Positive and Negative Effects of Adenovirus Type 5 Helper Functions on Adeno-Associated Virus Type 5 (AAV5) Protein Accumulation Govern AAV5 Virus Production

Abstract

Full replication of adeno-associated virus type 5 (AAV5) is sustained by adenovirus type 5 (Ad5) helper functions E1a, E1b, E2a, E4Orf6, and virus-associated (VA) RNA; however, their combined net enhancement of AAV5 replication was comprised of both positive and negative individual effects. Although Ad5 E4Orf6 was required for AAV5 genomic DNA replication, it also functioned together with E1b to degrade de novoexpressed, preassembled AAV5 capsid proteins and Rep52 in a proteosome-dependent manner. VA RNA enhanced accumulation of AAV5 protein, overcoming the degradative effects of E4Orf6, and was thus required to restore adequate amounts of AAV5 proteins necessary to achieve efficient virus production.Efficient replication of adeno-associated viruses (AAVs) requires helper functions that can be supplied by larger DNA viruses such as adenovirus (Ad) or herpes simplex virus (2). There are five adenovirus type 5 (Ad5) functions required to support both AAV2 replication and production of recombinant AAV (E1a, E1b, E2a, E4Orf6, and virus-associated [VA] RNA), and the roles these factors play during both Ad and AAV infection have been extensively characterized (2,3,24,25). Ad5 E2a provides single-stranded DNA (ssDNA) binding activity presumably required during the strand-displacement phase of AAV2 terminal repeat (TR)-mediated genome replication (20). E4Orf6 has been shown to be essential for second strand synthesis, both in vitro and in vivo (7), and likely plays other roles during AAV2 replication as well (22,24,26). VA RNA has been suggested to enhance AAV gene expression at the posttranscriptional level, most likely at the level of protein translation (22,26). The role of VA RNA in the translation of Ad5 proteins during Ad5 infection is well characterized (12, 13). Ad5 E1a and E1b have been reported to perform a number of essential roles during AAV gene expression, including promoter activation (3,11,15,28); however, other than the E1A's well-defined participation in the regulation of expression of AAV2 P5, the role that adenovirus plays in other aspects of AAV2 gene expression is as yet only partially understood (3, 25).Adeno-associated virus type 5 (AAV5), which was first isolated from a penile condylomatous lesion, is the most divergent of the AAV serotypes, sharing only 64% overall nucleotide identity with the prototype AAV2 (1, 8). While the basic transcription profile of AAV5 is similar to that of AAV2, there are also significant differences (17). In contrast to AAV2, RNAs generated from both the AAV5 P7 and P19 promoters are efficiently polyadenylated at a site lying within the intron in the center of the genome, and because these RNAs are not spliced, Rep78 and Rep52 are the only Rep proteins detected during AAV5 infection (17). Furthermore, unlike AAV2, neither the AAV5 Rep protein nor additional adenovirus gene products are required to achieve efficient AAV5 promoter activity and pre-mRNA splicing following transfection of an AAV5 rep/cap plasmid clone lacking the inverted terminal repeats (ITRs) into 293...

Adeno-Associated Virus Type 5 Utilizes Alternative Translation Initiation To Encode a Small Rep40-Like Protein

“…However, as shown in Fig. 1B, and as we have shown previously (7,8,(10)(11)(12), substantial levels of a Rep40-like protein product are generated during both AAV5 and goat AAV infections of 293 cells (Fig. 1B, lanes 1 and 2), despite the relatively low levels (Ͻ5%) of splicing from upstream transcripts (Fig.…”

“…Thus, these viruses would not be expected to generate significant quantities of Rep40. However, immunoblot analyses have consistently demonstrated the presence of significant levels of a Rep40-like (but not a Rep 68-like) protein during AAV5 and goat AAV coinfection with adenovirus, as well as during transient transfection (7,8,11). This suggested that AAV5 may generate a Rep40-like protein using a mechanism distinct from the alternative splicing utilized by AAV2.…”

E4Orf6-E1B-55k-Dependent Degradation of De Novo-Generated Adeno-Associated Virus Type 5 Rep52 and Capsid Proteins Employs a Cullin 5-Containing E3 Ligase Complex

“…In addition to these important functions, we have recently shown that E4Orf6 together with E1B-55k can promote the degradation of de novo-generated AAV5 capsid and small Rep proteins (9). We have also shown that one of the important roles that VA RNA plays in promoting AAV5 infection is to overcome the effect that E4Orf6-E1B-55k has in reducing the accumulated levels of AAV5 proteins (9).…”

“…E4Orf6-E1B-55k-dependent degradation of de novo-generated AAV5 capsid proteins and Rep52 is inhibited by both MG132 and lactacystin (9), suggesting that it occurs in a proteasome-dependent manner, which typically is mediated by ubiquitinylation of target proteins. As shown, E4Orf6-E1B-55k-dependent degradation of both AAV5 capsid proteins (Fig.…”

Glioma-Parvovirus Interactions: Molecular Insights and Therapeutic Potential

“…In the Adeno-Associated Virus type 5 (AAV5), a member of the Dependovirus genus, viral replication and protein synthesis was highly enhanced by the co-expression of an adenovirus VA I RNA (Nayak and Pintel 2007a) that, by binding to PKR, prevents its otherwise activation by a short RNA sequence of AAV5 messengers (Nayak and Pintel 2007b). The infection of untransformed fibroblast cells by MVM of the Parvovirus genus, activated PKR and subsequently phosphorylated eIF2α (Ventoso et al 2010).…”