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B Thimmapaya

Publications and source records attributed to B Thimmapaya.

33 records · Page 2Linked to original sources
PubMed1996

Transactivation of adenovirus E2-early promoter by E1A and E4 6/7 in the context of viral chromosome.

Transcription from adenovirus E2-early promoter is controlled by a unique array of four cis-acting elements which include an atypical TBP site, two E2F sites present in an inverted orientation relative to each other, and an ATF site. In virus-infected cells, this promoter is transactivated by E1A and the E4 6/7 proteins. In addition, it is also stimulated by the DNA-binding protein (DBP) in transient transfection assays. Here we describe a genetic analysis of the E2 transcriptional regulation in the context of the viral chromosome. By using genetically engineered mutant adenoviruses we have determined the interrelationship between the different cis-acting elements of the E2-early promoter during basal transcription, the extent to which E1A and E4 6/7 contribute to the E2 promoter activation and the E2 promoter elements that respond to these transactivators. We show that at eight hours following infection, E1A can transactivate the promoter about 21-fold whereas E4 6/7 can induce the promoter by only fivefold. DBP does not induce the promoter in the chromosomal context. Our mutational analysis suggests that the unique architecture of the E2-early promoter necessitates the concerted interaction of all three host transcription factors with their cognate recognition elements to form a stable and functional transcription complex. E1A mediated transactivation is dependent on this stable basal transcription complex and transactivation may involve simultaneous interaction of E1A with each of the three transcription factors present in the multicomponent basal transcription complex. The E4 6/7 protein can transactivate the E2-early promoter in the absence of ATF presumably by promoting the DNA binding capacity of transcription factor E2F and thereby stabilizing the basal transcription complex. We discuss some of the possible protein-protein interactions that may take place at the level of the multicomponent transcriptional complex at the E2-early promoter during transcriptional activation and the discrepancies that arise when a promoter is analyzed in infection versus transfection assays.

Adenovirus E1A Proteins↗
PubMed1996

Simian virus 40 large-T bypasses the translational block imposed by the phosphorylation of elF-2 alpha.

One of the cellular defense mechanisms against virus infection is mediated by activating the interferon-induced, double-stranded-RNA-activated protein kinase, PKR. Upon activation, PKR phosphorylates and thereby inactivates the protein synthesis initiation factor, elF-2, leading to cessation of protein synthesis. Viruses have evolved diverse strategies to counteract this cellular antiviral response. A majority of these strategies target PKR to prevent its activation. Recently, we showed that simian virus 40 (SV40) large-T antigen reverses PKR-mediated translational inhibition at a step downstream of PKR activation (Rajan et al., J. Virol. 69, 785--795, 1995). In this paper, we present evidence showing that SV40 can restore efficient translation in cells despite the elevated levels of phosphorylated elF-2 alpha resulting from PKR activation. Thus, SV40 large-T-mediated translational rescue occurs at a step downstream of elF-2 alpha phosphorylation.

Animals↗
PubMed1996

Repression of a matrix metalloprotease gene by E1A correlates with its ability to bind to cell type-specific transcription factor AP-2.

Adenovirus E1A 243-amino acid protein can repress a variety of enhancer -linked viral and cellular promoters. This repression is presumed to be mediated by its interaction with and sequestration of p3OO, a transcriptional coactivator. Type IV 72-kDa collagenase is one of the matrix metalloproteases that has been implicated in differentiation, development, angiogenesis, and tumor metastasis. We show here that the cell type-specific transcription factor AP-2 is an important transcription factor for the activation of the type IV 72-kDa collagenase promoter and that adenovirus E1A 243-amino acid protein represses this promoter by targeting AP-2. Glutathione S-transferase-affinity chromatography studies show that the E1A protein interacts with the DNA binding/dimerization region of AP-2 and that the N-terminal amino acids of E1A protein are required for this interaction. Further, E1A deletion mutants which do not bind to p3OO can repress this collagenase promoter as efficiently as the wildtype E1A protein. Because the AP-2 element is present in a variety of viral and cellular enhancers which are repressed by E1A, these studies suggest that E1A protein can repress cellular and viral promoter/enhancers by forming a complex with cellular transcription factors and that this repression mechanism may be independent of its interaction with p3OO.

