Herpes Simplex Virus Type 1 ICP0 Protein Mediates Activation of Adeno-Associated Virus Type 2 rep Gene Expression from a Latent Integrated Form

Abstract

Adeno-associated virus type 2 (AAV-2) is a human parvovirus that requires the presence of a helper virus, such as the herpes simplex virus type 1 (HSV-1) to accomplish a complete productive cycle. In the absence of helper virus, AAV-2 can establish a latent infection that is characterized by the absence of expression of viral genes. So far, four HSV-1 early genes, UL5/8/52 (helicase primase complex) and UL29 (single-stranded DNA-binding protein), were defined as sufficient for AAV replication when cells were transfected with a plasmid carrying the wild-type AAV-2 genome. However, none of these viral products was shown to behave as a transcriptional factor able to activate AAV gene expression. Our study provides the first evidence that the immediate-early HSV-1 protein ICP0 can promote rep gene expression in cells latently infected with wild-type AAV-2. This ICP0-mediated effect occurs at the transcriptional level and involves the ubiquitin-proteasome pathway. Furthermore, using deletion mutants, we demonstrate that the localization of ICP0 to ND10 and their disruption is not required for the activation of the rep promoter, whereas binding of ICP0 to the ubiquitinspecific protease HAUSP makes a significant contribution to this effect.Adeno-associated virus type 2 (AAV-2) is a nonpathogenic human parvovirus that establishes a latent infection in the absence of helper virus (1, 57). During latency, the viral genome persists in a largely repressed episomal or integrated form (14,54,63). Infection of latently infected cells with a helper virus such as herpes simplex virus (HSV) or adenovirus (Ad) leads to the reactivation of AAV gene expression, the rescue of the viral genome, and finally to the progression through a productive phase (1, 57).The 4.7-kb genome of AAV-2 contains two open reading frames (ORFs), rep and cap, flanked by two inverted terminal repeats (ITR) that constitute the cis-acting elements required for DNA replication. Two promoters at map positions 5 and 19 (p5 and p19, respectively) control the expression of the rep ORF leading to the synthesis of Rep78/68 and Rep52/40 proteins, respectively. These proteins are involved in many aspects of the viral life cycle and particularly in the regulation of AAV gene expression. The p40 promoter controls the synthesis of the three proteins (VP1, 2, and 3) that constitute the capsid (1).During latency, i.e., in the absence of helper virus, the silent state of the AAV promoters, particularly that of the p5 promoter, is generally attributed to repressive activity exerted by cellular factors and the Rep proteins. Indeed, the results of several studies with transient-transfection assays have reported that Rep78 and Rep68 act as site-specific DNA-binding proteins to shut down p5 and p19 transcription (52, 53). The Rep binding site involved in this repressive effect was identified both within the ITR and the p5 promoter (46, 53). In addition, silencing of the p5 promoter was shown to be mediated by its interaction with the cellular transcription factor YY1 bound at p...

Interactions Between AAV-2 and HSV-1: Implications for Hybrid Vector Design

“…Activation of the p5 promoter can therefore be considered the initiating event for productive AAV-2 replication. In analogy to Ad E1A, the HSV-1 IE gene product ICP0 was shown to transactivate transcription from the p5 promoter, resulting in Rep68 and Rep78 synthesis [135]. However, ICP0 also induced the synthesis of Rep40 and Rep52, which is controlled by the p19 promoter.…”

“…However, ICP0 also induced the synthesis of Rep40 and Rep52, which is controlled by the p19 promoter. It is currently not known if the induction of Rep40 and Rep52 synthesis by ICP0 involves a direct activation of the p19 promoter by ICP0 or if the effect is indirectly mediated by the Rep68 and Rep78 proteins [12,135]. Although ICP0 is considered a promiscuous transactivator of gene expression [136], the effect on rep expression seems specific, in that ICP0 does not activate cap expression [135].…”

“…It is currently not known if the induction of Rep40 and Rep52 synthesis by ICP0 involves a direct activation of the p19 promoter by ICP0 or if the effect is indirectly mediated by the Rep68 and Rep78 proteins [12,135]. Although ICP0 is considered a promiscuous transactivator of gene expression [136], the effect on rep expression seems specific, in that ICP0 does not activate cap expression [135]. The induction of rep expression is strictly dependent on the E3 ubiquitin ligase activity of the ICP0 RING-finger domain and on proteasome activity, Interactions between AAV-2 & HSV-1: implications for hybrid vector design Review suggesting that derepression of the p5 promoter involves the ICP0-induced degradation of one or several cellular factors [135,137].…”

“…Although ICP0 is considered a promiscuous transactivator of gene expression [136], the effect on rep expression seems specific, in that ICP0 does not activate cap expression [135]. The induction of rep expression is strictly dependent on the E3 ubiquitin ligase activity of the ICP0 RING-finger domain and on proteasome activity, Interactions between AAV-2 & HSV-1: implications for hybrid vector design Review suggesting that derepression of the p5 promoter involves the ICP0-induced degradation of one or several cellular factors [135,137]. Activation of the p5 promoter by ICP0 also involves the interaction of ICP0 with the deubiquitylating enzyme USP7/HAUSP, which, however, is not essential [135,137].…”

“…The induction of rep expression is strictly dependent on the E3 ubiquitin ligase activity of the ICP0 RING-finger domain and on proteasome activity, Interactions between AAV-2 & HSV-1: implications for hybrid vector design Review suggesting that derepression of the p5 promoter involves the ICP0-induced degradation of one or several cellular factors [135,137]. Activation of the p5 promoter by ICP0 also involves the interaction of ICP0 with the deubiquitylating enzyme USP7/HAUSP, which, however, is not essential [135,137]. It was recently shown that transactivation of rep expression by ICP0 can be further enhanced by the presence of two other HSV-1 IE proteins, ICP4 and ICP22 [138].…”