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Most human adenoviruses encode two virus-associated (VA) RNAs, VA RNAI and VA RNAII, that accumulate to high levels in the cytoplasm of infected cells. The function of VA RNAI in blocking the activation of the cellular kinase PKR is well known, but the role of VA RNAII is obscure. Herein we characterize and purify several human proteins that interact preferentially with VA RNAII in Northwestern blot assays. Two of these proteins were identified as RNA helicase A and NF90, a component of the heterodimeric nuclear factor of activated T cells (NFAT). They copurified with the smaller NFAT subunit, NF45, which did not bind VA RNAII, and with an unidentified protein, p97, which did bind VA RNAII. Both RNA helicase A and NF90 contain two copies of a double-stranded (ds) RNA binding motif and bind strongly to dsRNA. NF90 interacts with RNAs in the following order of affinity: dsRNA > VA RNAII > VA RNAI > single-stranded RNA. Furthermore, VA RNAII is more effective than VA RNAI as an inhibitor of RNA helicase activity. These data identify RNA helicase A and NF90 as cellular proteins with an affinity for dsRNA and other structured RNA molecules and suggest that their functions are subject to regulation by RNA ligands including VA RNAII.

The proliferating cell nuclear antigen (PCNA), a crucial component of eukaryotic cell cycle and DNA replication complexes, is induced by the adenovirus E1A 243R oncoprotein through a cis-acting element termed the PERE (PCNA-E1A responsive element). The PERE contains a sequence homologous to an activating transcription factor (ATF) motif, and ATF-1 is a major component of PERE-protein complexes. We have identified a second PERE-binding protein, the cAMP response element binding protein (CREB) transcription factor, which forms heterodimers with ATF-1 at this site. CREB, but not ATF-1, is able to mediate transactivation of a minimal PCNA-chloramphenicol acetyltransferase reporter by E1A 243R. Further analysis revealed that the transcriptional coactivator, the CREB-binding protein (CBP), associates with PERE-related complexes, and that CBP is able to mediate a strong transactivation response to E1A 243R at the PCNA promoter. Experiments conducted with mutants in the E1A or CREB components support a model whereby E1A 243R transactivates the PCNA promoter via a CBP-CREB-PERE pathway. These findings delineate a paradigm by which E1A 243R can target and transactivate specific DNA promoter sequences.

Proliferating-cell nuclear antigen (PCNA) is a DNA damage-inducible protein that performs an essential function in DNA replication and repair as an auxiliary factor for DNA polymerases delta and epsilon. Examination of the human PCNA promoter DNA sequence revealed a site with homology to the consensus DNA sequence bound by p53. PCNA promoter fragments with this site intact bound p53 in vitro and were transcriptionally activated by wild-type p53 in transient expression assays in SAOS-2 cells. The resident p53-binding site could be functionally substituted by a previously described p53-binding site from the ribosomal gene cluster. A plasmid expressing a mutated version of p53 derived from a patient with Li-Fraumeni syndrome failed to activate the PCNA promoter in the cotransfection assay. In different cell types, activation of the PCNA promoter by the p53-binding sequence correlated with the status of p53. Activation of the PCNA promoter by wild-type p53 depends upon the level of p53 expression. This concentration dependence and cell type specificity reconciles the observations presented here with prior results indicating that wild-type p53 represses the PCNA promoter. These findings provide a mechanism whereby p53 modulates activation of PCNA expression as a cellular response to DNA damage.

The yeast two-hybrid system and far-Western protein blot analysis were used to demonstrate dimerization of human double-stranded RNA (dsRNA)-dependent protein kinase (PKR) in vivo and in vitro. A catalytically inactive mutant of PKR with a single amino acid substitution (K296R) was found to dimerize in vivo, and a mutant with a deletion of the catalytic domain of PKR retained the ability to dimerize. In contrast, deletion of the two dsRNA-binding motifs in the N-terminal regulatory domain of PKR abolished dimerization. In vitro dimerization of the dsRNA-binding domain required the presence of dsRNA. These results suggest that the binding of dsRNA by PKR is necessary for dimerization. The mammalian dsRNA-binding protein TRBP, originally identified on the basis of its ability to bind the transactivation region (TAR) of human immunodeficiency virus RNA, also dimerized with itself and with PKR in the yeast assay. Taken together, these results suggest that complexes consisting of different combinations of dsRNA-binding proteins may exist in vivo. Such complexes could mediate differential effects on gene expression and control of cell growth.