Adenovirus E1A Proteins↗
PubMed1996

A novel simian virus 40 early-region domain mediates transactivation of the cyclin A promoter by small-t antigen and is required for transformation in small-t antigen-dependent assays.

At least three regions of the simian virus 40 small-t antigen (small-t) contribute to the protein's ability to enhance cellular transformation. As we showed previously for rat F111 cells, one region includes sequences from residues 97 to 103 that are involved in the binding and inhibition of protein phosphatase 2A. In the present study, the role of the protein phosphatase 2A binding region was confirmed in two additional small-t-dependent transformation systems. Second, small-t was found to provide a function previously identified as a large-T transformation domain. Mutations in residues 19 to 28 of large-T affected its transforming ability, but these mutations were complemented by a wild-type small-t. A third region of small-t was also required for efficient transformation. This region, the 42-47 region, is shared by large-T and small-t and contains a conserved HPDKGG hexapeptide. The 42-47 region function could be provided by either small-t or large-T in small-t-dependent systems. Mutations in the 42-47 region reduced the ability of small-t to transactivate the cyclin A promoter, of interest because small-t increased endogenous cyclin A mRNA levels in both human and monkey cells, as well as transactivating the promoter in transient assays.

Animals↗
PubMed1995

Activation of interferon-inducible 2'-5' oligoadenylate synthetase by adenoviral VAI RNA.

2'-5' oligoadenylate (2-5(A)) synthetase and protein kinase, RNA activated (PKR) are the only two known enzymes that bind double-stranded RNA (dsRNA) and get activated by it. We have previously identified their dsRNA binding domains, which do not have any sequence homology. Here, we report a profound difference between the two enzymes with respect to the structural features of the dsRNA that are required for their activation. The adenoviral virus-associated type I (VAI) RNA cannot activate PKR, although it binds to the protein and thereby prevents its activation by authentic dsRNA. In contrast, we observed that VAI RNA can both bind and activate 2-5(A) synthetase. Mutations in VAI RNA, which removed occasional mismatches present in its double-stranded stems, markedly enhanced its 2-5(A) synthetase-activating capacity. These mutants, however, are incapable of activating PKR. Other mutations, which disrupted the structure of the central stem-loop region of the VAI RNA, reduced its ability to activate 2-5(A) synthetase. These debilitated mutants could bind to the synthetase protein, although they fail to bind to PKR.

2',5'-Oligoadenylate Synthetase↗
PubMed1995

A novel translational regulation function for the simian virus 40 large-T antigen gene.

Cells use the interferon-induced, double-stranded-RNA-dependent protein kinase PKR as a defense against virus infections. Upon activation, PKR phosphorylates and thereby inactivates the protein synthesis initiation factor eIF-2, resulting in the cessation of protein synthesis. Viruses have evolved various strategies to counteract this cellular defense. In this paper, we show that simian virus 40 (SV40) large-T antigen can antagonize the translational inhibitory effect resulting from the activation of PKR in virus-infected cells. Unlike the situation with other virus-host cell interactions, SV40 large-T antigen does not block the activation of PKR, suggesting that SV40 counteracts the cellular antiviral response mediated by PKR at a step downstream of PKR activation. Mutational analysis of large-T antigen indicates that a domain located between amino acids 400 and 600 of large-T antigen is responsible for this function. These results define a novel translational regulatory function for the SV40 large-T antigen.

Adenosine Triphosphatases↗
PubMed1995

Effect of single-base substitutions in the central domain of virus-associated RNA I on its function.

