Inhibition of Herpes Simplex Virus Type 1 Replication by Adeno-Associated Virus Rep Proteins Depends on Their Combined DNA-Binding and ATPase/Helicase Activities

Abstract

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 Modulates the Host DNA Damage Response Induced by Herpes Simplex Virus 1 during Coinfection

“…Recently, we reported hyperphosphorylation of RPA32 at the DNA-PK target site S4/8 in cells transfected with a plasmid encoding the AAV2 Rep68 or Rep78 protein (30). In the present study, we detected phosphorylation of RPA32 at S4/8 and recruitment of RPA32-P-S4/8 into HSV-1-supported AAV2 RCs.…”

“…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).…”

Adeno-Associated Virus Type 2 Rep68 Can Bind to Consensus Rep-Binding Sites on the Herpes Simplex Virus 1 Genome

“…AAV2 has developed strategies to inhibit helper virus replication, likely to reduce competition (24,(28)(29)(30)(31)(32)(33). For example, expression of the AAV2 nonstructural proteins Rep68 and Rep78 alone leads to significant inhibition of 28). Specifically, we demonstrated previously that the AAV2 Rep protein domains responsible for the inhibition of HSV-1 DNA replication include the DNA-binding and the ATPase/helicase activities, while the endonuclease activity is not required (28).…”

“…In addition, the HSV-1 IE proteins ICP4 and ICP0, the E protein complex forming the HSV-1 polymerase (UL30 and UL42), and the US1 gene product strongly enhance AAV2 replication (26). AAV2 has developed strategies to inhibit helper virus replication, likely to reduce competition (24,(28)(29)(30)(31)(32)(33). For example, expression of the AAV2 nonstructural proteins Rep68 and Rep78 alone leads to significant inhibition of 28).…”

“…For example, expression of the AAV2 nonstructural proteins Rep68 and Rep78 alone leads to significant inhibition of HSV-1 DNA replication (24,28). Specifically, we demonstrated previously that the AAV2 Rep protein domains responsible for the inhibition of HSV-1 DNA replication include the DNA-binding and the ATPase/helicase activities, while the endonuclease activity is not required (28). We also showed that Repmediated inhibition of HSV-1 occurs even in the absence of AAV DNA and is not due to alterations of HSV-1 IE and E gene expression or to the Rep-mediated induction of toxic stress in the cell but rather occurs at the stage of HSV-1 DNA replication itself (28).…”