The catalytic subunit of the DNA polymerase of herpes simplex virus type 1 interacts specifically with the C terminus of the UL8 component of the viral helicase-primase complex

J Virol. 1997 Sep;71(9):6390-7. doi: 10.1128/JVI.71.9.6390-6397.1997.

Abstract

The herpes simplex virus type 1 (HSV-1) UL8 DNA replication protein is a component of a trimeric helicase-primase complex. Sixteen UL8-specific monoclonal antibodies (MAbs) were isolated and characterized. In initial immunoprecipitation experiments, one of these, MAb 804, was shown to coprecipitate POL, the catalytic subunit of the HSV-1 DNA polymerase, from extracts of insect cells infected with recombinant baculoviruses expressing the POL and UL8 proteins. Coprecipitation of POL was dependent on the presence of UL8 protein. Rapid enzyme-linked immunosorbent assays (ELISAs), in which one protein was bound to microtiter wells and binding of the other protein was detected with a UL8- or POL-specific MAb, were developed to investigate further the interaction between the two proteins. When tested in the ELISAs, five of the UL8-specific MAbs consistently inhibited the interaction, raising the possibility that these antibodies act by binding to epitopes at or near a site(s) on UL8 involved in its interaction with POL. The epitopes recognized by four of the inhibitory MAbs were approximately located by using a series of truncated UL8 proteins expressed in mammalian cells. Three of these MAbs recognized an epitope near the C terminus of UL8, which was subjected to fine mapping with a series of overlapping peptides. The C-terminal peptides were then tested in the ELISA for their ability to inhibit the POL-UL8 interaction: the most potent exhibited a 50% inhibitory concentration of approximately 5 microM. Our findings suggest that the UL8 protein may be involved in recruiting HSV-1 DNA polymerase into the viral DNA replication complex and also identify a potential new target for antiviral therapy.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Antibodies, Monoclonal / immunology
  • Catalysis
  • Cell Line
  • DNA Helicases / antagonists & inhibitors
  • DNA Helicases / genetics
  • DNA Helicases / metabolism*
  • DNA Primase
  • DNA-Directed DNA Polymerase / genetics
  • DNA-Directed DNA Polymerase / metabolism*
  • Enzyme-Linked Immunosorbent Assay
  • Epitope Mapping
  • Exodeoxyribonucleases / antagonists & inhibitors
  • Exodeoxyribonucleases / genetics
  • Exodeoxyribonucleases / metabolism*
  • Herpesvirus 1, Human / enzymology*
  • Herpesvirus 1, Human / genetics
  • Humans
  • Molecular Sequence Data
  • Nucleic Acid Synthesis Inhibitors
  • Peptides / metabolism
  • Peptides / pharmacology
  • Precipitin Tests
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / metabolism
  • Spodoptera / cytology
  • Viral Proteins / genetics
  • Viral Proteins / metabolism*

Substances

  • Antibodies, Monoclonal
  • Nucleic Acid Synthesis Inhibitors
  • Peptides
  • Recombinant Fusion Proteins
  • Viral Proteins
  • DNA Primase
  • DNA-Directed DNA Polymerase
  • Exodeoxyribonucleases
  • helicase-primase, Human herpesvirus 1
  • DNA polymerase, Simplexvirus
  • DNA Helicases