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

Abstract

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...

Novel Mutant AAV2 Rep Proteins Support AAV2 Replication without Blocking HSV-1 Helpervirus Replication

“…Compared to the positive control (wt Rep) both Rep mutants (K340H and D371Y) significantly rescued pHSVGFP titers by more than 10-fold and at comparable levels ( Fig 2C ). The helicase-null mutant Rep-K340H, which cannot support AAV2 replication, was previously known to allow HSV-1 replication [ 38 , 44 ]. As the mutant Rep-D371Y allowed HSV-1 replication, we expected that it may not support AAV2 DNA replication either.…”

“…We and others have demonstrated that the AAV2 Rep domains responsible for the inhibition of HSV-1 DNA replication include the combined DNA-binding and the ATPase/helicase domains [ 38 , 41 ]. Direct binding of the AAV2 Rep proteins to the HSV-1 DNA and the subsequent activity of the ATPase/helicase domain have been shown to account at least in part for the Rep-mediated inhibition of HSV-1 replication [ 44 ]. This interaction may cause DNA lesions which are not compatible with effective HSV-1 DNA replication.…”

“…1-rc was created by co-transfection of d27.1-lacZ infected cell DNA and a linearized integration plasmid harboring the rep and cap genes [ 79 ]. Since this type of HSV-1 vector production does not directly rely on HSV-1 DNA replication, it therefore may not be affected by the Rep-mediated inhibition of HSV-1 replication, which particularly involves blocking of the HSV-1 DNA amplification [ 38 , 44 ].…”

“…All cell cultures were kept at 37°C and 5% CO 2 . The wt HSV-1 (strain F) was grown in Vero cells and titers were determined as described previously [ 38 , 44 ]: briefly, confluent monolayers of Vero cells were infected with wt HSV-1 at a MOI of 0.1 and incubated until the cytopathic effect (CPE) reached 100%. Then, the cells were lysed by three freeze/thawing-cycles and cellular debris was separated from the virus stock by centrifugation for 10 min at 1,900 x g .…”

“…In previous studies, we have demonstrated that the expression of the AAV2 Rep68/78 proteins leads to a significant inhibition of HSV-1 DNA replication [ 37 , 38 ]. Moreover, we found 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 [ 38 , 44 ]. In those studies, we have utilized a set of mutant Rep proteins lacking distinct activities or domains to assess their effect on the production of infectious HSV-1 particles.…”