Cell Cycle-Dependent Expression of Adeno-Associated Virus 2 (AAV2) Rep in Coinfections with Herpes Simplex Virus 1 (HSV-1) Gives Rise to a Mosaic of Cells Replicating either AAV2 or HSV-1

Abstract

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/G 2 -phase cells, while HSV-1 DNA replication is restricted to G 1 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/G 2 -phase cells, suggesting that the preference for S/G 2 phase is independent of the nature of the viral genome. Interestingly, however, a substantial proportion of S/G 2 -phase cells transduced by the double-stranded but not the singlestranded 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/G 2 phase support AAV2 replication and inhibit HSV-1 replication. Conversely, low Rep protein levels in G 1 phase permit HSV-1 replication but are insufficient for AAV2 replication. This allows both viruses to productively replicate in distinct sets of dividing cells.KEYWORDS AAV2, HSV-1, Rep protein, biological niche, cell cycle, helper virus

NaCl and KCl mediate log increase in AAV vector particles and infectious titers in a specific/timely manner with the HSV platform

“…During this time frame we would expect that most of the infected cell population would have reached the G 1 or the S phase, which would also be in accordance with AAV and HSV active role in modulation of the cell cycle to their benefit. 14 , 15 …”

The Interplay between Adeno-Associated Virus and Its Helper Viruses

“…This raises the question of how those different viruses can coexist in a cell population. AAV and HSV-1 replicate during different cell cycle phases and hence replicate in two different ecological niches [ 232 ]. In the absence of AAV, HSV-1 replication is cell-cycle independent and blocks cell cycle progression in the G1 and G2 phases [ 233 , 234 , 235 , 236 ].…”

“…In the absence of AAV, HSV-1 replication is cell-cycle independent and blocks cell cycle progression in the G1 and G2 phases [ 233 , 234 , 235 , 236 ]. AAV gene expression and replication were found to preferentially occur in S/G2 phases of the cell cycle [ 232 ]. Restriction of AAV replication and gene expression to S/G2 was not dependent on second-strand synthesis as self-complementary rAAV showed the same cell cycle preference.…”

Genome-wide activation screens to increase adeno-associated virus production

“…30 For example, Rep78 arrests the cells in S-phase via multiple pathways, 31,32 and AAV2 genome replication occurs primarily in S/G2 phase. 33 We hypothesized SKA2 and/or ITPRIP may modulate the cell cycle in a manner that increases AAV genome replication, resulting in increased AAV titer. Flow cytometric analysis of 4 0 ,6-diamidino-2-phenylindole (DAPI) staining revealed that cell-cycle distribution was similar for normally propagating WT, SKA2-, and ITPRIP-expressing cell lines (Figure S6).…”