Association Of Virus Research Articles

Termination sequence requirements vary among genes transcribed by RNA polymerase III

RNA polymerase III (pol III) transcription generally terminates at a run of four or more thymidine (T) residues but some pol III genes contain runs of T residues that are not recognized as termination signals. Here, we investigate the terminal signal requirements that are operative in adenovirus virus-associated (VA) RNA genes. In the Xenopus 5 S RNA gene, efficient termination requires the T residues to be in a G + C-rich sequence context, but a run of five T residues in a G + C-rich context does not cause pol III termination when placed 30 nt downstream of the adenovirus-2 VA RNAIpromoter in a VA-Tat chimeric gene. The failure of pol III to recognize this putative termination signal is not due to the chimeric nature of the gene or to the proximity of the signal to the promoter, but to its sequence context. Termination at the VA RNA gene site requires a T-rich sequence and is inhibited by the proximity of G residues, but is insensitive to the presence of A residues. The T-rich sequence need not be uninterrupted, however. In the VA RNA gene of the avian adenovirus, CELO, the first of two tandem termination signals contains an interrupted run of T residues, TTATT, which functions as a terminator with high (although not complete) efficiency. These findings, together with a survey of sequences neighboring the terminal site of other pol III genes, lead to the conclusion that pol III termination signals are more complex than hitherto recognized, and that sequence context requirements differ between members of the class 1 and class 2 families of pol III genes.

Activities of adenovirus virus-associated RNAs: purification and characterization of RNA binding proteins.

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.

Ovine adenovirus (OAV287) lacks a virus-associated RNA gene.

Ovine adenovirus OAV287 (OAV) is the prototype of a virus group which is phylogenetically distinct from the mastadenoviruses and aviadenoviruses. The genome arrangement of OAV showed that virus-associated (VA) RNA genes were not located between the reading frames for p52/55K and terminal protein as these overlapped. To determine whether VA genes were located elsewhere, several approaches were used. Nuclear extracts containing RNA polymerase III activity were used to transcribe OAV genome fragments in vitro. A product of approximately 120 bp was produced from two widely separated coding regions of the genome. However, when these were subcloned and used as radiolabelled probes to analyse RNA from OAV-infected cells, no VA-like RNA was detected, although late mRNAs that were transcribed from the regions were identified. In addition, 5' radiolabelling of small RNA species in control- and OAV-infected cells followed by gel analysis did not identify candidate VA RNAs. Radiolabelling of proteins in control- and OAV-infected cells showed that there was little preferential translation of viral proteins compared with host polypeptides, in contrast to the situation for adenovirus 5 (Ad5). In addition, the kinetics of OAV infection were slower than observed for human adenoviruses. Collectively, the data suggest that OAV is unique in that it does not produce VA RNA during infection. This conclusion is supported by a comparison of the genomes of the phylogenetically related OAV and egg drop syndrome viruses which shows that the VA gene identified in the latter is located in a region absent from OAV.

Structure, function, and evolution of adenovirus-associated RNA: a phylogenetic approach.

To explore the structure and function of a small regulatory RNA, we examined the virus-associated (VA) RNA species of all 47 known human adenovirus serotypes and of one simian virus, SA7. The VA RNA gene regions of 43 human adenoviruses were amplified and sequenced, and the structures of 10 representative VA RNAs were probed by nuclease sensitivity analysis. Most human viruses have two VA RNA species, VA RNA, and VA RNAII, but nine viruses (19%) have a single VA RNA gene. Sequence alignments classified the RNAs into eight families, corresponding broadly to the known virus groups, and three superfamilies. One superfamily contains the single VA RNAs of groups A and F and the VA RNAI species of group C; the second contains the VA RNAI species of groups B1, D, and E and the unclassified viruses (adenovirus types 42 to 47), as well as the single VA RNAs of group B2; and the third contains all VA RNAII species. Fourteen regions of homology occur throughout the molecule. The longest of these correspond to transcription signals; most of the others participate in RNA secondary structure. The previously identified tetranucleotide pair, GGGU:ACCC, is nearly invariant, diverging slightly (to GGGU:ACCU) only in the two group F viruses and forming a stem in the central domain that is critical for VA RNA structure and function. Secondary structure models which accommodate the nuclease sensitivity data and sequence variations within each family were generated. The major structural features-the terminal stem, apical stem-loop, and central domain-are conserved in all VA RNAs, but differences exist in the apical stem and central domains, especially of the VA RNAII species. Sequence analysis suggests that an ancestral VA RNA gene underwent duplication during the evolution of viruses containing two VA RNA genes. Although the VA RNAII gene seems to have been lost or inactivated by secondary deletion events in some viruses, the high degree of homology among the VA RNAII species implies that this RNA may play an undiscovered role in virus survival. We speculate that the VA RNA genes originated from cellular sequences containing multiple tRNA genes.

