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.
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.
Marsden
H S
HS
MRC Virology Unit, University of Glasgow, United Kingdom. H.Marsden@vir.gla.ac.uk
McLean
G W
GW
Barnard
E C
EC
Francis
G J
GJ
MacEachran
K
K
Murphy
M
M
McVey
G
G
Cross
A
A
Abbotts
A P
AP
Stow
N D
ND
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D002384:Catalysis; D002460:Cell Line; D004265:DNA Helicases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D019915:DNA Primase; D004259:DNA-Directed DNA Polymerase / Q000235:genetics / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay; D018604:Epitope Mapping; D005090:Exodeoxyribonucleases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D018259:Herpesvirus 1, Human / Q000201:enzymology* / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D019384:Nucleic Acid Synthesis Inhibitors; D010455:Peptides / Q000378:metabolism / Q000494:pharmacology; D011233:Precipitin Tests; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D018411:Spodoptera / Q000166:cytology; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D019384:Nucleic Acid Synthesis Inhibitors; D010455:Peptides; D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; D019915:DNA Primase; D004259:DNA-Directed DNA Polymerase; D005090:Exodeoxyribonucleases; C062021:helicase-primase, Human herpesvirus 1; C043417:DNA polymerase, Simplexvirus; D004265:DNA Helicases