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AAV2 X increases AAV6 rep/cap-driven rAAV production.

ABSTRACT: We have recently identified a new gene, involved in DNA replication, at the far 3' end of the adeno-associated virus type 2 (AAV2) genome. The AAV type 6 (AAV6) genome has a disrupted X open reading frame (ORF) whose two halves, when combined, have full-length homology and comparable size to AAV2 X. Hypothesizing that AAV6 X is inactive, we assessed if AAV2 X augments recombinant (r)AAV2 DNA replication and virion production, but with rep and cap trans-functions of AAV6. Using AAV2 X expressing HEK293 cell lines we show AAV2 X significantly boosts rAAV DNA replication/virion production, driven by AAV6 rep/cap as it does the AAV2 rep/cap system. Protein BLAST search for homology between AAV2 X and various AAV Rep78 proteins suggests that X might be AAV8 Rep78-derived and have some of its activities. These data suggest that AAV2 X, and the corresponding X genes of other AAV types/clades, warrant further study.

SUBMITTER: Cao M

PROVIDER: S-EPMC4461464 | biostudies-literature | 2015 Aug

REPOSITORIES: biostudies-literature

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AAV2 X increases AAV6 rep/cap-driven rAAV production.

Cao M M Chiriva-Internati M M Hermonat P L PL

Virology 20150330

We have recently identified a new gene, involved in DNA replication, at the far 3' end of the adeno-associated virus type 2 (AAV2) genome. The AAV type 6 (AAV6) genome has a disrupted X open reading frame (ORF) whose two halves, when combined, have full-length homology and comparable size to AAV2 X. Hypothesizing that AAV6 X is inactive, we assessed if AAV2 X augments recombinant (r)AAV2 DNA replication and virion production, but with rep and cap trans-functions of AAV6. Using AAV2 X expressing ...[more]

PMID: 25838114

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Project description:Adeno-associated virus 2 (AAV2) depends on the simultaneous presence of a helper virus such as herpes simplex virus 1 (HSV-1) for productive replication. At the same time, AAV2 efficiently blocks the replication of HSV-1, which would eventually limit its own replication by diminishing the helper virus reservoir. This discrepancy begs the question of how AAV2 and HSV-1 can coexist in a cell population. Here we show that in coinfected cultures, AAV2 DNA replication takes place almost exclusively in S/G2-phase cells, while HSV-1 DNA replication is restricted to G1 phase. Live microscopy revealed that not only wild-type AAV2 (wtAAV2) replication but also reporter gene expression from both single-stranded and double-stranded (self-complementary) recombinant AAV2 vectors preferentially occurs in S/G2-phase cells, suggesting that the preference for S/G2 phase is independent of the nature of the viral genome. Interestingly, however, a substantial proportion of S/G2-phase cells transduced by the double-stranded but not the single-stranded recombinant AAV2 vectors progressed through mitosis in the absence of the helper virus. We conclude that cell cycle-dependent AAV2 rep expression facilitates cell cycle-dependent AAV2 DNA replication and inhibits HSV-1 DNA replication. This may limit competition for cellular and viral helper factors and, hence, creates a biological niche for either virus to replicate.IMPORTANCE Adeno-associated virus 2 (AAV2) differs from most other viruses, as it requires not only a host cell for replication but also a helper virus such as an adenovirus or a herpesvirus. This situation inevitably leads to competition for cellular resources. AAV2 has been shown to efficiently inhibit the replication of helper viruses. Here we present a new facet of the interaction between AAV2 and one of its helper viruses, herpes simplex virus 1 (HSV-1). We observed that AAV2 rep gene expression is cell cycle dependent and gives rise to distinct time-controlled windows for HSV-1 replication. High Rep protein levels in S/G2 phase support AAV2 replication and inhibit HSV-1 replication. Conversely, low Rep protein levels in G1 phase permit HSV-1 replication but are insufficient for AAV2 replication. This allows both viruses to productively replicate in distinct sets of dividing cells.

Dataset Information

AAV2 X increases AAV6 rep/cap-driven rAAV production.

Publications

AAV2 X increases AAV6 rep/cap-driven rAAV production.

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