Adeno-associated virus (AAV) has previously been shown to inhibit the replication of its helper virus herpes simplex virus type 1 (HSV-1), and the inhibitory activity has been attributed to the expression of the AAV Rep proteins. In the present study, we assessed the Rep activities required for inhibition of HSV-1 replication using a panel of wild-type and mutant Rep proteins lacking defined domains and activities. We found that the inhibition of HSV-1 replication required Rep DNA-binding and ATPase/helicase activities but not endonuclease activity. The Rep activities required for inhibition of HSV-1 replication precisely coincided with the activities that were responsible for induction of cellular DNA damage and apoptosis, suggesting that these three processes are closely linked. Notably, the presence of Rep induced the hyperphosphorylation of a DNA damage marker, replication protein A (RPA), which has been reported not to be normally hyperphosphorylated during HSV-1 infection and to be sequestered away from HSV-1 replication compartments during infection. Finally, we demonstrate that the execution of apoptosis is not required for inhibition of HSV-1 replication and that the hyperphosphorylation of RPA per se is not inhibitory for HSV-1 replication, suggesting that these two processes are not directly responsible for the inhibition of HSV-1 replication by Rep.Adeno-associated virus (AAV) is a widespread, nonpathogenic human parvovirus with a unique biphasic life cycle. In the absence of a helper virus, AAV establishes a latent infection in the host cell mediated either by site-specific integration of the viral genome into human chromosome 19 or by episomal persistence of circularized virus genomes (reviewed in reference 53). In the presence of helper viruses such as a herpesvirus, adenovirus (Ad), or papillomavirus, AAV is rescued from latency and undergoes lytic replication. The AAV genome is a single-stranded DNA (ssDNA) of 4,680 nucleotides, which is packaged into an icosahedral capsid with a diameter of 20 nm. The AAV genome harbors two open reading frames (ORFs), rep and cap, which are flanked by two inverted terminal repeats (ITRs) containing viral origins of DNA replication. The cap ORF is transcribed from the p40 promoter and encodes the capsid proteins VP1, VP2, and VP3, which differ in their N termini due to alternative start codons. The rep ORF encodes the Rep proteins, which are expressed in four different forms due to transcription from two different promoters, p5 and p19, and alternative splicing at an intron at the C-terminal end. The different Rep proteins are termed Rep40, Rep52, Rep68, and Rep78 according to their apparent molecular weight. The Rep proteins are involved in diverse processes in the viral life cycle, such as DNA replication, regulation of gene expression, genome packaging, and site-specific integration (reviewed in reference 56). The biochemical activities of Rep required for AAV DNA metabolism include site-specific DNA-binding and endonuclease activities, as well as non-site-sp...
Adeno-associated virus type 2 (AAV2) is a human parvovirus that relies on a helper virus for efficient replication. Herpes simplex virus 1 (HSV-1) supplies helper functions and changes the environment of the cell to promote AAV2 replication. In this study, we examined the accumulation of cellular replication and repair proteins at viral replication compartments (RCs) and the influence of replicating AAV2 on HSV-1-induced DNA damage responses (DDR). We observed that the ATM kinase was activated in cells coinfected with AAV2 and HSV-1. We also found that phosphorylated ATR kinase and its cofactor ATR-interacting protein were recruited into AAV2 RCs, but ATR signaling was not activated. DNA-PKcs, another main kinase in the DDR, was degraded during HSV-1 infection in an ICP0-dependent manner, and this degradation was markedly delayed during AAV2 coinfection. Furthermore, we detected phosphorylation of DNA-PKcs during AAV2 but not HSV-1 replication. The AAV2-mediated delay in DNA-PKcs degradation affected signaling through downstream substrates. Overall, our results demonstrate that coinfection with HSV-1 and AAV2 provokes a cellular DDR which is distinct from that induced by HSV-1 alone.A deno-associated virus type 2 (AAV2) is a small, nonenveloped parvovirus with a single-stranded DNA genome of 4.7 kb (52). In the absence of a helper virus, AAV2 establishes a latent infection characterized by site-specific integration of the viral genome into the AAVS1 site on human chromosome 19 (72). In the presence of a helper virus, AAV2 can replicate productively in the host cell nucleus. AAV2 DNA replication occurs at discrete sites in the nucleus, termed replication compartments (RCs). During the course of infection, several small RCs rapidly expand and fuse to large structures, which displace the cellular chromatin and fill the entire cell nucleus (28,35,37,79,91). AAV2 RCs contain AAV2 proteins, as well as defined helper virus proteins and cellular proteins (3,35,63,65,75,79,90,91). Replicating AAV2 has inhibitory effects on both the host cell (9,41,68,71,73,74,100,101) and the helper virus (5,30,31,34,40,44,61,84,100).One of the helper viruses for AAV2 replication is herpes simplex virus 1 (HSV-1) (14). The minimal HSV-1 helper factors for AAV2 replication from plasmid substrates include the helicaseprimase complex encoded by UL5, UL8, and UL52 and the major DNA binding protein ICP8 (3) (90). Besides viral helper factors, the fate of AAV2 replication also depends on cellular proteins. Recently, cellular proteins have been identified that interact with AAV2 Rep78/68 in adenovirus (Ad)-or HSV-1-supported AAV2 replication (63,65). Of these, the largest functional categories correspond to cellular proteins which are involved in DNA metabolism, including DNA replication, repair, and chromatin modification.There is accumulating evidence that the DNA damage response (DDR) pathways play central roles in viral replication (92). Control of DDR signaling may be a mechanism to prevent apoptosis and/or stop cell cycle progression (92)...