Transcription of adenovirus RNA polymerase III genes.

Eukaryotic RNA polymerase (RNAp) III is responsible for synthesis of abundant, low molecular mass RNA species, including tRNA, 5S rRNA, and U6 snRNA (see Roeder 1976; Geiduschek and Tocchini-Valentini 1988). Although they are noncoding, RNAp III transcripts participate in such essential processes as translation, RNA processing, and protein targeting (see Rich and Raj Bhandary 1976; Guthrie and Patterson 1988; Dreyfuss et al. 1988; Steitz et al. 1988; Wolin and Walter 1991; Young 1991). Transcription of a viral genome by RNAp III was first recognized 20 years ago, when it was established that a small RNA specific to adenovirus-infected cells, virus-associated (VA) RNA (Reich et al. 1966), is synthesized by this enzyme. All adenovirus genomes examined to date encode at least one VA RNA species. Investigation of the organization of the transcriptional control regions of these viral RNAp III genes made an important contribution to the unexpected discovery of the intragenic location of elements of RNAp III promoters. As discussed in this review, the VA RNA I promoter of subgroup C adenoviruses, which is exceptionally active in in vitro systems, has provided an important model for investigation of the mechanism of initiation of transcription by this RNA polymerase. Elucidation of the part played by VA RNA I during adenovirus infection has, moreover, identified a previously unknown mechanism by which viruses can evade cellular defence systems.

Structure, function, and evolution of adenovirus virus-associated RNAs.

One of the charms of adenovirus is its unique blend of orthodoxy and idiosyncrasy. Although the virus operates largely via host cell mechanisms, at least for transcriptional and translational purposes, it coopts these mechanisms by using a variety of viral products to modify cellular activities for its own benefit. Nowhere is this better illustrated than in the virus-associated (VA) RNA genes, which are transcribed by RNA polymerase III (pol III) while the rest of the viral genome is grist for pol II. More abundant in the infected cell than any messenger RNA, the VA RNAs encode no protein but are essential for efficient translation, a feat that is accomplished through a protein kinase that is regulated by highly structured RNA molecules. The protein kinase is a key element in the cellular antiviral defenses that are induced by interferon, and it is probable that one of the roles of the VA RNAs in natural infections is to spike the defensive guns.

The topology of the promoter of RNA polymerase II- and III-transcribed genes is modified by the methylation of 5'-CG-3' dinucleotides.

In eukaryotic cells, RNA polymerase II- and III-transcribed promoters can be inactivated by sequence-specific methylation. For some promoter motifs, the introduction of 5-methyldeoxycytidine (5-mC) residues has been shown to alter specific promoter motif-protein interactions. To what extent does the presence of 5-mC in promoter or regulatory DNA sequences affect the structure of DNA itself. We have investigated changes in DNA bending in three naturally occurring DNA elements, the late E2A promoter of adenovirus type 2 (Ad2) DNA, one of our main model systems, the VAI (virus-associated) RNA gene of Ad2 DNA, and an Alu element associated with the human angiogenin gene. Alterations in electrophoretic mobility of differently permuted promoter segments in non-denaturing polyacrylamide gels have been used as assay system. In the late E2A promoter of Ad2 DNA, a major and possibly some minor DNA bending motifs exist which cause deviations in electrophoretic mobility in comparison to coelectrophoresed marker DNA fragments devoid of DNA bending motifs. DNA elements have been specifically in vitro methylated by the HpaII (5'-CCGG-3'), the FnuDII (5'-CGCG-3'), or the CpG DNA methyltransferase from Spiroplasma species (M-SssI; 5'-CG-3'). Methylation by one of these DNA methyltransferases influences the electrophoretic mobility of the three tested promoter elements very strikingly, though to different extents. It cannot be predicted whether sequence-specific promoter methylation increases or decreases electrophoretic mobility; these changes have to be experimentally determined. Methylation of the E. coli dcm (5'-CCA/TGG-3') sites in some of the DNA constructs does not make a contribution to mobility changes. It is concluded that sequence-specific methylations in promoter or regulatory DNA elements can alter the bending of DNA very markedly. This parameter may contribute significantly to the silencing of promoters, probably via altering spatial relationships among DNA-bound transcription factors.

