DNA-binding protein associated with herpes simplex virus DNA polymerase.

Purified preparations of herpes simplex virus type 2 DNA polymerase made by many different laboratories always contain at least two polypeptides. The major one, of about 150,000 molecular weight, has been associated with the polymerase activity. The second protein, of about 54,000 molecular weight, which we previously designated ICSP 34, 35, has now been purified. The purified protein has been used to prepare antisera (both polyclonal rabbit serum and monoclonal antibodies). These reagents have been used to characterize the protein, to demonstrate its quite distinct map location from that of the DNA polymerase on the herpes simplex virus genome, and to demonstrate the close association between the two polypeptides.     

N-ethylmaleimide inhibition of the DNA-binding activity of the herpes simplex virus type 1 major DNA-binding protein.

The major herpes simplex virus DNA-binding protein, designated ICP8, binds tightly to single-stranded DNA and is required for replication of viral DNA. The sensitivity of the DNA-binding activity of ICP8 to the action of the sulfhydryl reagent N-ethylmaleimide has been examined by using nitrocellulose filter-binding and agarose gel electrophoresis assays. Incubation of ICP8 with N-ethylmaleimide results in a rapid loss of DNA-binding activity. Preincubation of ICP8 with single-stranded DNA markedly inhibits this loss of binding activity. These results imply that a free sulfhydryl group is involved in the interaction of ICP8 with single-stranded DNA and that this sulfhydryl group becomes less accessible to the environment upon binding. Agarose gel electrophoretic analysis of the binding interaction in the presence and absence of N-ethylmaleimide indicates that the cooperative binding exhibited by ICP8 is lost upon treatment with this reagent but that some residual noncooperative binding may remain. This last result was confirmed by equilibrium dialysis experiments with the 32P-labeled oligonucleotide dT10 and native and N-ethylmaleimide-treated ICP8.     

Characterization of two conformational forms of the major DNA-binding protein encoded by herpes simplex virus 1.

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.     

Intragenic complementation of herpes simplex virus ICP8 DNA-binding protein mutants.

The major DNA-binding protein, or infected-cell protein 8 (ICP8), of herpes simplex virus is required for viral DNA synthesis and normal regulation of viral gene expression. Previous genetic analysis has indicated that the carboxyl-terminal 28 residues are the only portion of ICP8 capable of acting independently as a nuclear localization signal. In this study, we constructed a mutant virus (n11SV) in which the carboxyl-terminal 28 residues of ICP8 were replaced by the simian virus 40 large-T-antigen nuclear localization signal. The n11SV ICP8 localized into the nucleus and bound to single-stranded DNA in vitro as tightly as wild-type ICP8 did but was defective for viral DNA synthesis and viral growth in Vero cells. Two mutant ICP8 proteins (TL4 and TL5) containing amino-terminal alterations could complement the n11SV mutant but not ICP8 gene deletion mutants. Cell lines expressing TL4 and TL5 ICP8 were isolated, and in these cells, complementation of n11SV was observed at the levels of both viral DNA replication and viral growth. Therefore, complementation between n11SV ICP8 and TL4 or TL5 ICP8 reconstituted wild-type ICP8 functions. Our results demonstrate that (i) the carboxyl-terminal 28 residues of ICP8 are required for a function(s) involved in viral DNA replication, (ii) this function can be supplied in trans by another mutant ICP8, and (iii) ICP8 has multiple domains possessing different functions, and at least some of these functions can complement in trans.     

Targeting Holliday junctions by origin DNA-binding protein of herpes simplex virus type 1

In the present paper, the interactions of the origin binding protein (OBP) of herpes simplex virus type 1 (HSV1) with synthetic four-way Holliday junctions (HJs) were studied using electrophoresis mobility shift assay and the FRET method and compared with the interactions of the protein with duplex and single-stranded DNAs. It has been found that OBP exhibits a strong preference for binding to four-way and three-way DNA junctions and possesses much lower affinities to duplex and single-stranded DNAs. The protein forms three types of complexes with HJs. It forms complexes I and II which are reminiscent of the tetramer and octamer complexes with four-way junction of HJ-specific protein RuvA of Escherichia coli. The binding approaches saturation level when two OBP dimers are bound per junction. In the presence of Mg²⁺ ions (≥2 mM) OBP also interacts with HJ in the stacked arm form (complex III). In the presence of 5 mM ATP and 10 mM Mg²⁺ ions OBP catalyzes processing of the HJ in which one of the annealed oligonucleotides has a 3′-terminal tail containing 20 unpaired thymine residues. The observed preference of OBP for binding to the four-way DNA junctions provides a basis for suggestion that OBP induces large DNA structural changes upon binding to Box I and Box II sites in OriS. These changes involve the bending and partial melting of the DNA at A+T-rich spacer and also include the formation of HJ containing Box I and Box II inverted repeats and flanking DNA sequences.     

