Characterization of human herpesvirus 8 ORF59 protein (PF-8) and mapping of the processivity and viral DNA polymerase-interacting domains

Human herpesvirus 8 (HHV-8) or Kaposi's sarcoma-associated herpesvirus (KSH V) ORF59 protein (PF-8) is a processivity factor for HHV-8 DNA polymerase ( Pol-8) and is homologous to processivity factors expressed by other herpesv iruses, such as herpes simplex virus type I UL42 and Epstein-Barr virus BMR F1. The interaction of UL42 and BMRF1 with their corresponding DNA polymera ses is essential for viral DNA replication and the subsequent production of infectious virus. Using HHV-8-specific monoclonal antibody 11D1, we have p reviously identified the cDNA encoding PF-8 and showed that it is an early- late gene product localized to HHV-8-infected cell nuclei (S. R. Chan, C. B loomer, and B. Chandran, Virology 240:118-126, 1998). Here, we have further characterized PF-8. This viral protein was phosphorylated both in vitro an d in vivo. PF-8 bound double-stranded DNA (dsDNA) and single-stranded DNA i ndependent of DNA sequence; however, the affinity for dsDNA was approximate ly fivefold higher. In coimmunoprecipitation reactions, PF-8 also interacte d with Pol-8. In in vitro processivity assays with excess poly(dA):oligo(dT ) as a template, PF-8 stimulated the production of elongated DNA products b y Pol-8 in a dose-dependent manner. Functional domains of PF-8 were determi ned using PF-8 truncation mutants. The carboxyl-terminal 95 amino acids (aa ) of PF-8 were dispensable for all three functions of PF-8: enhancing proce ssivity of Pol-8, binding dsDNA, and binding Pol-8. Residues 10 to 27 and 2 79 to 301 were identified as regions critical for the processivity function of PF-8. Interestingly, aa 10 to 27 were also essential for binding Pol-8, whereas aa 1 to 62 and aa 279 to 301 were involved in binding dsDNA, sugge sting that the processivity function of PF-8 is correlated with both the Po l-8-binding and the dsDNA-binding activities of PF-8.