Structural features of adenovirus 2 virus-associated RNA required for binding to the protein kinase DAI.

The double-stranded RNA activated protein kinase DAI contains an RNA binding domain consisting of two copies of a double-stranded RNA binding motif. We have investigated the role of RNA structure in the interaction between DAI and the structured single-stranded RNA, adenovirus VA RNAI, which inhibits DAI activation. Mutations in the apical stem, terminal stem, and central domain of the RNA were tested to assess the contribution of these elements to DAI binding in vitro. The data demonstrate that over half a turn of intact apical stem is required for the interaction and that there is a correlation between the binding of apical stem mutants and their ability to function both in vivo and in vitro. There was also evidence of preference for GC-rich sequence in the proximal region of the apical stem. In the central domain the correlation between binding and function of mutant RNAs was poor, suggesting that at least some of this region plays no direct role in binding to DAI, despite its functional importance. Exceptionally, central domain mutations that encroached on the phylogenetically conserved stem 4 of VA RNA disrupted binding, and complementary mutations in this sequence partially restored binding. Measurement of the binding of wild-type VA RNAI to DAI and p20, a truncated form of the protein containing the RNA binding domains alone, under various ionic conditions imply that the major interactions are electrostatic and occur via the protein's RNA binding domain. However, differences between full-length DAI and p20 in their binding to mutants in the conserved stem suggest that regions outside the RNA binding domain also participate in the binding. The additional interactions are likely to be non-ionic, and may be important for preventing DAI activation during virus infection.     

Interactions between the double-stranded RNA binding motif and RNA: definition of the binding site for the interferon-induced protein kinase DAI (PKR) on adenovirus VA RNA.

The protein kinase DAI, the double-stranded RNA activated inhibitor of translation (also known as PKR), regulates cell growth, virus infection, and other processes. DAI represents a class of proteins containing a recently recognized RNA binding motif, the dsRBM, but little is known about the contacts between these proteins and their RNA ligands. In adenovirus-infected cells, DAI activation is prevented by VA RNAI, a highly structured RNA that binds to the kinase. VA RNA contains three chief structural features: a terminal stem, an apical stem-loop, and a complex central domain. We used enzymatic and chemical footprinting to identify the interactions between DAI and VA RNAI. DAI protects the proximal part of the apical stem structure, an adjacent region in the central domain, and a region surrounding a conserved stem in the central domain from nuclease attack. During binding the RNA undergoes a conformational change that is mainly restricted to the central domain. A similar change is induced by magnesium ions alone. Footprinting and interference binding assays using base-specific chemical probes suggest that the protein does not make major contacts with RNA bases. On the other hand, footprinting with probes specific for the RNA backbone shows that DAI engages in a strong interaction with the minor groove of the apical stem and a weaker interaction in the central domain. A truncated form of DAI, p20, containing only the RNA binding domain, gives a similar protection pattern in the apical stem but protects the central domain less effectively. We conclude that the RNA binding domain of DAI interacts directly with the apical stem and central domain of VA RNA, and that other regions of the protein contribute to interactions with the central domain.     

Mutational analysis of the central domain of adenovirus virus-associated RNA mandates a revision of the proposed secondary structure.

Protein synthesis in adenovirus-infected cells is regulated during the late phase of infection. The rate of initiation is maintained by a small viral RNA, virus-associated (VA) RNAI, which prevents the phosphorylation of eukaryotic initiation factor eIF-2 by a double-stranded RNA-activated protein kinase, DAI. On the basis of nuclease sensitivity analysis, a secondary-structure model was proposed for VA RNA. The model predicts a complex stem-loop structure in the central part of the molecule, the central domain, joining two duplexed stems. The central domain is required for the inhibition of DAI activation and participates in the binding of VA RNA to DAI. To assess the significance of the postulated stem-loop structure in the central domain, we generated compensating, deletion, and substitution mutations. A substitution mutation which disrupts the structure in the central domain abolishes VA RNA function in vitro and in vivo. Base-compensating mutations failed to restore the function or structure of the mutant, implying that the stem-loop structure may not exist. To confirm this observation, we tested mutants with alterations in the hypothetical loop and short stem that constitute the main features of the wild-type model structure. The upper part of the hypothetical loop could be deleted without abolishing the ability of the RNA to block DAI activation in vitro, whereas other loop mutations were deleterious for function and caused major rearrangements in the molecule. Base-compensating mutations in the stem did not restore the expected base pairing, even though the mutant RNAs were still functional in vitro. Surprisingly, a mutant with a noncompensating substitution mutation in the stem was more effective than wild-type VA RNAI in DAI inhibition assays but was ineffective in vivo. The structural and functional consequences of these mutations do not support the proposed model structure for the central domain, and we therefore suggest an alternative structure in which tertiary interactions may play a significant role in shaping the specificity of VA RNA function in the infected cell. Discrepancies between the functionality of mutant forms of VA RNA in vivo and in vitro are consistent with the existence of additional roles for VA RNA in the cell.     

