Adeno-Associated Viruses Can Induce Phosphorylation of eIF2α via PKR Activation, Which Can Be Overcome by Helper Adenovirus Type 5 Virus-Associated RNA

Adeno-associated viruses (AAVs) are dependoviruses which require the helper virus functions of larger DNA viruses for efficient replication. Adenovirus type 5 (Ad5) has been shown to be the most efficient helper virus for AAV, and the Ad5 factors necessary to support AAV replication are E1A, E1B-55K, E4Orf6, E2a, and virus-associated (VA) RNA. The roles that these gene products play during Ad5 infection have been extensively characterized (1, 2, 16, 26, 28), and their roles in supporting AAV type 2 (AAV2) infection have been the object of study for many years (1, 2, 26, 28). The Ad5 E1A and E1B-55K proteins have been reported to perform a number of essential roles during AAV gene expression. E1A has a well-defined role in the regulation of the expression of AAV2 P5 and AAV5 P41 (2, 9, 17, 31); however, the role of E1B-55K is less clearly defined. Additional roles that Ad5 may play in other aspects of AAV gene expression are as yet only partially understood (2, 28). Ad5 E2a is a single-stranded DNA (ssDNA) binding protein, and that activity has been shown to function during the strand-displacement phase of AAV2 inverted terminal repeat (ITR)-mediated genome replication (24). The E4Orf6 protein of various Ad serotypes has been shown to be required for the successful completion of second-strand synthesis, both in vitro and in vivo (5, 6); however, the mechanism by which this is accomplished is not yet fully understood. E4Orf6 has been suggested to play other roles during AAV2 replication as well (25, 26, 29). Finally, the Ad VA RNA, a small RNA produced abundantly during Ad infection, had been suggested as enhancing AAV gene expression at the posttranscriptional level, most likely at the level of protein translation (8, 25, 29). VA RNA is not itself translated. It acts as a regulatory RNA during the Ad5 life cycle, and its role in enhancing the translation of Ad5 proteins during Ad5 infection is well characterized (11, 12). In normal cells, there are a number of innate mechanisms that play important roles in inhibiting productive viral replication. An important participant in this process is the serine-threonine kinase protein kinase R (PKR). In response to activation by double-stranded RNA, PKR undergoes autophosphorylation and brings about the phosphorylation of the translation initiation factor eIF2α (22, 30). Unphosphorylated eIF2α can participate in new rounds of translation when GDP, bound to eIF2α, can be exchanged for GTP in the presence of guanine exchange factor. However, when eIF2α is phosphorylated, it forms a tight complex with guanine exchange factor, so the GDP-GTP exchange cannot occur and translation is blocked (13). Many viruses are known to disturb this system in order to facilitate the protein expression needed for their replication. This is accomplished by a number of mechanisms. For example, the influenza virus protein NS1 inhibits the phosphorylation of PKR by sequestering double-stranded RNA (10); the hepatitis C NS5A protein inhibits PKR phosphorylation by directly binding to PKR (18); the herpes simplex virus (HSV) protein ICP34.5 inhibits protein phosphatase I kinase (PPI kinase), a modification which allows PPI to dephosphorylate eIF2α, preventing its inactivation (27); and the Ad5 VA I RNA binds to the RNA binding domain of PKR, thus competitively inhibiting it (4, 15). Ad 5 produces two distinct VA RNAs, VA I and VA II. They are 160-nucleotide (nt) RNA molecules which are transcribed by RNA polymerase III and not translated. VA I RNA is the more abundant species (13). It differs considerably at the nucleotide level from VA II, yet both form similar stable secondary structures (11). The secondary structure of VA I is composed of an apical stem-loop, central domain, and terminal stem (11). The regions of VA I RNA which bind to PKR have been identified, and nucleotide substitutions which disturb the integrity of the central domain have been shown to severely hamper the ability of VA I RNA to inhibit the phosphorylation of PKR in vitro (19, 21). We have recently shown that the Ad5 E4Orf6 protein, together with E1B-55K, can target AAV5 capsid and small Rep proteins for degradation via the proteasome and that at least part of the required help that VA I RNA provides during AAV5 infection is to increase the accumulated levels of viral proteins necessary for replication (16). Our work also showed that the accumulated levels of AAV5 capsid and small Rep proteins were enhanced by VA I RNA when their transcription units were expressed from plasmid expression vectors. These observations led us to suspect that expressed AAV5 RNA might induce cellular PKR, with subsequent phosphorylation of eIF2α, although this had not been previously reported for any parvovirus. In this scenario, Ad5 VA I RNA could increase the accumulated levels of AAV5 proteins by competitively inhibiting PKR. In this study, we found that VA I RNA mutants deficient in inhibiting the phosphorylation of PKR no longer function as helpers for AAV5 replication or capsid protein accumulation. Also, we report that a short region of the leader sequence of the AAV5 capsid gene RNA activated PKR, resulting in the phosphorylation of eIF2α. Both short interfering RNA (siRNA) directed against PKR and the addition of HSV ICP34.5 enhanced the accumulation of capsid protein in the presence of the PKR-inducing element, suggesting that during AAV5 infection, VA I RNA acts to overcome the direct AAV5-induced activation of PKR that leads to the phosphorylation of eIF2α.