LOCALIZATION OF THE ANTIGENIC SITES AND INTRINSIC PROTEIN-KINASE DOMAIN WITHIN A 300 AMINO-ACID SEGMENT OF THE RIBONUCLEOTIDE REDUCTASE LARGE SUBUNIT FROM HERPES-SIMPLEX VIRUS TYPE-2

The 140-kDa ribonucleotide reductase (RR1) protein of herpes simplex virus type 2 (HSV-2) functions as the large subunit of virus-specified RR1 and exhibits an intrinsic protein kinase (PK) activity at its unique NH2 terminal region. The N-terminal half of RR1 contains the protein and DNA functions of the morphological transforming region III (mtrIII) of HSV-2. In the present study, we have expressed a number of truncated RRi derivatives in a mammalian expression vector containing NH2 terminal RR1 gene fragments and amber mutants generated by site-specific mutagenesis. These derivatives, synthesized in transient expression assays, were used as test antigens to localize the epitopes of a panel of HSV-2 RR1-reactive monoclonal antibodies and to fine-map the PK catalytic domain. Our data show that the epitope for HSV-2-specific monoclonal antibody 6A-6 is located in a region of RR1 protein spanning as 72-350. The epitopes for cross-reactive antibodies to HSV RR1, i.e., 48S and 51 S, are formed predominantly by a stretch of amino acid residues specified by as 350-376 of the RRI molecule. The 6A-6 antibody utilized in conjunction with the RR1 amber mutants has allowed us to define a 278 as domain within the NH2 terminal half of the 140-kDa RR1 (aa 72-350) that is sufficient for PK activity. © 1992.