Adenoviruses use virus-associated RNA I (VAI RNA) to counteract the cellular antiviral response mediated by the interferon-induced, double-stranded-RNA-activated protein kinase PKR. VAI RNA is a highly structured small RNA which consists of two long duplex regions connected at the center by a complex, short stem-loop. This short stem-loop and the adjacent base-paired regions, referred to as the central domain, bind to PKR and inactivate it. Currently it is not known whether binding of VAI RNA to PKR is dependent solely on the secondary (and tertiary) structure of the central domain or whether nucleotide sequences in the central domain are also critical for this interaction. To address this question, 54 VAI mutants with single-base substitution mutations in the central domain of the RNA were constructed, and their capacities to inhibit the autophosphoryation of PKR in vitro were determined. It was found that although about half of the mutants inhibited PKR activity as efficiently as the wild type, a significant number of mutants lost the inhibitory activity substantially, without a perceptible change in their secondary structures. These results indicate that, in addition to secondary structure, at least some nucleotides in the central domain may be critical for the efficient function of VAI RNA.

Adenoviruses, Human↗
PubMed1994

In vitro analysis of virus-associated RNA I (VAI RNA): inhibition of the double-stranded RNA-activated protein kinase PKR by VAI RNA mutants correlates with the in vivo phenotype and the structural integrity of the central domain.

Adenoviruses use the virus-encoded virus-associated RNA (VAI RNA) as a defense against cellular antiviral response by blocking the activation of the interferon-induced, double-stranded RNA-activated protein kinase PKR. The structure of VAI RNA consists of two long, imperfectly base-paired duplex regions connected by a complex short stem-loop at the center, referred to as the central domain. By using a series of adenovirus mutants with linker-scan mutations in the VAI RNA gene, we recently showed that the critical elements required for function in the VAI RNA molecule are in the central domain and that these same elements of the central domain are also involved in binding to PKR. In virus-infected cells, VAI RNA interacts with latent kinase, which is bound to ribosomes; this interaction takes place in a complex milieu. To more fully understand the relationship between structure and function and to determine whether the in vivo phenotype of these mutants can be reproduced in vitro, we have now analyzed these mutant VAI alleles for their ability to block the activation of a partially purified PKR from HeLa cells. We have also derived the structure of these mutants experimentally and correlated the structure with function. Without exception, when the structure of the short stem-loop of the central domain was perturbed, the mutants failed to inhibit PKR. Structural disruptions elsewhere in the central domain or in the long duplex regions of the molecule were not deleterious for in vitro function. Thus, these results support our previous findings and underscore the importance of the elements present in the central domain of the VAI RNA for its function. Our results also suggest that the interaction between PKR and VAI RNA involves a precise secondary (and tertiary) structure in the central domain. It has been suggested that VAI RNA does not activate PKR in virus-infected cells because of mismatches in the imperfectly base-paired long duplex regions. We constructed mutant VAI genes in which the imperfectly base-paired duplex regions were converted to perfectly base-paired regions and assayed in vitro for the activation of PKR. As with the wild-type VAI RNA, these mutants failed to activate PKR in vitro, while they were able to block the activation of PKR better than did the wild type. These results suggest that the failure of VAI RNA to activate PKR is not the result of mismatches in the long duplex regions.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenoviruses, Human↗
PubMed1993

E2F site activates transcription in fission yeast Schizosaccharomyces pombe and binds to a 30-kDa transcription factor.

The mammalian transcription factor E2F binds to several cellular proteins including Rb, p107, cyclin A, cyclin E, and p33cdk2 protein kinase in a stage-specific manner during cell cycle. Its recognition sequence, TTTCGCGC, is present in two of the human adenovirus early promoters and in several promoters of cellular genes whose products are implicated in the control of cell proliferation. These observations suggest that E2F may play an important role in cell-cycle regulation and prompted us to ask whether E2F-like activities are present in yeast. We found that the E2F motif can function as an activating sequence in Schizosaccharomyces pombe when cloned upstream of a reporter gene. Consistent with this, the expression of adenovirus E2 promoter in S. pombe was dependent on both E2F motifs of this promoter. A protein, spE2F, that binds to the E2F site was partially purified from S. pombe using DNA-affinity chromatography. The binding specificity of this protein was compared to that of human E2F using a number of mutant E2F sites as competitors. These studies showed that spE2F recognizes a sequence closely related to the E2F site. Ultraviolet cross-linking and Southwestern blot studies indicated that the molecular size of spE2F is 30 kDa. Previous studies have shown that a cis-acting element, ACGCGTNA, also called MluI cell cycle box, or MCB, is critical for the regulated expression of cell cycle related genes both in fission and budding yeast. In S. pombe, the cdc10 gene product binds to this element and controls the cell cycle related genes. Electrophoretic mobility shift assays and molecular size determination studies indicated that spE2F is different from that encoded by cdc10. Thus, our studies suggest that spE2F is a novel transcription factor. We discuss these results in light of recent observations about the periodically expressed genes involved in the cell cycle progression in yeast.

