Identification of residues within the herpes simplex virus type 1 origin-binding protein that contribute to sequence-specific DNA binding.

Gene UL9 of herpes simplex virus type 1 encodes an 851-amino-acid protein which is essential for viral DNA synthesis and functions as a sequence-specific origin-binding protein and DNA helicase. We generated monoclonal antibodies against purified UL9 protein and identified one such antibody (MAb 13924) that can block the interaction of the UL9 C-terminal DNA-binding domain (amino acids 534-851) with its recognition sequence. MAb 13924 interacted with immobilized peptides containing residues 780-786 of UL9. Although the corresponding region of the homologous protein encoded by varicell-azoster virus differs at only a single position it was not recognized by MAb 13924. Site-directed mutagenesis experiments confirmed that residues within this region contribute to the epitope recognized by MAb 13924 and may be involved in sequence-specific DNA binding. In addition, all eight lysine residues within the DNA-binding domain were separately changed to alanine and the DNA-binding properties of the mutated proteins were examined. The results showed that lysine residues that are located close to the peptide recognized by MAb 13924 or lie within the region of the DNA-binding domain most highly conserved among homologous alphaherpesvirus proteins play a role in sequence-specific DNA binding. Moreover, alteration of a lysine residue 18 amino acids from the recognized peptide prevented the interaction of MAb 13924 with the UL9 C-terminal DNA-binding domain. Three helical segments are predicted to occur within the region containing mutations that affect sequence-specific binding and interaction with MAb 13924.

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.

The herpes simplex virus type 1 UL8 protein influences the intracellular localization of the UL52 but not the ICP8 or POL replication proteins in virus-infected cells.

We have developed a panel of 14 monoclonal antibodies (MAbs) to POL, the catalytic subunit of herpes simplex virus type 1 (HSV-1) DNA polymerase encoded by gene UL30, and one MAb to the UL52 protein, another of the seven proteins essential for replication of HSV DNA. The approximate locations of the epitopes of the polymerase-specific MAbs were identified using truncated polymerase molecules, and the antibodies were characterized in a number of immunological assays allowing eight different specificities to be recognized. These MAbs, together with a polyclonal antibody raised in rabbits against a third DNA replication protein, ICP8, were used to localize the respective proteins by immunofluorescence in cells infected with wild-type HSV-1 or the DNA replication-defective mutants ambUL8 or 2-2. In BHK cells infected with ambUL8, a mutant with an amber termination codon within the coding region of gene UL8, the UL52 protein did not enter the nucleus, although ICP8 and POL entered the nucleus in a normal fashion. The failure of the UL52 protein to be correctly transported to the nucleus was also observed in both HFL and Vero cells infected with ambUL8. In contrast, UL52 protein was transported to the nucleus in BHK cells infected with wild-type HSV-1 or with 2-2, a mutant lacking a functional UL9 protein.

The origin-binding domain of the herpes simplex virus type 1 UL9 protein is not required for DNA helicase activity.

The UL9 protein of herpes simplex virus type 1 binds to specific sequences within the viral origins of DNA replication and also functions as a DNA helicase. The C-terminal 317 amino acids of the 851 residue protein specify sequence-specific binding to the viral origins and the N-terminal 400 contain several motifs characteristic of many DNA and RNA helicases. To investigate whether the origin-binding domain is required for helicase function we have expressed a truncated version comprising amino acids 1-535 of UL9 using a recombinant baculovirus. Extracts were prepared from cells infected with the recombinant virus and chromatographed over ATP-agarose. DNA helicase, DNA-dependent ATPase and a novel single-stranded DNA-binding activity were present in fractions containing the truncated UL9 protein but not in corresponding gradient fractions from a control virus infection. These results indicate that the DNA helicase function of UL9 does not require the presence of the origin-binding domain and suggest that an interaction between the N-terminal domain and distorted or partially single-stranded regions of DNA may play a role in unwinding the origin region.

The herpes simplex virus type 1 origin-binding protein interacts specifically with the viral UL8 protein.

The products of herpes simplex virus type 1 (HSV-1) genes UL5, UL8 and UL52 form a complex in virus-infected cells that exhibits both DNA helicase and DNA primase activities. UL8 protein was purified from insect cells infected with a recombinant baculovirus and used to generate monoclonal antibodies (MAbs). MAb 0811 was shown to recognize the UL8 protein in both Western blots and immunoprecipitation assays and to co-precipitate the other two proteins in the complex from insect cells triply infected with recombinants expressing the UL5, UL8 and UL52 polypeptides. Experiments performed using extracts from doubly infected cells indicated that UL8 could interact separately with both the UL5 and UL52 proteins. Similar experiments using a recombinant virus that expressed the HSV-1 origin-binding protein (OBP), UL9, demonstrated a direct physical interaction between the helicase-primase complex and OBP which involved the UL8 subunit. The C-terminal DNA-binding domain of OBP is dispensable for this interaction, as evidenced by the ability of MAb 0811 to co-precipitate a truncated UL9 protein, containing only the N-terminal 535 amino acids, with UL8.