Varicella-zoster virus complements herpes simplex virus type 1 temperature-sensitive mutants.

Varicella-zoster virus (VZV) can complement temperature-sensitive mutants of herpes simplex virus. Of seven mutants tested, two, carrying mutations in the immediate-early ICP4 and ICP27 proteins, were complemented. This complementation was not seen in coinfections with adenovirus type 5 or cytomegalovirus. Following transfection into CV-1 cells, a DNA fragment containing the VZV short repeat sequence complemented the ICP4 mutant. These data demonstrate a functional relationship between VZV and herpes simplex virus and have allowed localization of a putative VZV immediate-early gene.     

Biochemical transformation by temperature-sensitive mutants of herpes simplex virus type 1.

Biochemical transformation assays of herpes simplex virus type 1 temperature-sensitive (ts) mutants distinguished three groups of mutants with regard to their thymidine kinase (TK) transforming ability: those incapable of transferring the TK gene at either the permissive or restrictive temperatures (group I); those resembling the wild-type virus, and therefore able to transform at both the permissive and nonpermissive temperatures (group II); and those that failed to transform or exhibited very low transformation frequencies at the permissive temperature but were able to transform at the nonpermissive temperature (group III). Two mutants in group II exhibited greatly enhanced transformation efficiency at the permissive temperature. The ts lesions in the majority of the mutants tested map between 0.30 and 0.60 units on the viral genome. Mutants with TK-positive (TK+), but DNA-negative, phenotypes at the nonpermissive temperature produced no TK+ transformants at the permissive temperature and only unstable transformants at the nonpermissive temperature. This suggests that a function which is required for viral DNA synthesis is also required to obtain stable expression or to transfer the TK+ gene or both when transfer is mediated by the entire viral genome.     

Genetic analysis of temperature-sensitive mutants which define the gene for the major herpes simplex virus type 1 DNA-binding protein.

We have assigned eight temperature-sensitive mutants of herpes simplex virus type 1 to complementation group 1-1. Members of this group fail to complement mutants in herpes simplex virus type 2 complementation group 2-2. The mutation of one member of group 1-1, tsHA1 of strain mP, has been shown to map in or near the sequence which encodes the major herpes simplex virus type 1 DNA-binding protein (Conley et al., J. Virol. 37:191-206, 1981). The mutations of five other members of group 1-1 map in or near the sequence in which the tsHA1 mutation maps, a sequence which lies near the center of UL between the genes for the viral DNA polymerase and viral glycoprotein gAgB. These mutants can be divided into two groups; the mutations of one group map between coordinates 0.385 and 0.398, and the mutations of the other group map between coordinates 0.398 and 0.413. At the nonpermissive temperature mutants in group 1-1 are viral DNA negative, and mutant-infected cells fail to react with monoclonal antibody to the 130,000-dalton DNA-binding protein. Taken together, these data indicate that mutants in complementation groups 1-1 and 2-2 define the gene for the major herpes simplex virus DNA-binding protein, an early gene product required for viral DNA synthesis.     

Characterization of a major DNA-binding domain in the herpes simplex virus type 1 DNA-binding protein (ICP8).

We have studied the major DNA-binding protein (ICP8) from herpes simplex virus type 1 to identify its DNA-binding site. Since we obtained our protein from a cell line carrying multiple chromosomally located copies of the ICP8 gene, we first analyzed this protein to assess its similarity to the corresponding viral protein. Our protein resembled the viral protein by molecular weight, response to antibody, preference for binding single-stranded DNA, and ability to lower the melting temperature of poly(dA-dT). To define the DNA-binding domain, we subjected the protein to limited trypsin digestion and separated the peptide products on a sodium dodecyl sulfate-polyacrylamide gel. These fragments were then transferred to a nitrocellulose membrane, renatured in situ, and tested for their ability to bind DNA. From this assay, we identified four fragments which both bound DNA and exhibited the expected binding preference for single-stranded DNA. The sequence of the smallest of these fragments was determined and corresponds to a polypeptide spanning residues 300 to 849 in the intact protein. This peptide contains several regions which may be important for DNA binding based on sequence similarities in single-stranded DNA-binding proteins from other herpesviruses and, in one case, on a conserved sequence found in more distant procaryotic and eucaryotic proteins.     

