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

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 M. 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.

The Epstein-Barr Virus Lytic Transactivator Zta Interacts with the Helicase-Primase Replication Proteins

“…The potential exists for multiple contacts within the replication complex. Contacts between the HSV single-stranded DNA binding protein and polymerase and the origin binding protein, UL9, have also been documented (3,51), as have contacts between UL8 and polymerase (47). The information available for assembly of the EBV replication complex is much more limited, but the present study provides parallels with the HSV assembly model in that initial steps in assembly seem to involve tethering of the helicase-primase complex at the origin by interaction with DNA-bound Zta and entry of the single-stranded DNA binding protein, BALF2, through interaction with the helicase-primase proteins.…”

Herpes Simplex Virus Processivity Factor UL42 Imparts Increased DNA-Binding Specificity to the Viral DNA Polymerase and Decreased Dissociation from Primer-Template without Reducing the Elongation Rate

“…HSV does not utilize auxiliary protein complexes to guide the polymerase to the primer-template junction, as do other organisms (27,32,33,50). However, as recently reported, Pol and UL8, a component of the viral helicase-primase complex, can interact, at least in vitro (37). Such interactions between the primase and the DNA polymerase, combined with de-creases in interaction of Pol/UL42 with DNA that is not in a primer-template configuration, might help guide polymerase to the primer-template junction.…”

“…In the other direction, interaction of E. coli polymerase III DNA polymerase and helicase increases the rate of unwinding 10-fold (28). Since HSV Pol can interact with a helicase-primase subunit (37), it would be of interest to determine if HSV helicase activity is stimulated by Pol/UL42 or if the rate of unwinding limits fork movement instead.…”

An Intertypic Herpes Simplex Virus Helicase-Primase Complex Associated with a Defect in Neurovirulence Has Reduced Primase Activity

“…Marker rescue of R13-1 with the HSV-1 UL5 gene produced a neurovirulent rescuant that still retains the HSV-2 UL8 gene (3). Moreover, UL8 is known to interact not only with the other two subunits of the helicase-primase but also with the originbinding protein UL9 (29), the polymerase accessory protein, UL42 (27), and the ssDNA-binding protein, ICP8 (14). Nevertheless, even though the HSV-2 and HSV-1 UL8 proteins have a smaller degree of sequence identity than that of the corresponding UL5 polypeptides (80 versus 89% [27a]), the presence of a HSV-2 UL8 gene in an otherwise HSV-1 genetic background is tolerated with no known phenotypic consequences.…”