Vol. 44, No. 2 JOURNAL OF VIROLOGY, Nov. 1982, P. 736-741 0022-538X/82/110736-06$02.00/0 Copyright © 1982, American Society for Microbiology Characterization of Two Conformational Forms of the Major DNA-Binding Protein Encoded by Herpes Simplex Virus 1 DAVID M. KNIPE,* MARGARET P. QUINLAN, AND ANNE E. SPANG Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, Massachusetts 02115 Received 3 May 1982/Accepted 13 July 1982 We have resolved two electrophoretic species of the major DNA-binding protein, infected cell polypeptide 8 (ICP8), encoded by herpes simplex virus 1. In pulse-chase experiments, we observed the conversion of the ICP8a form, the slower migrating species, to the faster migrating form, ICP8b. Thus, the two species appear to be related as precursor-product. The conversion was not due to proteolytic cleavage, because higher concentrations of reducing agents in the sample buffer shifted the faster moving form to the slower moving species. Also, the two forms have identical peptide patterns as analyzed by partial proteolysis in sodium dodecyl sulfate. Thus, the faster moving species appears to be a conformational isomer containing intramolecular disulfide bonds. The functional significance of the two forms of the protein is discussed. Herpes simplex virus (HSV) encodes a major DNA-binding protein (2, 14, 15, 18) as a beta or delayed early gene product (8, 9). This protein has been called ICP8 (infected cell polypeptide 8), VP130 (viral protein of molecular weight 130,000), or ICSP 11/12 (infected cell-specific protein 11/12) (2, 8, 9, 11, 12, 14, 15, 18). Two lines of evidence indicate that the protein plays a role in the replication of viral DNA: (i) mutant viruses which encode a defective DNA-binding protein or whose temperature-sensitive lesions map in or near the ICP8 gene are defective for DNA replication (4, 14); and (ii) monospecific antiserum to the HSV type 2 (HSV-2) protein inhibits DNA replication in vitro but does not neutralize the viral DNA polymerase activity (14). The HSV-2 protein ICSP 11/12 has been reported to bind more tightly to single-stranded DNA than to double-stranded DNA (16), and the purified HSV-1 and HSV-2 DNA binding pro- teins can enhance the denaturation of a poly- deoxyadenylic acid-polydeoxythymidylic acid duplex (14). In infected cells, the protein is localized through a series of stages to the nucle- us, where it finally binds to viral DNA (6, 10, 13). Thus, ICP8 appears to be involved in the replication of HSV DNA in the cell nucleus. No posttranslational modifications of this pro- tein have been reported. It appears to lack phosphorylation (18). Others have reported two closely migrating protein bands near the size of ICP8 in the bound fraction from a DNA-cellu- lose column when extracts from HSV-2-infected cells were used (16, 18). In this communication, we define the relationship between two electro- phoretic forms of the HSV-1 ICP8 protein mole- cule. We observed a pair of closely migrating bands in the region of a polyacrylamide gel where ICP8, the major DNA-binding protein encoded by HSV, migrates (Fig. 1). The two bands were present in extracts prepared from cells labeled at 4 h postinfection. The molecular weights of the two bands were determined to be 127,000 and 125,000 by comparison with the migration of proteins of known molecular weight (not shown). Because we believe these two bands to be two different forms of ICP8, we have named the slower migrating band ICP8a and the faster migrating band ICP8b. The two bands were both present in extracts of cells immediately after a 5-min pulse label (Fig. 1). During the 2-h chase period, there was a progressive conversion to the ICP8b form so that by 2 h of chase, the ICP8b form was the predominant form. Figure 2 shows the micro- densitometer tracing of the ICP8 region of a gel from a similar pulse-chase experiment. During the chase period, the amount of the ICP8a form decreased, and the amount of the ICP8b form increased. Thus, it appeared that the ICP8a form was the precursor to the ICP8b form, although the conversion was never complete. The time of expression of the two bands corresponded to that for a beta or delayed early gene in that both were labeled at maximal rates at 3 to 6 h postinfection (not shown). Also, the period of expression of the two species was extended to at least 10 h postinfection by the incubation of infected cells in medium contain- 736