Adenoviruses, Human↗
PubMed1993

Activation of a dual adenovirus promoter containing nonconsensus TATA motifs in Schizosaccharomyces pombe: role of TATA sequences in the efficiency of transcription.

The role of TATA elements in the expression of a mammalian promoter was investigated in the fission yeast Schizosaccharomyces pombe, by studying the human adenovirus E2-early promoter. This is a unique dual promoter with two nonconsensus TATA elements directing transcription from two cap sites, +1 and -26. A sequence TTAAGA provides the TATA box function for the +1 promoter, whereas a sequence TAAATT, with a closer resemblance to the consensus (TATAA/TA) provides this function for the -26 promoter. Yet, in human cells, the +1 promoter is transcribed about 20 fold more efficiently than the -26 promoter. We found that both promoters are transcribed faithfully in S. pombe with start sites identical or close to those found in human cells. Surprisingly, the relative ratio of expression for the +1 and -26 promoters was exactly reversed in S. pombe cells. This reversal appeared to be due to the relatively weak binding of S. pombe TATA binding protein to the TTAAGA motif, rather than to its rate of dissociation. Furthermore, we show that in S. pombe, promoter expression correlates well with the nucleotide sequence of the TATA element rather than the context in which it is placed. By contrast, it is the context of the TATA element, rather than its nucleotide sequence that appears to be critical for promoter expression in human cells. Our data suggest the existence of one or more additional factors in human cells that permit the utilization of nonconsensus TATA elements. S. pombe appears to lack these factors.

Adenoviruses, Human↗
PubMed1991

Binding of the adenovirus VAI RNA to the interferon-induced 68-kDa protein kinase correlates with function.

In human cells infected with adenovirus, the virus-associated RNA VAI blocks the activation of the interferon-induced double-stranded-RNA-dependent 68-kDa protein kinase (p68) and maintains normal levels of protein synthesis at late times after infection. VAI antagonizes the kinase activity by binding to p68. The structure of VAI consists of two long, base-paired stems connected by a complex short stem-loop structure. Previous work using a series of adenovirus mutants showed that the structural determinants of the VAI RNA that are essential for function reside in the central complex short stem-loop structure and adjacent base-paired regions (functional domain); the long duplex regions were found to be dispensable for function. To determine whether binding of VAI to p68 correlates with function and whether the structural determinants that are essential for function are also essential for binding, we studied the interaction of wild-type and several mutant VAI RNAs with p68 in whole cells. The p68-VAI complexes from mutant- and wild-type-infected cells were immunoprecipitated by an anti-p68 monoclonal antibody. The mutant RNAs that functioned efficiently in the cells bound to p68 efficiently in the cells, whereas functionally impaired mutants failed to bind to p68, indicating that the binding of the VAI RNA to p68 correlates well with function. In vitro binding assays with immunopurified p68 confirmed these observations. Secondary-structure analysis of several mutant VAI RNAs suggests that the binding does not depend on the long duplex regions but requires all the elements of the functional domain. We propose that the functional domain and the p68-binding domain of the VAI RNA are identical.

Adenoviruses, Human↗
PubMed1991

Simian virus 40 small-t does not transactivate RNA polymerase II promoters in virus infections.

