Abstract
ABSTRACTAdeno-associated virus serotype 2 (AAV2) can efficiently replicate in cells that have been infected with helper viruses, such as adenovirus or herpesvirus. However, in the absence of helper virus infection, AAV2 establishes latency by integrating its genome site specifically into PPP1R12C, a gene located on chromosome 19. This integration target site falls into one of the most gene-dense regions of the human genome, thus inviting the question as to whether the virus has evolved mechanisms to control this complex transcriptional environment in order to facilitate integration, maintain an apparently innocuous latency, and/or establish conditions that are conducive to the rescue of the integrated viral genome. The viral replication (Rep) proteins control and direct every known aspect of the viral life cycle and have been shown to tightly control all AAV2 promoters. In addition, a number of heterologous promoters are repressed by the AAV2 Rep proteins. Here, we demonstrate that Rep proteins efficiently repress expression from the target site PPP1R12C promoter. We find evidence that this repression employs mechanisms similar to those described for Rep-mediated AAV2 p5 promoter regulation. Furthermore, we show that the repression of the cellular target site promoter is based on two distinct mechanisms, one relying on the presence of a functional Rep binding motif within the 5′ untranslated region (UTR) of PPP1R12C, whereas the second pathway requires only an intact nucleoside triphosphate (NTP) binding site within the Rep proteins, indicating the possible reliance of this pathway on interactions of the Rep proteins with cellular proteins that mediate or regulate cellular transcription.IMPORTANCE The observation that repression of transcription from the adeno-associated virus serotype 2 (AAV2) p5 and integration target site promoters is mediated by shared mechanisms highlights the possible coevolution of virus and host and could lead to the identification of host factors that the virus exploits to navigate its life cycle.
Highlights
Adeno-associated virus serotype 2 (AAV2) can efficiently replicate in cells that have been infected with helper viruses, such as adenovirus or herpesvirus
To determine if AAV2 infection leads to changes in the expression levels of the target site PPP1R12C promoter, 293T and HeLa cells were infected with wt AAV2 using increasing MOIs in the presence and absence of adenovirus
In contrast to nonpermissive conditions, PPP1R12C expression is clearly repressed in wt AAV2- and adenovirus-coinfected cells compared to control cells, and this was observed for both cell lines (Fig. 1A, top row)
Summary
Adeno-associated virus serotype 2 (AAV2) can efficiently replicate in cells that have been infected with helper viruses, such as adenovirus or herpesvirus. The observation that repression of transcription from the adeno-associated virus serotype 2 (AAV2) p5 and integration target site promoters is mediated by shared mechanisms highlights the possible coevolution of virus and host and could lead to the identification of host factors that the virus exploits to navigate its life cycle. The large Rep proteins activate transcription from the p5 promoter through binding to the Rep binding site (RBS) present in the inverted terminal repeat (ITR) and mediate p5 repression by binding to the RBS in the p5 promoter This repression can be partially lifted by the small Rep proteins and contributes to an autoregulatory loop, which maintains constant ratios of the p5 and p19 transcripts [16].
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