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 RR1 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 aa 72-350. The epitopes for cross-reactive antibodies to HSV RR1, i.e., 48S and 51S, are formed predominantly by a stretch of amino acid residues specified by aa 350-376 of the RR1 molecule. The 6A-6 antibody utilized in conjunction with the RR1 amber mutants has allowed us to define a 278 aa domain within the NH2-terminal half of the 140-kDa RR1 (aa 72-350) that is sufficient for PK activity.
Ali
M A
MA
Viral Carcinogenesis and Immunology Laboratories, Linus Pauling Institute of Science and Medicine, Palo Alto, California 94306.
Prakash
S S
SS
Jariwalla
R J
RJ
D001483:Base Sequence; D003001:Cloning, Molecular; D000939:Epitopes / Q000235:genetics / Q000276:immunology; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011494:Protein Kinases / Q000235:genetics / Q000276:immunology*; D015183:Restriction Mapping; D012264:Ribonucleotide Reductases / Q000235:genetics / Q000276:immunology*; D018139:Simplexvirus / Q000201:enzymology / Q000235:genetics / Q000276:immunology*
D000939:Epitopes; D012264:Ribonucleotide Reductases; D011494:Protein Kinases