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
e Adeno-associated virus type 2 is known to inhibit replication of herpes simplex virus 1 (HSV-1). This activity has been linked to the helicase-and DNA-binding domains of the Rep68/Rep78 proteins. Here, we show that Rep68 can bind to consensus Repbinding sites on the HSV-1 genome and that the Rep helicase activity can inhibit replication of any DNA if binding is facilitated. Therefore, we hypothesize that inhibition of HSV-1 replication involves direct binding of Rep68/Rep78 to the HSV-1 genome.A deno-associated virus type 2 (AAV2) is a nonpathogenic human parvovirus with a unique biphasic life cycle. In the absence of a helper virus, AAV2 establishes a latent infection, while in the presence of a helper virus, such as adenovirus type 2 (AdV2), herpes simplex virus 1 (HSV-1), or human papillomavirus 16 (HPV-16), it undergoes lytic replication (1-4). The AAV2 genome is a single-stranded DNA (ssDNA) molecule of 4,680 nucleotides, which is packaged into an icosahedral capsid with a diameter of approximately 20 nm (5). The genome harbors two clusters of genes, rep and cap, which are flanked by inverted terminal repeats (ITRs). The ITRs form hairpin structures and contain a Rep-binding site (RBS) and a terminal resolution site (trs), which together act as the viral origin of DNA replication (6, 7). The cap gene is transcribed from the p40 promoter and encodes the capsid proteins VP1, VP2, and VP3, which differ in their N termini due to alternative start codons (8, 9). In addition, a nested open reading frame (ORF) within the cap gene encodes a protein designated assembly-activating protein (AAP), which is believed to be required for AAV2 capsid assembly in the nucleolus (10, 11). The rep gene encodes the Rep proteins, which are synthesized in four different forms due to transcription from two different promoters, p5 and p19, and alternative splicing of an intron near the C-terminal end (12). The different Rep proteins are termed Rep40, Rep52, Rep68, and Rep78 according to their apparent molecular weights. The Rep proteins are involved in diverse processes during the viral life cycle, such as DNA replication, regulation of gene expression, genome packaging, and site-specific genomic integration (13-18).HSV-1 belongs to the subfamily of the Alphaherpesvirinae and is the reagent causing mucosal eruptions at the site of infection, which can reoccur at the same site upon reactivation from latency (19,20). The HSV-1 virion is built up by three structural components, the capsid, the tegument, and the surrounding envelope. The viral genome is a linear double-stranded DNA (dsDNA) molecule 152 kb in size and has a unique structure. It is divided into two covalently joined segments, which contain unique segments (unique long [U L ] and unique short [U S ]) and inverted repeat regions (TR L , IR L , IR S , and TR S ). The IR sequences link the L and S segments (Fig. 1A). HSV-1 gene expression and replication occur in a temporally regulated cascade: immediate early (IE), early (E), and late. IE proteins exhibit mainly regulatory...
Adeno-associated virus 2 (AAV2) is a helpervirus-dependent parvovirus with a bi-phasic life cycle comprising latency in absence and lytic replication in presence of a helpervirus, such as adenovirus (Ad) or herpes simplex virus type 1 (HSV-1). Helpervirus-supported AAV2 replication takes place in replication compartments (RCs) in the cell nucleus where virus DNA replication and transcription occur. RCs consist of a defined set of helper virus-, AAV2-, and cellular proteins. Here we compare the profile of cellular proteins recruited into AAV2 RCs or identified in Rep78-associated complexes when either Ad or HSV-1 is the helpervirus, and we discuss the potential roles of some of these proteins in AAV2 and helpervirus infection.
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