The adeno-associated virus (AAV) is a small, nonpathogenic parvovirus, which depends on helper factors to replicate. Those helper factors can be provided by coinfecting helper viruses such as adenoviruses, herpesviruses, or papillomaviruses. We review the basic biology of AAV and its most-studied helper viruses, adenovirus type 5 (AdV5) and herpes simplex virus type 1 (HSV-1). We further outline the direct and indirect interactions of AAV with those and additional helper viruses.

Inflammasomes are immune complexes that induce an inflammatory response upon sensing of different stress signals. This effect is mainly mediated by activation and secretion of the proinflammatory cytokines proIL-1β and -18. Here we report that infection of human primary keratinocytes with the double-stranded DNA viruses modified vaccinia virus Ankara (MVA) or herpes simplex virus type 1 (HSV-1)-induced secretion of mature IL-1β and -18. This secretion was dependent on several inflammasome complexes; however, the absent in melanoma 2 (AIM2) inflammasome, which is activated by binding of double-stranded DNA, played the most important role. Whereas prestimulation of keratinocytes with IFN-γ moderately increased MVA-induced IL-1β and IL-18 secretion, it was essential for substantial secretion of these cytokines in response to herpes simplex virus type 1 infection. IFN-γ partially restored HSV-1 suppressed proIL-1β expression and was also required for inflammasome activation. Most importantly, IFN-γ strongly suppressed virus replication in keratinocytes in vitro and ex vivo, which was independent of inflammasome activation. Our results suggest that, similar to Herpesviridae infection in mice, HSV-1 replication in human skin is controlled by a positive feedback loop of keratinocyte-derived IL-1/IL-18 and IFN-γ expressed by immune cells.

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)...

Herpes Simplex Virus Type-1 (HSV-1) forms progeny in the nucleus within distinct membrane-less inclusions, the viral replication compartments (VRCs), where viral gene expression, DNA replication, and packaging occur. The way in which the VRCs maintain spatial integrity remains unresolved. Here, we demonstrate that the essential viral transcription factor ICP4 is an intrinsically disordered protein (IDP) capable of driving protein condensation and liquid–liquid phase separation (LLPS) in transfected cells. Particularly, ICP4 forms nuclear liquid-like condensates in a dose- and time-dependent manner. Fluorescence recovery after photobleaching (FRAP) assays revealed rapid exchange rates of EYFP-ICP4 between phase-separated condensates and the surroundings, akin to other viral IDPs that drive LLPS. Likewise, HSV-1 VRCs revealed by EYFP-tagged ICP4 retained their liquid-like nature, suggesting that they are phase-separated condensates. Individual VRCs homotypically fused when reaching close proximity and grew over the course of infection. Together, the results of this study demonstrate that the HSV-1 transcription factor ICP4 has characteristics of a viral IDP, forms condensates in the cell nucleus by LLPS, and can be used as a proxy for HSV-1 VRCs with characteristics of liquid–liquid phase-separated condensates.