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Journal of Virology, December 2000, p. 10920-10929, Vol. 74, No. 23
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Characterization of Human Herpesvirus 8 ORF59 Protein (PF-8) and Mapping of the Processivity and Viral DNA Polymerase-Interacting Domains

Szeman Ruby Chan and Bala Chandran*

Department of Microbiology, Molecular Genetics, and Immunology, University of Kansas Medical Center, Kansas City, Kansas 66160-7700

Received 5 June 2000/Accepted 5 September 2000

Human herpesvirus 8 (HHV-8) or Kaposi's sarcoma-associated herpesvirus (KSHV) ORF59 protein (PF-8) is a processivity factor for HHV-8 DNA polymerase (Pol-8) and is homologous to processivity factors expressed by other herpesviruses, such as herpes simplex virus type 1 UL42 and Epstein-Barr virus BMRF1. The interaction of UL42 and BMRF1 with their corresponding DNA polymerases is essential for viral DNA replication and the subsequent production of infectious virus. Using HHV-8-specific monoclonal antibody 11D1, we have previously 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. Bloomer, and B. Chandran, Virology 240:118-126, 1998). Here, we have further characterized PF-8. This viral protein was phosphorylated both in vitro and in vivo. PF-8 bound double-stranded DNA (dsDNA) and single-stranded DNA independent of DNA sequence; however, the affinity for dsDNA was approximately fivefold higher. In coimmunoprecipitation reactions, PF-8 also interacted 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 by Pol-8 in a dose-dependent manner. Functional domains of PF-8 were determined 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 processivity of Pol-8, binding dsDNA, and binding Pol-8. Residues 10 to 27 and 279 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, suggesting that the processivity function of PF-8 is correlated with both the Pol-8-binding and the dsDNA-binding activities of PF-8.

* Corresponding author. Mailing address: Department of Microbiology, Molecular Genetics, and Immunology, University of Kansas Medical Center, Kansas City, KS 66160. Phone: (913) 588-7043. Fax: (913) 588-7295. E-mail: bchandra{at}kumc.edu.

Journal of Virology, December 2000, p. 10920-10929, Vol. 74, No. 23
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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