Characterization of a single SA7-like VA RNA gene in subgroup F adenoviruses.

The virus-associated (VA) RNA gene regions of the human subgroup F adenoviruses (types 40 and 41) were amplified using primers corresponding to flanking open reading frames of human and simian (SA7) adenovirus sequences previously published. The subgroup F adeno-viruses, like human Ad12 (subgroup A) and SA7, were found to have only one VA RNA gene at this locus (map unit 30). The type 40 and type 41 VA RNA genes have primary sequence characteristics in common with other known VA RNA genes, have high cross-identity (93%), and show appreciably higher identity to the single VA RNA gene present in SA7 (77% and 81%, respectively) than to any of the VA RNA genes of Ad2, Ad7 or Ad12 (< 61%). These findings may have implications for explaining the evolution and growth peculiarities of human subgroup F adenoviruses.

Repression of RNA polymerase III transcription by adenovirus E1A

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 show that efficient VA RNA transcription requires expression of both E1A and the E1B-19K protein during virus infection.

Probing DNA-protein interactions in vitro with the CpG DNA methyltransferase.

A sensitive method was devised to monitor the in vitro binding of nuclear proteins from HeLa cells presumably to the major groove of DNA. Upon the incubation of DNA with nuclear extracts, the complexed DNA was incubated with the CpG DNA methyltransferase from Spiroplasma species. Subsequently, the DNA was repurified, and the location of the methylated cytidine residues was determined by the hydrazine reaction of the DNA sequencing method. By using as DNA substrate the VAI (virus associated) region of human adenovirus type 2 (Ad2) DNA or specific Alu sequences associated with a number of human genes, it was documented that those segments of DNA that were protected by bound proteins against the reaction with DNasel also escaped in vitro methylation by the CpG DNA methyltransferase. This new footprinting method provides a sensitive indicator for in vitro DNA--protein interactions which are specific for the major groove of DNA.

Activation of protein kinase C inhibits adenovirus VA gene transcription in vitro.

We report here that activation of protein kinase C (PKC) results in the inhibition of adenovirus virus-associated (VA) gene transcription in vitro. The involvement of PKC in this inhibition is supported by the fact that the addition of PKC inhibitors to transcription reactions in which the PKC cofactor phosphatidyl serine (PS) was present resulted in increased levels of transcription compared to those in reactions in which PKC activity was stimulated in the absence of PKC inhibitors. Furthermore, based on these in vitro studies we propose that the inhibition of VA gene transcription is possibly due to the inability of the VA gene to form an active transcription complex following the activation of PKC. This conclusion was drawn from in vitro data which demonstrated that PS stimulation of endogenous PKC present in cell extracts resulted in failure to isolate a fully initiated, stable transcription complex.

Role of the apical stem in maintaining the structure and function of adenovirus virus-associated RNA.

Adenovirus virus-associated (VA) RNAI maintains efficient protein synthesis during the late phase of infection by preventing the activation of the double-stranded-RNA-dependent protein kinase, DAI. A secondary structure model for VA RNAI predicts the existence of two stems joined by a complex stem-loop structure, the central domain. The structural consequences of mutations and compensating mutations introduced into the apical stem lend support to this model. In transient expression assays for VA RNA function, foreign sequences inserted into the apical stem were fully tolerated provided that the stem remained intact. Mutants in which the base of the apical stem was disrupted retained partial activity, but truncation of the apical stem abolished the ability of the RNA to block DAI activation in vitro, suggesting that the length and position of the stem are both important for VA RNA function. These results imply that VA RNAI activity depends on secondary structure at the top of the apical stem as well as in the central domain and are consistent with a two-step mechanism involving DAI interactions with both the apical stem and the central domain.

Permanent cell lines that show temperature-dependent expression of adenovirus virus-associated RNA.