Secondary and tertiary structure in the central domain of adenovirus type 2 VA RNA I.

The small (160 nt) adenovirus RNA, VA RNAI, antagonizes the activation of the cellular protein kinase PKR (also known as DAI), a key regulator of gene expression. VA RNA consists of two stems separated by a complex region, the central domain, that is essential for its function. A notable feature of the central domain is a pair of tetranucleotides, GGGU and ACCC, which are mutually complementary and phylogenetically conserved. To investigate their role in the structure and function of VA RNA, we generated three sets of mutations designed to disrupt the putative stem and to restore it with different nucleotides. Substitutions in either of the tetranucleotides abrogated VA RNA function in two independent PKR-based assays, demonstrating the importance of these sequences in vivo. Compensating mutants restored function, indicating that the two tetranucleotides pair in the cell, but all of the compensating mutants were less active than wild-type VA RNA. The effects of the mutations on RNA structure were probed by nuclease sensitivity analysis. Pronounced changes in two loops in the central domain correlated closely with the formation and disruption of the stem, suggesting that the tetranucleotide stem defines a critical element in the structure of the central domain through tertiary interactions with the two loops. A model for the central domain is presented that accommodates these findings and also accounts for the known sites of PKR interaction.     

Interaction of adenovirus VA RNAl with the protein kinase DAI: nonequivalence of binding and function

Adenovirus VA RNAL maintains protein synthesis by preventing activation of the double-stranded RNA (dsRNA)-dependent protein kinase DAI. There appears to be a single binding site for dsRNA on DAI, and this site is blocked by VA RNAl. VA RNAl binds to purified DAI and can be cross-linked to the enzyme by UV irradiation. To determine the relationship between DAI binding and VA RNAl structure and function, we examined the binding abilities of wild-type and mutant VA RNAs. In several cases, the ability to bind DAI efficiently in vitro did not correlate with function in vivo. Secondary structure analysis suggested that efficient binding requires an apical stem-loop structure, whereas inhibition of DAI activation requires the central domain of the VA RNA molecule. We propose that the duplex stem permits VA RNA to interact with the dsRNA binding site on DAI and inhibits activation by juxtaposing the central domain of the RNA with the enzyme's active site.     

Purification and activation of the double-stranded RNA-dependent eIF-2 kinase DAI.

The double-stranded RNA (dsRNA)-dependent protein kinase DAI (also termed dsI and P1) possesses two kinase activities; one is an autophosphorylation activity, and the other phosphorylates initiation factor eIF-2. We purified the enzyme, in a latent form, to near homogeneity from interferon-treated human 293 cells. The purified enzyme consisted of a single polypeptide subunit of approximately 70,000 daltons, retained its dependence on dsRNA for activation, and was sensitive to inhibition by adenovirus VA RNAI. Autophosphorylation required a suitable concentration of dsRNA and was second order with respect to DAI concentration, which suggests an intermolecular mechanism in which one DAI molecule phosphorylates a neighboring molecule. Once autophosphorylated, the enzyme could phosphorylate eIF-2 but seemed unable to phosphorylate other DAI molecules, which implies a change in substrate specificity upon activation. VA RNAI blocked autophosphorylation and activation but permitted the activated enzyme to phosphorylate eIF-2. VA RNAI also blocked the binding of dsRNA to the enzyme. The data are consistent with a model in which activation requires the interaction of two molecules of DAI with dsRNA, followed by intermolecular autophosphorylation of the latent enzyme. VA RNAI would block activation by preventing the interaction between DAI and dsRNA.     

Removal of double-stranded contaminants from RNA transcripts: synthesis of adenovirus VA RNAI from a T7 vector

Bacteriophage RNA polymerases are widely used to synthesize defined RNAs on a large scale in vitro. Unfortunately, the RNA product contains a small proportion of contaminating RNAs, including complementary species, which can lead to errors of interpretation. We cloned the gene encoding Ad2 VA RNAI into a vector containing a T7 RNA polymerase promoter in order to generate large quantities of VA RNA for the study of its interaction with the dsRNA-dependent protein kinase DAI. Exact copies of VA RNAI were synthesized efficiently, but were contaminated with small amounts of dsRNA which activated DAI and confounded interpretation of kinase assays. We therefore developed a method to remove the dsRNA contaminants, allowing VA RNAI and mutants to be tested for their ability to activate or inhibit DAI. This method appears to be generally applicable.     

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.     

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.     

