Adenovirus VAI RNA prevents phosphorylation of the eukaryotic initiation factor 2 alpha subunit subsequent to infection (original) (raw)
Related papers
Journal of virology, 1992
Interferon resistance of vaccinia virus is mediated by specific inhibition of phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF-2 alpha) by the double-stranded-RNA-activated (DAI) protein kinase. Vaccinia virus encodes a homolog of eIF-2 alpha, K3L, the deletion of which renders the virus sensitive to interferon treatment. We have studied the mechanism by which this protein product elicits interferon resistance in a transient DNA transfection system designed to evaluate regulators of eIF-2 alpha phosphorylation. In this system, translation of a reporter gene mRNA is inefficient because of eIF-2 phosphorylation mediated by the DAI protein kinase. Cotransfection of the K3L gene enhances translation of the reporter mRNA in this system. The K3L protein inhibits eIF-2 alpha phosphorylation and DAI kinase activation, apparently without being phosphorylated itself. Inhibition of protein synthesis, elicited by expression of a mutant Ser-51----Asp eIF-2 alpha design...
Journal of Virology, 1991
We have previously demonstrated that a purified bacterially synthesized E1A 289-amino-acid protein is capable of stimulating transcription from the promoters of genes transcribed by RNA polymerase II in vitro (R. Spangler, M. Bruner, B. Dalie, and M. L. Harter, Science 237:1044-1046, 1987). In this study, we show that this protein is also capable of transactivating in vitro the adenovirus virus-associated (VA1) RNA gene transcribed by RNA polymerase III. Pertinent to the transcription of this gene is the rate-limiting component, TFIIIC, which appears to be of two distinct forms in uninfected HeLa cells. The addition of an oligonucleotide containing a TFIIIC binding site to HeLa whole-cell extracts inhibits VA1 transcription by sequestering TFIIIC. However, the addition of purified E1A to extracts previously challenged with the TFIIIC oligonucleotide restores the level of VA1 transcription. When included in the same reaction, an E1A-specific monoclonal antibody reverses the restorati...
Journal of Biological Chemistry, 1989
Interferon treatment of mammalian cells induces a double-stranded (as) RNA-dependent protein kinase known as DAI. When activated, DAI phosphorylates the a-subunit of eukaryotic initiation factor eIF-2, impairing its ability to be recycled and leading to the inhibition of protein synthesis. We have identified a novel DAI substrate in the ribosomal salt wash of rabbit reticulocyte lysates. This substrate is a 90-kDa polypeptide which has been purified to apparent homogeneity. It can be cross-linked by ultraviolet irradiation to adenovirus VA RNAI, a small RNA polymerase I11 transcript RNA which acts as an inhibitor of DAI. As assayed by a nitrocellulose filter binding assay, the 90-kDa polypeptide is also able to associate with authentic double-stranded RNA, but not singlestranded RNA, made in vitro. Thus, this newly identified substrate of DAI appears to have affinity for dsRNA structures and may be involved in dsRNAregulated processes in the reticulocyte. Polyclonal and monoclonal antibodies directed against the 90-kDa polypeptide co-precipitate DAI, suggesting that these two proteins may exist as a complex.
1991
We have previously demonstrated that a purified bacterially synthesized ElA 289-amino-acid protein is capable of stimulating transcription from the promoters of genes transcribed by RNA polymerase II in vitro (R. Spangler, M. Bruner, B. Dalie, and M. L. Harter, Science 237:1044-1046, 1987). In this study, we show that this protein is also capable of transactivating in vitro the adenovirus virus-associated (VA1) RNA gene transcribed by RNA polymerase UI. Pertinent to the transcription of this gene is the rate-limiting component, TFIIIC, which appears to be of two distinct forms in uninfected HeLa cells. The addition of an oligonucleotide containing a TFIIIC binding site to HeLa whole-cell extracts inhibits VAT transcription by sequestering TFIIIC. However, the addition of purified ETA to extracts previously challenged with the TFIIIC oligonucle-otide restores the level of VAT transcription. When included in the same reaction, an ElA-specific monoclonal antibody reverses the restorati...
A novel adenovirus-2 E1A mRNA encoding a protein with transcription activation properties
The EMBO Journal
Two novel adenovirus-2 early region 1A mRNAs, designated 1OS and 11S, have been characterized. They differ from the previously described 9S, 12S and 13S mRNAs by having an additional intron removed during mRNA maturation. The 10S and 11S mRNAs encode proteins with mol. wts of 30 and 35 kd. These proteins are encoded in the same translational reading frame as the 12S and 13S mRNA products and differ by lacking 72 amino acids between position 27 and 98. A functional analysis showed that both the 10S and 11S mRNA products are non-essential for lytic virus growth, and, furthermore, defective in cellular transformation. Interestingly the 11S mRNA product functioned as an efficient transcriptional activator in transient expression assays but was very ineffective as a gene activator during virus growth. Moreover, the virus expressing the 11S cDNA failed to block host cell gene expression although substantial amounts of late proteins were expressed. From the biological properties of the ElA cDNA mutants it was possible to localize two functional domains in the ElA proteins; one region required for transcriptional activation (amino acids 140-185), and a second domain required for adenovirus transformation and the control of viral and cellular gene expression during a lytic infection (amino acids 27-98).
Repression of RNA polymerase III transcription by adenovirus E1A
Journal of Virology, 1993
Adenovirus E1A encodes two major proteins of 289 and 243 amino acids (289R and 243R), which both have transcription regulatory properties. E1A-289R is a transactivator whereas E1A-243R primarily functions as a repressor of transcription. Here we show that E1A repression is not restricted to RNA polymerase II genes but also includes the adenovirus virus-associated (VA) RNA genes. These genes are transcribed by RNA polymerase III and have previously been suggested to be the target of an E1A-289R-mediated transactivation. Surprisingly, we found that during transient transfection both E1A proteins repressed VA RNA transcription. E1A repression of VA RNA transcription required both conserved regions 1 and 2 and therefore differed from the E1A-mediated inhibition of simian virus 40 enhancer activity which primarily required conserved region 1. The repression was counteracted by the E1B-19K protein, which also, in the absence of E1A, enhanced the accumulation of VA RNA. Importantly, we sho...