Temperature-sensitive COS cells, clone E540, have been stably transformed at a restrictive temperature with plasmid pVA1, which contains the adenovirus type 5 virus-associated (VA) genes in addition to the Neor marker. Transformed cell clones, named EVA cells, contained adenovirus DNA in an integrated form while grown at restrictive temperature but accumulated up to 100 to 200 copies of the input plasmid per cell after temperature shift down. Concomitant with this gene amplification, an accumulation of VA RNA was observed, reaching average concentrations of 10(4) to 10(5) copies per cell. The VA RNA synthesized in EVA cells is functional, as judged by inhibition of in vitro eucaryotic initiation factor-2 phosphorylation and enhancement of reporter gene expression. These EVA cell lines may be of use to study the mechanism of VA RNA function in the absence of adenovirus infection.

Defect of adenovirus type 12 replication in hamster cells: absence of transcription of viral virus-associated and L1 RNAs.

The nonpermissive interaction of hamster cells with human adenovirus type 12 (Ad12) is characterized by a total block of Ad12 DNA replication and late transcription, whereas most of the early functions of Ad12 DNA can be transcribed. Ad2 can replicate in hamster cells. The replication and late transcription defects of Ad12 DNA can be complemented to a certain extent by the E1B functions of Ad2 DNA. This complementation fails, however, to lead to the synthesis of the late Ad12 proteins and to the assembly of infectious virions. It will now be demonstrated that the Ad12 L1 (late genes of group 1) and virus-associated (VA) RNAs are not transcribed in hamster cells. Synthesis of these RNAs in productively infected human cells or Ad2-infected hamster cells is readily detectable by S1 nuclease protection experiments and Northern (RNA) blotting. Similarly, the Ad2-transformed hamster cell line BHK-Ad2E1 fails to complement L1 and VA RNA syntheses after superinfection with Ad12. However, Ad12 infection of the Ad5-transformed hamster cell line BHK297-C131 leads to the transcription of the Ad12 L1 and VA segments. This difference in complementation by the two transformed hamster cell lines might be accounted for by functions in the segment of Ad5 DNA extending between map units 30 and 40 and persisting in the Ad5-transformed hamster cells or by hamster host cell functions which might be operative in cell line BHK297-C131 but not in BHK-Ad2E1 or BHK-21 hamster cells.

Effects of mutations in stem and loop regions on the structure and function of adenovirus VA RNAI.

Adenovirus virus-associated (VA) RNAI is required for efficient protein synthesis at late times of adenoviral infection, and in some other situations where double-stranded RNA (dsRNA) is present. It prevents inhibition of protein synthesis by a dsRNA-activated protein kinase and the secondary structure of VA RNAI is though to be important for its activity. To test this idea and to define structures and sequences responsible for VA RNAI activity, we constructed several mutant VA RNA genes and tested them in a transient expression assay. Activity is unaffected by deletions within a small region near the center of the gene, nt 72-85, but it is greatly diminished by deletion or substitution of sequences on the 3' side of this region. The structures of wild-type and mutant RNAs were examined by nuclease-sensitivity analysis. We propose a model for wild-type VA RNAI which differs from that predicted to be the most stable structure. Surprisingly disruption of the longest duplex region in the molecule is tolerated, provided that adjacent structural elements are not rearranged. However, perturbations of elements located in the center of the structure correlate well with loss of function.

DNA-binding properties and characterization of human transcription factor TFIIIC2.

Interaction between the B-block region of adenovirus VA1 DNA and the human RNA polymerase III transcription factor (TFIII) C2 was analyzed using a gel DNA-binding assay. The retarded band corresponding to the specific complex between TFIIIC2 and the regulatory B-block region was identified by DNase I footprint analysis, competition experiments, and gel shift assays using mutated and truncated virus-associated (VA) 1 DNA probes. The equilibrium constants for the binding reaction with the complete VA1 gene were determined. TFIIIC2 was found to bind to non-specific DNA sequences with a relatively low affinity (equilibrium constant Kn = 6 x 10(4) M-1), and to the B-block sequence with a high affinity (specific constant Ks = 2 x 10(11) M-1). Assuming one site per molecule, the total concentration of binding sites [C0] in the TFIIIC2-containing fractions ranged between 0.6 and 1.6 x 10(-10) M. This corresponded to 1500 TFIIIC2 molecules extracted per 293 cell. Sedimentation analysis of TFIIIC2 on a sucrose gradient showed both VA1 DNA binding activity and in vitro transcription activity cosedimenting with an apparent coefficient of 17-18 S, consistent with a molecular weight of 400-500 kDa. UV cross-linking of a 5-bromo 2'-deoxyuridine-containing, 32P-labeled VA1 probe with the TFIIIC2 fraction, followed by DNase I digestion and analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, revealed a single 32P-labeled band migrating as a 250-kDa polypeptide. Compared with the sedimentation data, this result suggests that native TFIIIC2 may be a dimer of the approximately 250-kDa polypeptide.