DNA synthesis and DNA polymerase activity of herpes simplex virus type 1 temperature-sensitive mutants.

Fifteen temperature-sensitive mutants of herpes simplex virus type 1 were studied with regard to the relationship between their ability to synthesize viral DNA and to induce viral DNA polymerase (DP) activity at permissive (34 C) and nonpermissive (39 C) temperatures. At 34 C, all mutants synthesized viral DNA, while at 39 C four mutants demonstrated a DNA+ phenotype, three were DNA+/-, and eight were DNA-. DNA+ mutants induced levels of DP activity similar to thhose of the wild-type virus at both temperatures, and DNA+/- mutants induced reduced levels of DP activity at 39 C but not at 34 C. Among the DNA- mutants three were DP+, two were DP+/-, and three showed reduced DP activity at 34 C with no DP activity at 39 C. DNA-, DP- mutants induced the synthesis of a temperature-sensitive DP as determined by in vivo studies.     

Cellular protein interactions with herpes simplex virus type 1 oriS.

The herpes simplex virus type 1 (HSV-1) origin of DNA replication, oriS, contains an AT-rich region and three highly homologous sequences, sites I, II, and III, identified as binding sites for the HSV-1 origin-binding protein (OBP). In the present study, interactions between specific oriS DNA sequences and proteins in uninfected cell extracts were characterized. The formation of one predominant protein-DNA complex, M, was demonstrated in gel shift assays following incubation of uninfected cell extracts with site I DNA. The cellular protein(s) that comprises complex M has been designated origin factor I (OF-I). The OF-I binding site was shown to partially overlap the OBP binding site within site I. Complexes with mobilities indistinguishable from that of complex M also formed with site II and III DNAs in gel shift assays. oriS-containing plasmid DNA mutated in the OF-I binding site exhibited reduced replication efficiency in transient assays, demonstrating a role for this site in oriS function. The OF-I binding site is highly homologous to binding sites for the cellular CCAAT DNA-binding proteins. The binding site for the CCAAT protein CP2 was found to compete for OF-I binding to site I DNA. These studies support a model involving the participation of cellular proteins in the initiation of HSV-1 DNA synthesis at oriS.     

The major herpes simplex virus type-1 DNA-binding protein is a zinc metalloprotein.

The primary amino acid sequence of the major herpes simplex virus type 1 (HSV-1)-infected cell polypeptide 8 (ICP8) deduced from the DNA sequence of the unique long open reading frame 29 (UL29 ORF) contains a potential metal-binding domain of the form Cys-X2-5-Cys-X2-15-A-X2-4-A where A may be either histidine or cysteine and X is any amino acid. The putative metal-binding sequence in ICP8 encompasses residues 499-512 as follows: C-N-L-C-T-F-D-T-R-H-A-C-V-H-. Atomic absorption analysis of several preparations of ICP8 indicates the presence of 1 mol of zinc/mol of protein. The zinc is resistant to removal by dialysis against concentrations of EDTA which deplete zinc from alcohol dehydrogenase. The bound zinc can be removed by reaction with the reversible sulfhydryl reagent p-hydroxymercurimethylsulfonate and the zinc-depleted protein transiently retains DNA binding activity. Digestion of both native and zinc-depleted ICP8 with V8 protease indicates that the bound zinc is required for the structural integrity of the protein.     

In vitro characterization of a thermolabile herpes simplex virus DNA-binding protein.

The major herpes simplex virus DNA-binding protein, ICP8, was purified from cells infected with the herpes simplex virus type 1 temperature-sensitive strain tsHA1. tsHA1 ICP8 bound single-stranded DNA in filter binding assays carried out at room temperature and exhibited nonrandom binding to single-stranded bacteriophage fd DNA circles as determined by electron microscopy. The filter binding assay results and the apparent nucleotide spacing of the DNA complexed with protein were identical, within experimental error, to those observed with wild-type ICP8. Thermal inactivation assays, however, showed that the DNA-binding activity of tsHA1 ICP8 was 50% inactivated at approximately 39 degrees C as compared with 45 degrees C for the wild-type protein. Both wild-type and tsHA1 ICP8 were capable of stimulating viral DNA polymerase activity at permissive temperatures. The stimulatory effect of both proteins was lost at 39 degrees C.     