Suppression of RNA interference by adenovirus virus-associated RNA

We show that human adenovirus inhibits RNA interference (RNAi) at late times of infection by suppressing the activity of two key enzyme systems involved, Dicer and RNA-induced silencing complex (RISC). To define the mechanisms by which adenovirus blocks RNAi, we used a panel of mutant adenoviruses defective in virus-associated (VA) RNA expression. The results show that the virus-associated RNAs, VA RNAI and VA RNAII, function as suppressors of RNAi by interfering with the activity of Dicer. The VA RNAs bind Dicer and function as competitive substrates squelching Dicer. Further, we show that VA RNAI and VA RNAII are processed by Dicer, both in vitro and during a lytic infection, and that the resulting short interfering RNAs (siRNAs) are incorporated into active RISC. Dicer cleaves the terminal stem of both VA RNAI and VA RNAII. However, whereas both strands of the VA RNAI-specific siRNA are incorporated into RISC, the 3' strand of the VA RNAII-specific siRNA is selectively incorporated during a lytic infection. In summary, our work shows that adenovirus suppresses RNAi during a lytic infection and gives insight into the mechanisms of RNAi suppression by VA RNA.     

Control of translation in adenovirus-infected cells.

The initiation of protein synthesis in adenovirus-infected cells is regulated during the late phase in two ways, which may be related. The overall translation rate is maintained by a small viral RNA, VA RNAI, which prevents the phosphorylation of initiation factor eIF-2 by a double-stranded RNA-activated protein kinase, DAI. In addition, the relative efficiency of translation of host cell and viral mRNA populations is regulated in the infected cell during the late phase such that viral mRNAs are selectively utilized. Three viral elements have been implicated in this process: the 5' leader present on most late viral mRNAs; the late protein, 100K; and VA RNA. This article reviews the mechanisms underlying these translational control phenomena.     

Functional dissection of adenovirus VAI RNA.

During the course of adenovirus infection, the VAI RNA protects the translation apparatus of host cells by preventing the activation of host double-stranded RNA-activated protein kinase, which phosphorylates and thereby inactivates the protein synthesis initiation factor eIF-2. In the absence of VAI RNA, protein synthesis is drastically inhibited at late times in infected cells. The experimentally derived secondary structure of VAI RNA consists of two extended base-paired regions, stems I and III, which are joined by a short base-paired region, stem II, at the center. Stems I and II are joined by a small loop, A, and stem III contains a hairpin loop, B. At the center of the molecule and at the 3' side, stems II and III are connected by a short stem-loop (stem IV and hairpin loop C). A fourth, minor loop, D, exists between stems II and IV. To determine sequences and domains critical for function within this VAI RNA structure, we have constructed adenovirus mutants with linker-scan substitution mutations in defined regions of the molecule. Cells infected with these mutants were analyzed for polypeptide synthesis, virus yield, and eIF-2 alpha kinase activity. Our results showed that disruption of base-paired regions in the distal parts of the longest stems, I and III, did not affect function, whereas mutations causing structural perturbations in the central part of the molecule containing stem II, the proximal part of stem III, and the central short stem-loop led to loss of function. Surprisingly, one substitution mutant, sub742, although dramatically perturbing the integrity of the structure of this central portion, showed a wild-type phenotype, suggesting that an RNA with an alternate secondary structure is functional. On the basis of sensitivity to single-strand-specific RNases, we can derive a novel secondary structure for the mutant RNA in which a portion of the sequences may fold to form a structure that resembles the central part of the wild-type molecule, which suggests that only the short stem-loop located in the center of the molecule and the adjoining base-paired regions may define the functional domain. These results also imply that only a portion of the VAI RNA structure may be recognized by the host factor(s).     

Two functionally distinct RNA-binding motifs in the regulatory domain of the protein kinase DAI.

The RNA-binding domain of the protein kinase DAI, the double-stranded RNA inhibitor of translation, contains two repeats of a motif that is also found in a number of other RNA-binding proteins. This motif consists of 67 amino acid residues and is predicted to contain a positively charged alpha helix at its C terminus. We have analyzed the effects of equivalent single amino acid changes in three conserved residues distributed over each copy of the motif. Mutants in the C-terminal portion of either repeat were severely defective, indicating that both copies of the motif are essential for RNA binding. Changes in the N-terminal and central parts of the motif were more debilitating if they were made in the first motif than in the second, suggesting that the first motif is the more important for RNA binding and that the second motif is structurally more flexible. When the second motif was replaced by a duplicate of the first motif, the ectopic copy retained its greater sensitivity to mutation, implying that the two motifs have distinct functions with respect to the process of RNA binding. Furthermore, the mutations have the same effect on the binding of double-stranded RNA and VA RNA, consistent with the existence of a single RNA-binding domain for both activating and inhibitory RNAs.