Herpes simplex virus 1 ICP8 mutant lacking annealing activity is deficient for viral DNA replication

Abstract

Most DNA viruses that use recombination-dependent mechanisms to replicate their DNA encode a single-strand annealing protein (SSAP). The herpes simplex virus (HSV) single-strand DNA binding protein (SSB), ICP8, is the central player in all stages of DNA replication. ICP8 is a classical replicative SSB and interacts physically and/or functionally with the other viral replication proteins. Additionally, ICP8 can promote efficient annealing of complementary ssDNA and is thus considered to be a member of the SSAP family. The role of annealing during HSV infection has been difficult to assess in part, because it has not been possible to distinguish between the role of ICP8 as an SSAP from its role as a replicative SSB during viral replication. In this paper, we have characterized an ICP8 mutant, Q706A/F707A (QF), that lacks annealing activity but retains many other functions characteristic of replicative SSBs. Like WT ICP8, the QF mutant protein forms filaments in vitro, binds ssDNA cooperatively, and stimulates the activities of other replication proteins including the viral polymerase, helicase–primase complex, and the origin binding protein. Interestingly, the QF mutant does not complement an ICP8-null virus for viral growth, replication compartment formation, or DNA replication. Thus, we have been able to separate the activities of ICP8 as a replicative SSB from its annealing activity. Taken together, our data indicate that the annealing activity of ICP8 is essential for viral DNA replication in the context of infection and support the notion that HSV-1 uses recombination-dependent mechanisms during DNA replication.

The Interplay between Adeno-Associated Virus and Its Helper Viruses

“…Early reports hypothesized that genome circularization takes place prior to DNA replication, thus enabling a rolling circle amplification, which inherently generates concatemers [ 114 ]. More recent work suggests that concatemers are formed by direct recombination mediated by ICP8 and UL12 [ 115 , 116 , 117 ].…”

Mass Spectrometric Characterization of HSV-1 L-Particles From Human Dendritic Cells and BHK21 Cells and Analysis of Their Functional Role

“…Despite the hypothesis that H- and L-particles share a similar way of maturation, the non-infectious particles can also be produced in the absence of DNA replication and packaging ( Dargan et al, 1995 ; Russell et al, 2018 ). Two important proteins regarding DNA replication are, e.g., ICP8, a single-stranded DNA binding protein ( Zhou and Knipe, 2002 ; Weerasooriya et al, 2019 ), and the viral thymidine kinase UL23, which phosphorylates thymidine and thus prepares nucleotides for viral DNA synthesis ( Chen et al, 1998 ; Pilger et al, 1999 ; Xie et al, 2019 ). Our mass spectrometric data of BHK21 cell-derived HSV-1 particles revealed ICP8 and UL23 to be present in L- and H-particles, representing the major hits among non-structural proteins ( Table 2 and Figure 3 ).…”

HSV-1 DNA Replication—Coordinated Regulation by Viral and Cellular Factors

“…Like other replicative single-stranded DNA binding proteins, ICP8 forms filaments and binds cooperatively to single-stranded DNA. In addition, ICP8 has strand annealing activity, which may indicate a role in viral DNA recombination [ 39 , 40 ]. ICP8 stimulates the viral DNA polymerase and helicase–primase complex and interacts with other viral and cellular proteins to promote viral DNA replication [ 41 , 42 , 43 , 44 , 45 , 46 ].…”