Summary auto-generated
This study examined how herpes simplex virus type 1 (HSV-1) proteins interact during viral DNA replication, specifically focusing on ICP8, a single-stranded DNA-binding protein, and the helicase-primase complex (UL5/8/52). Researchers used purified viral proteins in a coupled primase-polymerase DNA synthesis system to investigate protein-protein interactions. They found that while heterologous DNA-binding proteins (E. coli SSB and T4 gene 32 protein) could stimulate DNA polymerase activity on pre-primed templates, only ICP8 specifically stimulated polymerase activity when primers were synthesized by the viral helicase-primase complex. Direct biochemical assays showed that ICP8 stimulated both primer synthesis and ATP hydrolysis (ATPase activity) of the UL5/8/52 complex, but crucially, this stimulation required the UL8 subunit. ICP8 did not stimulate the UL5/52 subassembly lacking UL8. Further analysis indicated that stimulation occurred through direct protein-protein interaction rather than by secondary structure removal, as ICP8 activated the helicase-primase on homopolymer DNA templates and increased the catalytic rate constant while not reducing DNA binding affinity.
Key findings
- ICP8 specifically stimulates HSV DNA polymerase activity only when primers are synthesized by the viral helicase-primase complex, unlike heterologous SSB proteins that non-specifically stimulate polymerase on pre-primed templates
- ICP8 stimulation of the helicase-primase complex requires the UL8 subunit; the UL5/52 subassembly without UL8 is not stimulated by ICP8
- ICP8 increases both primer synthesis and ATP hydrolysis rates of the UL5/8/52 complex through a direct functional interaction mediated by UL8
- Stimulation occurs through increased enzymatic catalytic rates rather than improved DNA binding or secondary structure removal
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Abstract
The herpes simplex virus type 1 (HSV) single-stranded DNA-binding protein (SSB, ICP8) stimulates the viral DNA polymerase (Pol) on an oligonucleotide-primed single-stranded DNA template. This stimulation is non-specific since other SSBs also increase Pol activity. However, only ICP8 was stimulatory when Pol activity was dependent upon priming by the viral helicase-primase complex. ICP8 also specifically stimulated the primer synthesis and ATPase activities of the helicase- primase. The mechanism of stimulation was different from that of Pol; helicase-primase stimulation required much lower amounts of ICP8 than the amount that saturates the DNA and optimally stimulates Pol. Furthermore, ICP8 did not act by removing secondary structure as stimulation also occurred on homopolymer templates. While the UL8 component of the helicase-primase is not required for enzymatic activities by a subassembly of the UL5 and UL52 proteins, only the holoenzyme (UL5/8/52) was stimulated by ICP8. These results identify a unique, functional interaction between the ICP8 SSB and the helicase- primase complex, mediated by the UL8 subunit.