and posterior promoter elements of an Alu family repeat.

Similar to tRNA genes and the VAI gene, the Alu family repeats are transcribed by RNA polymerase III and contain a split intragenic promoter. Results of our previous studies have shown that when the anterior, box A-containing promoter element (5'-Pu-Pu-Py-N-N-Pu-Pu-Py-G-G-3' in which Pu is any purine, Py is any pyrimidine, and N is any nucleotide) of a human Alu family repeat is deleted, the remaining box B-containing promoter element (5'-G-A/T-T-C-Pu-A-N-N-C-3') is still capable of directing weak transcriptional initiation at approximately 70 base pairs (bp) upstream from the box B sequence. This is different from the tRNA genes in which the box A-containing promoter element plays the major role in the positioning of the transcriptional initiation site(s). To account for this difference, we first carried out competition experiments in which we show that the posterior element of the Alu repeat competes with the VAI gene effectively for the transcription factor C in HeLa cell extracts. We then constructed a series of contraction and expansion mutants of the Alu repeat promoter in which the spacing between boxes A and B was systematically varied by molecular cloning. In vitro transcription of these clones in HeLa cell extracts was analyzed by RNA gel electrophoresis and primer extension mapping. We show that when the box A and box B promoter sequences are separated by 47 to 298 bp, the transcriptional initiation sites remain 4 to 5 bp upstream from box A. However, this positioning function by the box A-containing promoter element was lost when the spacing was shortened to only 26 bp or increased to longer than 600 bp. Instead, transcriptional initiation occurred approximately 70 bp upstream from box B, similar to that in the clones containing only the box B promoter element. All the mutant clones were transcribed less efficiently than was the wild type. An increase in the distance between boxes A and B also activated a second box A-like element within the Alu family repeat. We compare these results with the results of tRNA gene studies. We also discuss this comparison in terms of the positioning function of the split class III promoter elements and the evolutionary conservation of the spacing between the two promoter elements for optimum transcriptional efficiency.

Competitive and cooperative functioning of the anterior and posterior promoter elements of an Alu family repeat.

Similar to tRNA genes and the VAI gene, the Alu family repeats are transcribed by RNA polymerase III and contain a split intragenic promoter. Results of our previous studies have shown that when the anterior, box A-containing promoter element (5'-Pu-Pu-Py-N-N-Pu-Pu-Py-G-G-3' in which Pu is any purine, Py is any pyrimidine, and N is any nucleotide) of a human Alu family repeat is deleted, the remaining box B-containing promoter element (5'-G-A/T-T-C-Pu-A-N-N-C-3') is still capable of directing weak transcriptional initiation at approximately 70 base pairs (bp) upstream from the box B sequence. This is different from the tRNA genes in which the box A-containing promoter element plays the major role in the positioning of the transcriptional initiation site(s). To account for this difference, we first carried out competition experiments in which we show that the posterior element of the Alu repeat competes with the VAI gene effectively for the transcription factor C in HeLa cell extracts. We then constructed a series of contraction and expansion mutants of the Alu repeat promoter in which the spacing between boxes A and B was systematically varied by molecular cloning. In vitro transcription of these clones in HeLa cell extracts was analyzed by RNA gel electrophoresis and primer extension mapping. We show that when the box A and box B promoter sequences are separated by 47 to 298 bp, the transcriptional initiation sites remain 4 to 5 bp upstream from box A. However, this positioning function by the box A-containing promoter element was lost when the spacing was shortened to only 26 bp or increased to longer than 600 bp. Instead, transcriptional initiation occurred approximately 70 bp upstream from box B, similar to that in the clones containing only the box B promoter element. All the mutant clones were transcribed less efficiently than was the wild type. An increase in the distance between boxes A and B also activated a second box A-like element within the Alu family repeat. We compare these results with the results of tRNA gene studies. We also discuss this comparison in terms of the positioning function of the split class III promoter elements and the evolutionary conservation of the spacing between the two promoter elements for optimum transcriptional efficiency.

Adenovirus stimulation of transcription by RNA polymerase III: evidence for an E1A-dependent increase in transcription factor IIIC concentration.

Human cells expressing adenovirus E1A proteins transcribe transfected tRNA and adenovirus VAI genes at greater than 10-fold higher levels than uninfected HeLa cells. Here we show that the increased transcription observed in vivo is reflected in the in vitro transcriptional activity of cell extracts. Depletion of E1A protein from these extracts by immunoprecipitation with a monoclonal antibody did not diminish the activity, suggesting that E1A proteins do not stimulate transcription directly. Fractionation of the extracts by chromatography on phosphocellulose suggests that the higher activity of extracts of adenovirus-infected cells was due to increased activity of the transcription factor (TF) which is the limiting component required for specific initiation of tRNA and VAI transcription in extracts of uninfected HeLa cells, i.e. TFIIIC. Template commitment titrations further suggest that the increased TFIIIC activity was due to an increase in the concentration of active TFIIIC. On the basis of these results and recent genetic analyses of early adenovirus promoters, we suggest that E1A proteins stimulate transcription of adenovirus genes indirectly by increasing the effective in vivo concentration of the limiting cellular transcription factors required for their transcription.

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.

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.

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.

Differential activation of RNA polymerase III-transcribed genes by the polyomavirus enhancer and the adenovirus E1A gene products.

