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

Abstract

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.

FIG. 1

Phosphorylation of HHV-8 PF-8. (A) IVT PF-8 is phosphorylated. PF-8 was expressed from pCI-neo expression vector with an in vitro transcription-translation (TNT) system in the presence of [³⁵S]methionine (lane 4). TNT lysate programmed with pCI-neo vector (V) is shown in lane 3. [³⁵S]Met-labeled IVT PF-8 was incubated with (lane 2) or without (lane 1) λPPase for 3 h. Samples were resolved by SDS–9% PAGE. Protein products synthesized by TNT due to the presence of the pCI-neo vector itself are indicated by asterisks. (B) PF-8 is phosphorylated in BCBL-1 cells. [³²P]orthophosphate-labeled BJAB (lane 1), uninduced BCBL-1 (lane 2), and TPA-induced BCBL-1 (lane 3) lysates were immunoprecipitated with MAb 11D1 and resolved by SDS–9% PAGE. (C) PF-8 is expressed in BCBL-1 cells, but not in BJAB cells. [³⁵S]Met/Cys-labeled BJAB (lane 1), uninduced BCBL-1 (lane 2), and TPA-induced BCBL-1 (lane 3) lysates were immunoprecipitated with MAb 11D1 and resolved by SDS–9% PAGE. The sizes of protein markers are shown to the left of each gel. The position of PF-8 is indicated at right. IP, immunoprecipitation.

FIG. 2

dsDNA-binding activity of PF-8. IVT radiolabeled PF-8 was applied to 500 μl of equilibrated unmodified cellulose (A), dsDNA-cellulose (B), or ssDNA-cellulose (C) in columns. The columns were extensively washed (lane 2) and eluted stepwise with increasing concentrations of NaCl (0.1, 0.2, 0.3, 0.4, 0.5, 1, and 2 M in binding buffer). Thirty percent of the first fraction from each NaCl concentration was precipitated and resolved by SDS–9% PAGE (lanes 3 to 9). Five percent of column input is shown in lane 1. The sizes of protein markers are shown at left.

FIG. 3

Coimmunoprecipitation of IVT Pol-8 and IVT PF-8 by MAb 11D1. Fifty percent PF-8 and 10% of Pol-8 used in the coimmunoprecipitation reactions are shown in lanes 2 and 3, respectively. PF-8 alone (lanes 4 and 5), Pol-8 alone (lanes 6 and 7), or PF-8 plus Pol-8 (lanes 8 and 9) was incubated with protein A-Sepharose alone (A) (lanes 4, 6, and 8) or with protein A-Sepharose and MAb 11D1 (lanes 5, 7, and 9). The immune complexes were extensively washed and resolved by SDS–10% PAGE. The sizes of protein markers are shown at left. The positions of Pol-8 and PF-8 are indicated at right. V, TNT lysate programmed with pCI-neo vector.

FIG. 4

Stimulation of the processivity of Pol-8 by PF-8 on poly(dA):oligo(dT)₁₆ template. TNT lysate programmed with pCI-neo vector (V) (lane 3), Pol-8 (lane 4), Pol-8 plus increasing amounts of PF-8 (lanes 5 to 7), PF-8 (lane 11), or Pol-8 plus glycoprotein K8.1A (lane 12) were assayed for DNA synthesis on poly(dA):oligo(dT)₁₆. Pol-8 and PF-8 were incubated with poly(dA):oligo(dT)₁₆ for 10, 30, or 60 min (lanes 8, 9, or 10, respectively). DNA products were fractionated by 7 M urea–15% PAGE. Poly(dA):oligo(dT)₁₆ template was electrophoresed in lane 2 to show the size of the primers (16 bases). Nonspecific DNA products synthesized by pCI-neo-programmed TNT lysate are indicated by an asterisk. The sizes of DNA markers (lane 1) are shown at left.

FIG. 5

Schematic representation and summary of properties of full-length PF-8 and PF-8 truncation mutants. Ability of full-length (FL) PF-8 and PF-8 mutants to enhance Pol-8 processivity and to interact with Pol-8 and dsDNA (as assessed in Fig. 7A, Fig. 8, and Fig. 9) are scored as follows: positive (+), negative (−), or weak (+/−). For dsDNA-binding activity, +∗ indicates an altered elution profile of the polypeptide from the dsDNA-cellulose column. Abbreviations: M, Met; ND, not determined.

FIG. 6

Expression of IVT PF-8 truncation mutants. TNT lysate programmed with pCI-neo vector (V) (lane 1), C-terminal truncation mutants (panel A, lanes 2 to 7), N-terminal truncation mutants (panel B, lanes 2 to 5), and full-length PF-8 (panel A, lane 8, and panel B, lane 6) were resolved by SDS–12% PAGE. The apparent molecular masses of the following PF-8 mutants are as indicated: ΔC191–396, 20 kDa; ΔC234–396, 25 kDa; ΔC279–396, 30 kDa; ΔC302–396, 32 kDa; ΔC323–396, 34 kDa; ΔC359–396, 38 kDa; ΔN1–9, 49 kDa; ΔN1–27, 47 kDa; ΔN1–62, 45 kDa; ΔN1–127, 38 kDa. The sizes of protein markers are shown at left. FL, full-length.

FIG. 7

Effects of PF-8 truncation mutants on the processivity of Pol-8. (A) TNT lysate programmed with pCI-neo vector (V) (lane 3), Pol-8 (lane 4), Pol-8 plus PF-8 mutants (lanes 5 to 14), or Pol-8 plus full-length PF-8 (lane 15) was incubated with components of the processivity assay for 1 h and DNA products were resolved by 7 M urea–15% PAGE. Poly(dA):oligo(dT)₁₆ template alone is in lane 2. The sizes of DNA markers (lane 1) are shown at left. Nonspecific DNA products are indicated by an asterisk. (B and C) PF-8 mutants are stable under the processivity assay conditions. To monitor protein stability under assay conditions, processivity assays were carried out without the addition of BSA, dTTP, and template, and proteins were resolved by SDS–12% PAGE. (B) C-terminal truncation mutants. (C) N-terminal truncation mutants. The sizes of protein markers are shown at left.

FIG. 8

dsDNA-binding activities of PF-8 truncation mutants. [³⁵S]Met-labeled IVT PF-8 mutants were applied to dsDNA-cellulose columns. DNA-cellulose chromatography was carried out as described in the legend to Fig. 2.

FIG. 9

Coimmunoprecipitation of IVT Pol-8 and IVT PF-8 mutants by MAb 11D1. IVT C-terminal or N-terminal PF-8 truncation mutants were mixed with IVT Pol-8 and incubated with MAb 11D1 and protein A-Sepharose. The immune complexes were washed and resolved by SDS–11% PAGE. The sizes of protein markers are shown at left. The positions of Pol-8 and full-length PF-8 are indicated by arrows, and the positions of PF-8 mutants are indicated by open circles.