Characterization of a low-molecular-weight virus-associated (VA) RNA encoded by simian adenovirus type 7 which functionally can substitute for adenovirus type 5 VA RNAI

Human adenoviruses (Ads), like Ad type 2 (Ad2) and Ad5, encode a low-molecular-weight RNA (designated virus-associated [VA] RNAI) which is required for the efficient translation of viral mRNAs late after infection. We cloned and characterized a VA RNA gene from simian adenovirus type 7 (SA7) which appears to have biological activity analogous to that of Ad2 VA RNAI. Thus, SA7 VA RNA stimulates protein synthesis in a transient expression assay and can also functionally substitute for VA RNAI during lytic growth of human Ad5. The SA7 genome encodes only one VA RNA species, in contrast to human Ad2, which encodes two distinct species. This RNA is transcribed by RNA polymerase III in the rightward direction from a gene located at about coordinate 30 on the viral genome, like its Ad2 counterparts. SA7 VA RNA shows only a limited primary sequence homology with the Ad2 VA RNAs (approximately 55%); the flanking sequences, in fact, are better conserved than the VA RNA gene itself. The predicted secondary structure of SA7 VA RNA is, however, very similar to that of Ad2 VA RNAI, inferring that the double-stranded nature rather than the primary sequence of VA RNA is important for its biological activity.

Defective RNA splicing in the absence of adenovirus-associated RNAI.

We have analyzed late gene expression in 293 cells infected with an adenovirus type 5 mutant dl331, which is defective in production of the low molecular weight virus-associated (VA) RNAI. The results show that several steps in late gene expression are affected. In addition to the previously characterized defect in late mRNA translation, mutant infected cells also show an aberrant selection of RNA splice sites and a substantially reduced L2, L3, and L5 mRNA accumulation. Normal or even slightly elevated amounts of mRNA from region L1 are produced. However, the L1 pre-mRNA is spliced only to generate the mRNA encoding the Mr 52,000-55,000 polypeptide and no detectable mRNA for polypeptide IIIa. Cotransfection of a plasmid encoding VA RNAI complemented the splicing defect in trans, suggesting that the abnormalities are due to the absence of VA RNAI, rather than to a cis-acting change in the nuclear precursor RNA. In a HeLa cell variant, which allows late protein synthesis also in the absence of VA RNAI, because of a lack of eukaryotic initiation factor 2 alpha kinase expression, a normal repertoire of late mRNA was produced. We conclude that a soluble factor, most likely a late viral protein, controls differential RNA splicing and late mRNA accumulation during an adenovirus infection.

Translational control by adenovirus: lack of virus-associated RNAI during adenovirus infection results in phosphorylation of initiation factor eIF-2 and inhibition of protein synthesis.

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.

Identification of the components necessary for adenovirus translational control and their utilization in cDNA expression vectors.

A transient expression system was used to study the role of the adenovirus late and simian virus 40 (SV40) early mRNA leader sequences and adenovirus virus-associated (VA) RNAs in mRNA translation. Hybrid transcription units containing the adenovirus late and SV40 early promoters fused to various coding regions were introduced into monkey COS cells on plasmids containing a SV40 origin of replication. The translational efficiencies of the mRNAs produced from these plasmids were determined after alterations in the viral leader sequences or in the presence of VA RNAs provided by adenovirus infection of the transfected cells or by cotransfection with plasmids containing the VA genes. Efficient translation of mRNA with either adenovirus or SV40 leader sequences is dependent upon the presence of VA RNA. Translational stimulation by VA RNA of mRNAs containing the adenovirus tripartite leader sequences is dramatically reduced if leader exons 2 and 3 are removed or if their orientation is altered. Sequence analysis has indicated a homology between the nontranslated 5' end of SV40 early mRNA and sequences at the border of the 2nd and 3rd tripartite leader exons, which may be responsible for the increased translation of these mRNAs in the presence of VA RNA.