Identification of the gene encoding the 65-kilodalton DNA-binding protein of herpes simplex virus type 1

Abstract

Hybrid arrest of in vitro translation was used to localize the region of the herpes simplex virus type 1 genome encoding the 65-kilodalton DNA-binding protein (65KDBP) to between genome coordinates 0.592 and 0.649. Knowledge of the DNA sequence of this region allowed us to identify three open reading frames as likely candidates for the gene encoding 65KDBP. Two independent approaches were used to determine which of these three open reading frames encoded the protein. For the first approach a monoclonal antibody, MAb 6898, which reacted specifically with 65KDBP, was isolated. This antibody was used, with the techniques of hybrid arrest of in vitro translation and in vitro translation of selected mRNA, to identify the gene encoding 65KDBP. The second approach involved preparation of antisera directed against oligopeptides corresponding to regions of the predicted amino acid sequence of this gene. These antisera reacted specifically with 65KDBP, thus confirming the gene assignment. Many proteins which bind to DNA play some role in regulation of transcription or DNA replication. Herpes simplex virus types 1 and 2 (HSV-1 and HSV-2) induce the expression of up to 20 DNA-binding proteins (DBPs) as determined by their affinity for DNA-containing matrices (3, 36). Only a few have been studied in detail: the most abundant of these is the major DBP, infected-cell polypeptide 8 (ICP8), or ICSP11,12 (8, 36, 45), of Mr 128,000 (1,196 amino acids [37]). ICP8 binds preferentially to singlestranded DNA (18, 32), is able to destabilize poly(dA-dT) helices (31), and is required for the synthesis of HSV DNA (11, 20). The HSV DNA polymerase and the alkaline exonuclease also bind to DNA, and analysis of temperaturesensitive mutants with lesions in these genes demonstrates that they too are essential for viral DNA replication (16, 28, 35). ICSP34,35 is another distinct protein which binds to DNA and copurifies with the HSV-2 DNA polymerase (43). The function of ICSP34,35, however, is not known. Interestingly, these four DBPs may interact in vivo, since immunoabsorbent columns containing monoclonal antibodies (MAbs) directed to the major DBP, the alkaline exonuclease, or ICSP 34,35 bind at least two of these four proteins. These interactions have been postulated to reflect the existence of replication complexes (42). Recently, we partially characterized a 65-kilodalton DBP (65KDBP) of HSV-1, which was the major protein species in high-salt eluates from double-stranded DNA-cellulose columns used to fractionate HSV nuclear proteins (22). Whether the protein is intrinsically DNA binding or whether its binding is mediated by some other protein is not known. We demonstrated that it was distinct from the 65-kilodalton virion trans-inducing factor (65KTIF), which stimulates transcription from immediate-early genes (2, 5, 30), and showed that its gene mapped to between coordinates 0.574 and 0.682 on the HSV-1 genome (22). These coordinates were based on published restriction enzyme and transcript maps (collated * Corresponding author. in refere...

Cloning, sequencing, and functional characterization of the two subunits of the pseudorabies virus DNA polymerase holoenzyme: evidence for specificity of interaction

“…The major translation product of the PRV pol gene corresponded to a protein with an apparent molecular weight of 110,000 (lane 2), whereas the major translation product produced from the HSV-1 gene migrated as a 130-kDa peptide (lane 4). The major translation product of the HSV-1 UL42 gene has an apparent molecular mass of 62 kDa (lane 5) as previously described (55), whereas that of the PRV homolog is considerably smaller, migrating as a 42-kDa polypeptide (lane 3). The sizes of both the PRV pol and pap gene products are in good agreement with the 115.3 and 40.3 kDa predicted from the ORFs.…”

“…This polypeptide possesses inherent enzymatic properties including DNA chain elongation (17,30,48), 3Ј-5Ј exonuclease (41,48), and RNase H (9,48) activities. It has been shown to be tightly associated with a 65-kDa double-stranded DNA-binding protein encoded by the UL42 gene of HSV-1 (9,23,29,35,55,68), a gene that is essential for DNA synthesis and virus replication (38,47). The product of the UL42 gene stimulates the basal activity and processivity of Pol (22,29,35), properties which appear to be essential for HSV-1 replication (13,14,59,63).…”

Kinetics of expression of the gene encoding the 65-kilodalton DNA-binding protein of herpes simplex virus type 1

“…Plasmid pTZTK3 was derived from the plasmid pTK1 (35) and contains the 503base-pair BglII-to-SstI fragment, which overlaps the 5' end of the thymidine kinase (tk) transcript, cloned into pTZ18U. MAb 6898 has been described previously (26) and was used to specifically detect 65KDBP. MAb 39S, which recognizes the HSV-1 major DNA-binding protein ICP8 (36), was kindly provided by Martin Zweig.…”

“…The gene encoding 65KDBP maps in the HindIII L fragment and corresponds to UL42 (26). Sev_ral pieces of evidence indicate that 65KDBP is involved in DNA replication: (i) UL42 is one of only seven HSV-1 genes required for the replication of plasmids containing an HSV origin of replication (44); (ii) a temperature-sensitive mutant with a lesion which maps to coordinates occupied by UL42 does not synthesize viral DNA at the nonpermissive temperature (20); and (iii) 65KDBP is closely associated with the viral DNA polymerase (Pol), suggesting that it is a component of replication complexes (9,42).…”

“…Hybridization was performed at 65°C in 3 x SSC-0.5% SDS-2x Denhardt solution-2.5 p.g of sonicated and denatured salmon sperm DNA per ml for 36 h, and the blots were successively washed at 65°C in decreasing salt concentrations to lx SSC-0.1% SDS-0.1% sodium pyrophosphate prior to autoradiography.RESULTSLocalization of 65KDBP. To examine the intracellular distribution of 65KDBp at various times p.i., we used MAb 6898, specific for 65KDBP(26), in indirect immunofluorescence tests. For comparison with a virus-encoded protein of known localization pattern and kinetic class, we analyzed parallel samples with MAb 39S, which recognizes the early cells, pockets of higher concentrations of 65KDBP accumulated at the inner periphery of the nuclear membrane…”