Transcriptional stimulatory properties of virus-encoded transactivators appear to be critical for viral gene expression and may be linked to cellular transformation in certain cases. Recently, the simian virus 40 (SV40) 17-kDa small-t antigen was shown to stimulate transcription of polymerase II and III genes in transient transfection assays. In experiments performed in our laboratory, two of the polymerase II promoters of the adenovirus genome, namely, the EII-early and EIII promoters, were transactivation, we examined the transient transfection assays. To further elucidate the mechanism of this transactivation, we examined the ability of small-t to transactivate the adenovirus type 5 EII-early and EIII promoters in CV-1 cells under conditions in which the small-t gene or the reporter genes were introduced into the cells through transfection and other routes. In one approach, we used established CV-1 cell lines which constitutively express the small-t gene, and study of the EII-early promoter was afforded by infection of an EIA-negative adenovirus type 5 variant. For the second approach, a recombinant adenovirus was constructed in which small-t was expressed from a replication origin-negative SV40 early promoter in the EIA region of an adenovirus vector (Ad-SV-t). The effect of small-t on adenovirus EII-early and EIII promoter expression was studied in coinfection or single-infection experiments. In both cases, transcription of the adenovirus early promoters was not stimulated by small-t. These and other results indicate that transactivation of polymerase II promoters by small-t occurs only when the target gene is in a transiently transfected state. Thus, small-t-mediated transactivation of polymerase II promoters is dependent on the type of assay system used and may be mechanistically different from that of the widely studied EIA.

Adenoviruses, Human↗
PubMed1990

The adenovirus EII early promoter has multiple EIA-sensitive elements, two of which function cooperatively in basal and virus-induced transcription.

The mechanism by which the adenovirus-encoded nuclear oncogene EIA activates transcription of several viral and host promoters is an important issue in the regulation of eucaryotic gene expression and virus-host cell interactions. Identification of cis-acting elements of the promoters and the cognate host transcription factors that are targets for EIA action is crucial for our understanding of the EIA-mediated control of coordinately regulated genes. The adenovirus EII early promoter has a complex architecture and contains two overlapping promoters with start sites at +1 (major promoter) and -26 (minor promoter). The major promoter responds strongly to virus-encoded trans activators EIA and EIV and contains four elements: a TAGA motif analogous to the TATA box, two EIIF sites present in an inverted orientation, and an ATF/CREB site. To determine precisely the roles played by these cis-acting elements in both basal and virus-induced transcription when the promoter is situated in its natural context, we investigated the phenotype of a series of linker scan promoter substitution mutants inserted into the viral chromosome. Promoter constructs harboring linker scan mutations in each element were rebuilt into a novel EIA- adenovirus vector, and transcriptional activity was monitored in virus-infected cells. In the absence of virus-encoded trans activators, basal activity in vivo was dependent on all four cis-acting elements. Surprisingly, a promoter mutant with only one of the two EIIF sites intact could not promote transcription in vivo, suggesting that the two EIIF sites function cooperatively even in basal transcription. Promoters harboring mutations in either of these two EIIF sites also failed to bind to an infection-specific form of EIIF in gel shift assays and competed only very weakly for EIIF binding with the wild-type promoter fragment. The dramatic cooperativity shown by the two inverted EIIF sites of the EII promoter both in vivo and in vitro could reflect simultaneous contact of both sites by the transcription factor EIIF. Furthermore, promoter mutants with mutations in the TAGA motif, the two EIIF sites, and the single ATF site all failed to respond to virus-encoded trans activators. Whereas recent results demonstrate that EIIF activity can be modulated independently by EIV, leading to transactivation of this promoter, our results and those published previously strongly indicate that the three different transcription factors that bind to TAGA, EIIF, and ATF motifs of the EII early promoter are all targets for EIA regulation in vivo. Thus, strong transactivation of the EII early promoter through these multiple EIA-sensitive elements and independently by the recently discovered EIV pathway suggests that the EII early promoter is stringently regulated in virus-infected cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenovirus Early Proteins↗