The interferon-induced, dsRNA-activated human protein kinase (PKR) exerts antiviral and antiproliferative effects through inhibition of protein synthesis. Studies of structure-function relationships in PKR have shown that two dsRNA binding motifs are important for its autophosphorylation and activation by dsRNA in vitro. To correlate these findings with the activity of PKR in vivo, we examined the function of various PKR deletion mutants in cultured cells by using an inducible expression system. In a reporter gene assay, mutant forms of the kinase lacking amino acids 1-97 (delta 1-97) and 104-157 (delta 104-157), which are required for dsRNA binding in vitro, retained full activity in vivo. Deletion of amino acids 233-271 (delta 233-271), however, abolished the translational inhibitory activity of the kinase and prevented its phosphorylation. Moreover, cells infected with vaccinia virus recombinants expressing wild-type PKR, the mutant delta 104-157, delta 186-222), developed almost complete inhibition of both viral and cellular protein synthesis was upon induction of PKR. This inhibition of viral protein synthesis was not observed in cells infected with a recombinant expressing delta 233-271 mutant PKR. Our findings establish that the region encompassing amino acids 233-271 of PKR is critical for kinase activity in vivo, whereas its dsRNA binding domain is dispensable.

The Tat-responsive region (TAR) sequence is present at the 5' end of human immunodeficiency virus 1 mRNAs and as a cytoplasmic form of 58-66 nucleotides. TAR RNA blocks the activation and autophosphorylation of the double-stranded RNA-activated protein kinase in vitro. We show here that TAR RNA also prevents the double-stranded RNA-mediated inhibition of translation in a cell-free system. Mutagenic and structural analyses of TAR RNA indicate that a stem of at least 14 base pairs is required for this activity, whereas the loop and bulge required for transactivation by Tat are dispensable. Truncation of the RNA to 68 nucleotides results in the loss of translational rescue ability, suggesting that the short cytoplasmic TAR RNA produced by viral transcription in vivo may not have the capability to suppress activation of the kinase. However, because longer TAR transcripts stimulate expression in a transient assay in vivo, the TAR structure at the 5' end of viral mRNAs could still exert this function in cis.

We examined the activation of transcription from the human immunodeficiency virus type 1 (HIV-1) promoter by the viral Tat protein in a transient expression system. Plasmids contained a HIV-reporter gene cassette and a simian virus 40 origin of DNA replication. Run-on assays of transcription complex distribution and analysis of cytoplasmic RNA accumulation confirmed that Tat is able to activate transcription by two mechanisms: by increasing the rate of transcriptional initiation and the efficiency of transcriptional elongation. The degree to which Tat stimulated initiation is determined by the basal level of HIV-directed transcription, which is influenced by the presence [corrected] of the simian virus 40 replication origin. Tat functions primarily to increase the efficiency of elongation when the origin is present and the basal level of transcription is high [corrected]. On the other hand, Tat functions primarily to increase the rate of initiation when the origin is absent [corrected] and the basal level of transcription is 10-fold lower. These studies suggest that the site of integration of the virus into the cellular genome may significantly affect the level of expression from the HIV promoter and consequently the pathobiology of the virus.

Transcription from the human immunodeficiency virus type 1 promoter gives rise to short cytoplasmic transcripts of approximately 60 nucleotides as well as to longer mRNAs. These RNAs contain the Tat-responsive region sequence, which is capable of assuming a stem-loop structure and has been implicated in the regulation of both transcription and translation. It has been reported that Tat-responsive region RNA inhibits translation in vitro through activation of an interferon-induced protein kinase, the double-stranded-RNA-activated inhibitor, which phosphorylates eukaryotic initiation factor 2. We show that the activation property is due to double-stranded RNA that often contaminates RNA synthesized in vitro using bacteriophage RNA polymerases. After purification, high concentrations of Tat-responsive region RNA inhibit the activation of double-stranded RNA-activated inhibitor, suggesting that it may serve to protect human immunodeficiency virus type 1 infection from a cellular defense mechanism.

A DNA sequence, 552 base pairs in length, encoding the two "virus-associated" (VA) RNAs of adenovirus type 2 is presented. Comparison of the oligonucleotide maps of VA RNAI and VA RNII with the established sequence permits identification of the genes for these RNAs. VA RNAI is 157-160 nucleotides long and VA RNAII 158-163 nucleotides long, depending on the exact length of their heterogeneous 3' end. The genes are separated by a spacer of about 98 nucleotides. The RNAs exhibit scattered regions of primary sequence homology and can adopt secondary structures which resemble each other closely in their configuration and stability. VA RNAII is also capable of assuming a different configuration that is energetically more favorable. The data suggest that the two RNA genes may have arisen by duplication of an ancestral gene and that the folding of the RNA chain may be of importance for the function of VA RNAs. Hypothetical RNA polymerase III recognition sequences and the coding potential of the region are discussed.

