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

Michael Seyffert,Hildegard Büning,Elisabeth M Schraner,Mathias Ackermann,Anna-Paula De Oliveira,Cornel Fraefel,Jorge Mansilla-Soto,R Michael Linden,Bernd Vogt,Daniel L Glauser,John A Chiorini

doi:10.1371/journal.pone.0170908

Michael Seyffert, Hildegard Büning + Show 9 more

Open Access

https://doi.org/10.1371/journal.pone.0170908

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Journal: PloS one	Publication Date: Jan 26, 2017
Citations: 5	License type: CC BY 4.0

Affiliation: University of Zurich, King's College London

Abstract

As their names imply, parvoviruses of the genus Dependovirus rely for their efficient replication on the concurrent presence of a helpervirus, such as herpesvirus, adenovirus, or papilloma virus. Adeno-associated virus 2 (AAV2) is such an example, which in turn can efficiently inhibit the replication of each helpervirus by distinct mechanisms. In a previous study we have shown that expression of the AAV2 rep gene is not compatible with efficient replication of herpes simplex virus 1 (HSV-1). In particular, the combined DNA-binding and ATPase/helicase activities of the Rep68/78 proteins have been shown to exert opposite effects on the replication of AAV2 and HSV-1. While essential for AAV2 DNA replication these protein activities account for the Rep-mediated inhibition of HSV-1 replication. Here, we describe a novel Rep mutant (Rep-D371Y), which displayed an unexpected phenotype. Rep-D371Y did not block HSV-1 replication, but still supported efficient AAV2 replication, at least when a double-stranded AAV2 genome template was used. We also found that the capacity of Rep-D371Y to induce apoptosis and a Rep-specific DNA damage response was significantly reduced compared to wild-type Rep. These findings suggest that AAV2 Rep-helicase subdomains exert diverging activities, which contribute to distinct steps of the AAV2 life cycle. More important, the novel AAV2 mutant Rep-D371Y may allow deciphering yet unsolved activities of the AAV2 Rep proteins such as DNA second-strand synthesis, genomic integration or packaging, which all involve the Rep-helicase activity.

Highlights

Adeno-associated virus 2 (AAV2) is a helper virus-dependent human parvovirus with a unique biphasic life cycle
The DNA-binding domain is responsible for binding to double-strandedDNA templates at specific Rep binding site (RBS) motifs consisting of the minimal consensus sequence GAGYGAGC [16], which are located within the AAV2 inverted terminal repeats (ITRs) and the p5 promoter region
We demonstrated that Rep68, Rep78 and Rep68/78-Y156F, which has a mutation within the RCR2 motif required for endonuclease activity [17, 45, 46], reduce titers of infectious herpes simplex virus 1 (HSV-1) particle stocks by approximately 100-fold, whereas Rep52, which lacks the DNA binding domain, and Rep68/ 78-K340H, which is deficient for helicase activity [47], do not [38]

Summary

Introduction

Adeno-associated virus 2 (AAV2) is a helper virus-dependent human parvovirus with a unique biphasic life cycle. At the very N-terminal region of the rep open-reading frame (ORF) the combined DNA-binding and endonuclease domains are located [13,14,15] (Fig 1A). The DNA-binding domain (map position 1–200) is responsible for binding to double-stranded (ds)DNA templates at specific Rep binding site (RBS) motifs consisting of the minimal consensus sequence GAGYGAGC [16], which are located within the AAV2 ITRs and the p5 promoter region. At the C-terminal end of the rep ORF a protein kinase A (PKA) binding site is located (map position 526–621) and consists of a Zn-finger motif [33, 34] and a PKA inhibitor (PKI)-like motif [35] (Fig 1A).

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