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-speci fic monoclonal antibodies (MAbs) were isolated and characterized, In i nitial immunoprecipitation experiments, one of these, MAb 804, was sho wn to coprecipitate POL, the catalytic subunit of the HSV-1 DNA polyme rase, from extracts of insect cells infected with recombinant baculovi ruses expressing the POL and UL8 proteins, Coprecipitation of POL was dependent on the presence of UL8 protein, Rapid enzyme-linked immunoso rbent assays (ELISAs), in which one protein was bound to microtiter we lls and binding of the other protein was detected with a UL8- or POL-s pecific MAb, were developed to investigate further the interaction bet ween the two proteins, When tested in the ELISAs, five of the UL8-spec ific MAbs consistently inhibited the interaction, raising the possibil ity that these antibodies act by binding to epitopes at or near a site (s) on UL8 involved in its interaction with POL, The epitopes recogniz ed by four of the inhibitory, MAbs were approximately located by using a series of truncated UL8 proteins expressed in mammalian cells, Thre e of these MAbs recognized an epitope near the C terminus of UL8, whic h was subjected to fine mapping with a series of overlapping peptides, The C-terminal peptides were then tested in the ELISA for their abili ty to inhibit the POL-UL8 interaction: the most potent exhibited a 50% inhibitory concentration of approximately 5 mu M. Our findings sugges t that the UL8 protein may be involved in recruiting HSV-1 DNA polymer ase into the viral DNA replication complex and also identify a potenti al new target for antiviral therapy.