Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 1998 Jul;72(7):6228-32.
doi: 10.1128/JVI.72.7.6228-6232.1998.

Cloning and functional analysis of Kaposi's sarcoma-associated herpesvirus DNA polymerase and its processivity factor

K Lin  1 , C Y Dai, R P Ricciardi
Affiliations

Cloning and functional analysis of Kaposi's sarcoma-associated herpesvirus DNA polymerase and its processivity factor

K Lin et al. J Virol. 1998 Jul.

Abstract

Kaposi's sarcoma-associated herpesvirus (KSHV), or human herpesvirus 8, is a newly identified virus with tumorigenic potential. Here, we cloned and expressed the DNA polymerase (Pol-8) of KSHV and its processivity factor (PF-8). Pol-8 bound specifically to PF-8 in vitro. Moreover, the DNA synthesis activity of Pol-8 was shown in vitro to be strongly dependent on PF-8. Addition of PF-8 to Pol-8 allowed efficient synthesis of fully extended DNA products corresponding to the full-length M13 template (7,249 nucleotides), whereas Pol-8 alone could incorporate only several nucleotides. The specificity of PF-8 and Pol-8 for each other was demonstrated by their inability to be functionally replaced by the DNA polymerases and processivity factors of herpes simplex virus 1 and human herpesvirus 6.

PubMed Disclaimer

Figures

FIG. 1
FIG. 1
Cloning and expression of Pol-8 and PF-8. (A) The coding regions of Pol-8 and PF-8 were amplified by PCR from two KSHV genomic DNA clones, GB11 and GB21, respectively, with oligonucleotides containing appended NcoI and SacI sites, and introduced into pTM1 expression vectors under the control of a T7 promoter. (B) Pol-8 and PF-8 were expressed in vitro with the Promega T7-TNT coupled transcription-translation system. [35S]methionine-labeled Pol-8 and PF-8 proteins were fractionated on an SDS–7.5% polyacrylamide gel and examined by autoradiography. The molecular mass (MW) of the protein markers is indicated.
FIG. 2
FIG. 2
Sequence alignment of PF-8, HVS ORF59, and EBV BMRF1. The amino acid sequences of three herpesvirus processivity factors, PF-8 of KSHV, ORF59 of HVS, and BMRF1 of EBV, were aligned by the ClustalW 1.7 multiple sequence alignment program. Identical residues are shaded, and conserved basic residues are indicated by asterisks (∗).
FIG. 3
FIG. 3
Pol-8 binds to PF-8 in vitro. (A) Pol-8 binds to PF-8 in the presence of ethidium bromide. [35S]methionine-labeled in vitro-translated Pol-8 (∗Pol-8) was incubated with GST or GST–PF-8 in the GST-binding assay in the absence (lanes 2 and 3) or presence (lane 4) of 200 μg of ethidium bromide (EtBr) per ml. Bound proteins were eluted and analyzed by SDS-polyacrylamide gel electrophoresis and autoradiography. (B) The binding of Pol-8 to PF-8 is specific. The binding assay was performed as described for panel A in the presence of ethidium bromide. [35S]methionine-labeled in vitro-translated DNA polymerase of HSV-1 (∗UL30) failed to bind GST–PF-8 (lane 5). Labeled proteins were also loaded directly onto the gels representing 20% (A) or 10% (B) of the input used in the binding assays.
FIG. 4
FIG. 4
DNA synthesis activity of Pol-8 strongly depends on the presence of PF-8. The DNA synthesis activities of mock-translated protein, Pol-8, PF-8, or Pol-8 and PF-8 together were analyzed in the DNA synthesis assay by measuring the incorporation of [α-32P]dCTP (in disintegrations per minute [dpm]) into synthesized DNA products. Mock represents translation from the pTM1 vector lacking a coding insert.
FIG. 5
FIG. 5
PF-8 specifically enables Pol-8 to synthesize fully extended DNA. The DNA synthesis assays were performed for 1 h with 2 μl of each indicated protein, except that in lanes 6 and 7 reaction mixtures were incubated for 5 and 15 min, respectively, and in lanes 9 and 11, reaction mixtures contained either 1 or 5 μl of both Pol-8 and PF-8, respectively. The synthesized DNA products, labeled by [α-32P]dCTP incorporation, were fractionated on a 1.3% alkaline agarose gel which was analyzed by autoradiography. The sizes of 32P-labeled M13 and λ markers are indicated. Mock represents translation from the pTM1 vector lacking a coding insert. The translation efficiencies of all proteins tested in the assay were similar. The bracket at lower left indicates short products.
FIG. 6
FIG. 6
Pol-8 alone has significant catalytic activity but very limited processivity. The DNA synthesis activities of Pol-8, PF-8, and Pol-8 and PF-8 together were compared on a 15% urea-polyacrylamide gel. The DNA synthesis assay employed M13 ssDNA template with an annealed 5′-end 32P-labeled primer and contained different combinations of unlabeled dNTPs. The extended sequence represents the actual M13 sequence immediately following the 3′ end of the primer. Only the bottom of the gel is shown here, since no products were visualized in the top portion of the gel except in lane 14, in which most of the high-molecular-weight products remained in the well.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Agulnick A D, Thompson J R, Iyengar S, Pearson G, Ablashi D, Ricciardi R P. Identification of a DNA-binding protein of human herpesvirus 6, a putative DNA polymerase stimulatory factor. J Gen Virol. 1993;74:1003–1009. - PubMed
    1. Albrecht J-C, Nicholas J, Biller D, Cameron K R, Biesinger B, Newman C, Wittmann S, Craxton M A, Coleman H, Fleckenstein B, Honess R W. Primary structure of the herpesvirus saimiri genome. J Virol. 1992;66:5047–5058. - PMC - PubMed
    1. Brooks L A, Wilson A J, Crooks T. Kaposi’s sarcoma-associated herpesvirus (KSHV)/human herpesvirus 8 (HHV-8)—a new human tumor virus. J Pathol. 1997;182:262–265. - PubMed
    1. Cesarman E, Chang Y, Moore P S, Said J W, Knowles D M. Kaposi’s sarcoma-associated herpesvirus-like DNA sequences in AIDS-related body-cavity-based lymphomas. N Engl J Med. 1995;332:1186–1191. - PubMed
    1. Chang C K, Balachandran N. Identification, characterization, and sequence analysis of a cDNA encoding a phosphorprotein of human herpesvirus 6. J Virol. 1991;65:2884–2894. - PMC - PubMed

Publication types

MeSH terms

LinkOut - more resources