Abstract
During lytic infection, herpes simplex virus (HSV) DNA is replicated by a mechanism involving DNA recombination. For instance, replication of the HSV-1 genome produces X- and Y-branched structures, reminiscent of recombination intermediates. HSV-1's replication machinery includes a trimeric helicase-primase composed of helicase (UL5) and primase (UL52) subunits and a third subunit, UL8. UL8 has been reported to stimulate the helicase and primase activities of the complex in the presence of ICP8, an HSV-1 protein that functions as an annealase, a protein that binds complementary single-stranded DNA (ssDNA) and facilitates its annealing to duplex DNA. UL8 also influences the intracellular localization of the UL5/UL52 subunits, but UL8's catalytic activities are not known. In this study we used a combination of biochemical techniques and transmission electron microscopy. First, we report that UL8 alone forms protein filaments in solution. Moreover, we also found that UL8 binds to ssDNAs >50-nucletides long and promotes the annealing of complementary ssDNA to generate highly branched duplex DNA structures. Finally, UL8 has a very high affinity for replication fork structures containing a gap in the lagging strand as short as 15 nucleotides, suggesting that UL8 may aid in directing or loading the trimeric complex onto a replication fork. The properties of UL8 uncovered here suggest that UL8 may be involved in the generation of the X- and Y-branched structures that are the hallmarks of HSV replication.
Highlights
During lytic infection, herpes simplex virus (HSV) DNA is replicated by a mechanism involving DNA recombination
During Herpes simplex virus type 1 (HSV-1) infection, the viral genome is released from the capsid into the nucleus and is replicated to form concatemeric DNA that is recognized by the packaging machinery to produce infectious virus
ICP8 and UL12 have been implicated in singlestrand annealing, and in this paper we focused on previously unrecognized properties of the UL8 subunit of the helicase– primase complex
Summary
HSV-1 UL8 is a subunit of the heterotrimeric helicase–primase complex; no catalytic activities have been identified. Based on its molecular weight, a UL8 monomer would be predicted to have a diameter of ϳ5 nm, and we conclude that UL8 at this protein concentration is predominantly monomeric with some oligomeric forms. Examination of fields of molecules after incubation at increasing protein concentration revealed three distinct forms: irregular particles, regular oligomeric rings, and linear protein filaments (Table 1). At concentrations Ͻ9 nM, UL8 appeared predominantly monomeric with some irregularly shaped oligomeric forms (5–15 nm) similar to the ones seen in the absence of Mg2ϩ and with 150 mM NaCl (Fig. 2A). At 10 –50 nM with 5 mM MgCl2 at room temperature or on ice, UL8 formed regular, donut-shaped structures (oligomeric rings) 20 nm in diameter (Fig. 2B). The independence of the indicated parameter from the physical state of UL8 is designated as (Ϫ)/(ϩ)
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