THE HERPES-SIMPLEX VIRUS TYPE-1 DNA-POLYMERASE ACCESSORY PROTEIN, UL42, CONTAINS A FUNCTIONAL PROTEASE-RESISTANT DOMAIN

Herpes simplex virus type 1 encodes its own DNA polymerase (Pol), the product of the UL30 gene, and a polymerase accessory subunit, the prod uct of the UL42 gene, both of which are required for viral DNA replica tion. Pol and the UL42 protein associate to form a heterodimeric compl ex (Pol/UL42) which is more active and has a higher processivity than the Pol catalytic subunit alone. The Pol/UL42 complex has been reconst ituted by mixing together highly purified Pol and UL42 subunits obtain ed from recombinant baculovirus-infected cells. We have used polymeras e activity on poly(dA): oligo(dT20), a template that the Pol subunit u tilizes with low efficiency, to measure the formation of the Pol/UL42 complex. Our data indicate that the association constant for the Pol/U L42 complex is 1 x 10(8) M-1. Proteolytic digestions of UL42 were perf ormed to determine whether structural domains of UL42 could be disclos ed by differential amino acid accessibilities. The ability of these pr otease-resistant domains to form a functional complex with Pol was det ermined by measuring their ability to stimulate Pol activity on poly(d A):oligo(dT20). We have found that trypsin digestion of UL42 in the pr esence of DNA generates protease-resistant fragments of 28K and 8K whi ch co-elute from a MonoQ column and are able to stimulate Pol activity on poly(dA): oligo(dT20). Complex formation of the 28K and 8K tryptic fragments with Pol was also shown by their co-immunoprecipitation wit h antibody to Pol. It was determined that the 28K fragment of UL42 com prised amino acids 1 to 245 or 1 to 254 of UL42, whereas the 8K fragme nt started at amino acid 255. Thus. controlled proteolysis of UL42 rev ealed two closely contiguous structural domains that retained the abil ity to complex with Pol and stimulate Pol activity.