DNA-binding properties of a herpes simplex virus immediate early protein

The herpes simplex virus alpha, or immediate early, protein ICP4 has been shown to be central to the control of the early stages of virus replication. The detailed mechanism of this control is unknown. In this communication we show that purified ICP4 was unable to bind to DNA even though the protein was capable of such activity in a crude extract. Addition of either infected- or uninfected-cell extracts to the purified protein restored its DNA-binding activity. These results suggest that ICP4 binds to DNA only via a component of uninfected cells.     

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

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.     

Isolation of a protein kinase induced by herpes simplex virus type 1

We have isolated a new cyclic AMP-independent protein kinase activity induced in HeLa cells by infection with herpes simplex virus type 1. Induction of the enzyme does not occur in cells treated with cycloheximide at the time of infection, or in cells infected with UV-inactivated herpes simplex virus type 1. The amount of enzyme induced in infected cells is dependent upon the multiplicity of infection. An enzyme with identical properties to the appearing in infected HeLa cells is also induced by herpes simplex virus type 1 in BHK cells.     

Identification of dominant-negative mutants of the herpes simplex virus type 1 immediate-early protein ICP0.

ICP0 is a 110,000-molecular-weight immediate-early protein of herpes simplex virus type 1 (HSV-1) which is encoded by three exons. It has been shown to function as a promiscuous transactivator of a variety of different HSV-1 and non-HSV-1 promoters in transient expression assays. Analysis of mutations which truncated the carboxy-terminal end of this 775-amino-acid (aa) protein demonstrated that a polypeptide which contained only aa 1 to 553 still possessed significant transactivation potential. Additional carboxy-terminal truncations which sequentially removed aa 245 to 553 and thus the remainder of the third exon resulted in the eventual loss of transactivation capability in these mutants. However, further analysis of these truncated derivatives demonstrated that they behaved as dominant-negative mutants to the wild-type polypeptide. Moreover, one of the mutants was found to act as a promiscuous repressor, in that it could dramatically inhibit a variety of HSV-1 promoters, non-HSV-1 promoters, and heterologous transactivator proteins in transient expression assays, despite having lost almost the entire third exon. These results indicate that a domain encoded by the first two exons probably interacts with, and can effectively titrate, the unknown cellular factor(s) through which ICP0 mediates transactivation.     

Stimulation of expression of a herpes simplex virus DNA-binding protein by two viral functions.

We examined the expression and localization of herpesvirus proteins in monkey cells transfected with recombinant plasmids containing herpes simplex virus (HSV) DNA sequences. Low levels of expression of the major HSV DNA-binding protein ICP8 were observed when ICP8-encoding plasmids were introduced into cells alone. ICP8 expression was greatly increased when a recombinant plasmid encoding the HSV alpha (immediate-early) ICP4 and ICP0 genes was transfected with the ICP8 gene. Deletion and subcloning analysis indicated that two separate functions capable of stimulating ICP8 expression were encoded on the alpha gene plasmid. One mapped in or near the ICP4 gene, and one mapped in or near the ICP0 gene. Their stimulatory effects were synergistic when introduced on two separate plasmids. Thus, two separate viral functions can activate herpesvirus early gene expression in transfected cells.     

DNA-binding proteins induced by herpes simplex virus type 2 in HEp-2 cells.

Affinity chromatography on single-stranded and double-stranded DNA-cellulose indicates that 12 proteins previously identified from herpes simplex virus type 2-infected cells, ranging in molecular weight from 28 X 10(3) to 186 X 10(3), bind to DNA-cellulose. The DNA-binding proteins found in infected cells differed in relative binding strengths for denatured DNA-cellulose. The virus specificity of these DNA-binding proteins was further studied by comparison with DNA-binding proteins isolated from mock-infected cells, and by immunoprecipitation of infected-cell DNA-binding proteins with antisera specific for viral antigens. The promise this technique holds for the purification and study of polypeptides involved in virus DNA replication, recombination, or repair is discussed.     

Mutation spectra of herpes simplex virus type 1 thymidine kinase mutants

To examine whether the exonuclease activity intrinsic to the polymerase (Pol) of herpes simplex virus type 1 can influence the mutational spectra, we applied the denaturing gradient gel electrophoresis (DGGE) system combined with sequencing to characterize thymidine kinase mutants isolated from both the wild-type virus and a mutant deficient in exonuclease activity, Y7. Wild-type viruses produced predominantly frameshift mutations (67%), whereas Y7 replicated a significantly lower proportion of frameshifts (21%; P < 0.005). Furthermore, the majority of substitutions were transitional changes in both groups, although they distributed differently. The implications of these findings are discussed.