We have compared the effect of the polyomavirus cis-acting transcriptional enhancer and the adenovirus trans-acting E1A gene on expression of RNA polymerase III-transcribed genes (the adenovirus VAI gene and a bacterial tRNA gene) using DNA transfection and transient expression assays. The polyomavirus enhancer has little effect upon transcription of the VAI gene by RNA polymerase III in any cell type tested (murine, hamster, or human). In contrast, expression of the E1A gene within adenovirus infected cells stimulates transcription of RNA polymerase III-transcribed genes from co-transfected DNAs. Human 293 cells, which constitutively produce adenovirus E1A gene products, also express high levels of RNA polymerase III transcripts from transfected DNAs.

Proximal and distal domains that control in vitro transcription of the adenovirus IVa2 gene.

The adenovirus IVa2 gene, which is expressed at an intermediate time in the viral infectious cycle, is separated from the adenovirus major late promoter (MLP) 5' start site by 210 base pairs and is transcribed from the opposite strand. In contrast to the MLP, the IVa2 gene does not contain a "TATA" box upstream from its 5' start sites. By using a series of deletion mutants, two upstream control regions that are rich in cytidine residues, one proximal to the cap site at nucleotide positions -39 to -48 and a distal domain between nucleotide positions -152 and -242 have been identified as essential for IVa2 transcription (IVa2 cap site is nucleotide position + 1). Transcription efficiency is decreased by 70-90% after the deletion of a proximal C-rich domain when either linear or supercoiled DNAs were used as template. However, distal sequences functioned as transcriptional control domains only with covalently closed DNA templates. The deletion of both the proximal and distal regions from covalently closed DNA templates reduces the levels of IVa2 transcription by a factor of 100-150. When the plasmid pAd242 that contains the 5' start sites of adenovirus MLP and IVa2 is transcribed, there is essentially a complete suppression of transcription of the adenovirus IVa2 gene. The transcription efficiency of IVa2 is increased 10-fold after deletion of the MLP cap site. A model based on a shared entry site for RNA polymerase II and competition between major late and IVa2 promoters is proposed to explain the in vitro transcriptional results.

Specific transcription of an adenoviral gene that possesses no TATA sequence homology in extracts of HeLa cells.

Transcription of the adenovirus type 2 (Ad2) IVa2 gene, which contains no TATA-like sequence in the region immediately upstream of the IVa2 cap sites (Baker, C. C., and Ziff, E. B. (1981) J. Mol. Biol. 149, 189-221), has been examined in extracts of HeLa cells (Manley, J. L., Fire, A., Cano, A., Sharp, P. A., and Gefter, M.L. (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 3855-3859). Run-off transcripts of the predicted length of those initiated at the IVa2 cap sites were synthesized from different Ad2 DNA templates, each of which also contained the major late transcriptional control region. Mapping of the 5' ends of the RNA made from one template by a nuclease protection assay established the fidelity of initiation of IVa2 transcription in vitro. The efficiency of IVa2 expression in whole HeLa extracts was influenced quite dramatically by monovalent and divalent metal ion concentrations and the concentration of extract protein present in the reaction mixture. Under certain conditions, IVa2 run-off transcripts were made almost as efficiently as those from the Ad2 major late transcriptional control region. However, conditions promoting optimal IVa2 transcription in vitro did not favor recognition of the major late transcriptional control region, and vice versa: the synthesis of IVa2 and major late run-off transcripts responded differently to all parameters tested.

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.

Point mutations in the regulatory region of the human adenoviral VAI gene.

The VA RNA genes of human adenoviruses are transcribed by RNA polymerase III and, like other RNA polymerase III transcriptional units, contain internal regulatory regions. By means of a scheme for directed mutagenesis which employs a deletion mutation to define the target, point mutations have been induced in the regulatory region of a cloned VAI gene. One mutant plasmid which fails to direct VAI transcription and several others which direct the synthesis of normal amounts of RNA of aberrant electrophoretic mobilities were identified using an in vitro transcription assay. The nucleotide sequences of the regulatory regions of three of these plasmids have been determined. All three contain mutations which fall in a block of nucleotides whose sequence (GGTTCGANNCC) is highly conserved in RNA polymerase III promoters (Galli, G., Hofstetter, H., and Birnstiel, M. L. (1981) Nature (Lond.) 294, 626-631). The VAI-negative mutant (pRR5028) contains a G leads to A transition mutation at the second position, which is invariant in functional RNA polymerase III promoters. One mobility variant carries three point mutations, including one G leads to A base change at the position adjacent on the 5' side to that altered in pRR5028. The third mutant contains a single C leads to T transition mutation, in the final position of the conserved block.

The nucleotide sequence of the gene encoding protein IVa2 in human adenovirus type 7

The nucleotide sequences of cloned DNA segments encoding the IVa2 gene from Ad7 and a portion of Ad12 (group B and group A human adenoviruses, respectively) have been determined. When compared to Ad5, a group C adenovirus, these sequences have been found to be 80% homologous. Most changes are transitions or transversions. This high degree of nucleotide homology results in a high degree of amino acid conservation in the predicted polypeptides encoded from these genes; most nucleotide changes occur at the third position in the codon. The predicted polypeptide contains 448 amino acids and has a calculated M r-value of 50 700. The positions of the 5′ end of the mRNA and of the donor and acceptor splice sites of Ad7 and Ad12 can be inferred by analogy to those of Ad5. A long open reading frame starting upstream from the IVa2 gene overlaps the N-terminal portion of the polypeptide but is encoded in a different reading frame. Within this overlapping region, the long open reading frame is more conserved in amino acid sequence than is the presumed IVa2 polypeptide, suggesting that evolutionary pressure was exerted on the longer protein, a product of viral early region 2B. The high degree of conservation of this E2B region within the overlapping segment suggests that its activities must be more important for adenovirus infection than are the functions encoded in the amino-terminus of the IVa2 gene.