The human immunodeficiency virus 1 (HIV-1) long terminal repeat (LTR) sequences were inserted into adenovirus in place of the E1 region. The HIV-1 LTR contained in this recombinant adenovirus responds to trans-activation by tatIII in a HeLa cell line constitutively expressing that HIV-1 gene product. In addition, the HIV-1 LTR is activated by the adenovirus E1A 13S, but not 12S or 9S, gene product when it is supplied in trans by a coinfecting wild-type adenovirus. The Rous sarcoma virus LTR, in a similar recombinant adenovirus, is insensitive to tatIII but is also trans-activated by the E1A 13S protein. The action of the 13S E1A and tatIII proteins are additive for the HIV-1 LTR in the context of adenovirus and they appear to act at the transcriptional level. As in HeLa cells, the adenovirus-borne HIV-1 LTR is inactive in the absence of a trans-activator in H9 and Jurkat cells, two human leukemic T-cell lines. This suggests that recombinant adenoviruses have diagnostic potential for the detection of trans-activators of the HIV-1 LTR that are present in circulating human lymphocytes.

The dl331 mutant of adenovirus serotype 5 fails to produce virus-associated (VA) RNAI, and cells infected with this mutant do not synthesize proteins efficiently at late times in infection. The translational defect occurs at the level of polypeptide chain initiation, and cell-free extracts prepared from dl331-infected cells exhibit the defect observed in vivo. Addition of either eukaryotic initiation factor 2 (eIF-2) or guanine nucleotide exchange factor (GEF) to these cell-free extracts restores translational activity, with GEF functioning more efficiently in this regard. These results suggest that cells infected with the dl331 mutant develop a translational block at the level of GEF-catalyzed guanine nucleotide exchange and that this block is most likely established through phosphorylation of the alpha subunit of eIF-2. In the present investigation we show that endogenous HeLa cell GEF activity is significantly reduced in cells infected with the dl331 mutant. Further, in contrast to cells infected with wild-type serotype 2 adenovirus, dl331-infected cells contain increased eIF-2 alpha kinase activity. These results indicate that VA RNAI plays a role in suppressing eIF-2 alpha kinase activity during adenovirus infection of HeLa cells.

The small adenovirus-encoded VA RNAs occur as ribonucleoprotein (RNP) particles in association with a cellular protein antigen, La, recognized by the anti-La class of lupus sera [Lerner, M. R., Boyle, J. A., Hardin, J. A. & Steitz, J. A. (1981) Science 211, 400-402]. We have tentatively identified the La antigen as a HeLa cell phosphoprotein of Mr approximately equal to 45,000, present in infected and uninfected cells. The antigen appears not to be required for the transcription of VA RNAs in vitro. RNP particles that contain newly synthesized VA RNAs assemble rapidly in transcription extracts making VA RNA and also can be reconstituted from purified VA RNA and a source of La antigen. Variant forms of VA RNAI with sequence deletions and substitutions bind to the La antigen, suggesting that the recognition site includes the RNA termini or the sequences corresponding to the internal control region (promoter), or both. Upon reconstitution with fragments of VA RNAI, oligonucleotides from both the 5' and 3' termini bind to the antigen, but those from the control region do not. The terminal oligonucleotides of wild-type VA RNA can form a basepaired stem, but structures of comparable stability cannot be formed by the chimeric variant molecules. Therefore, the recognition site is probably the terminal nucleotides themselves rather than the stem structure.

Concentrations of double-stranded RNA above about 0.1 mug/ml inhibit translation of encephalo-myocarditis viral RNA and mouse globin messenger RNA in extracts of Krebs II ascites cells. Protein synthesis initially proceeds at the control rate, then abruptly shuts off in a manner similar to that observed in reticulocyte lysates [Hunt, T. & Ehrenfeld, E. (1971) Nature New Biol. 230, 91-94]. Substantially higher concentrations of double-stranded RNA are required to give this effect in ascites extracts. Subcellular fractions of Krebs II ascites cells contain a nucleolytic activity capable of digesting several natural and synthetic double-stranded RNAs. This nuclease is most active under conditions of protein synthesis, and part of the activity remains associated with ribosomes upon sedimentation. It is probably because of digestion of double-stranded RNA by this nuclease that higher concentrations of double-stranded RNA are required for inhibition of protein synthesis in Krebs cell extracts than in reticulocyte lysates.