Howard S. Marsden

Identification of the herpes simplex virus protein kinase as the product of viral gene US3

Previous work has shown that a novel protein kinase is induced after infection of cultured cells with herpes simplex virus type 1 (HSV-1). Separately, it has been reported that the protein encoded by HSV-1 gene US3 shows similarity in its amino acid sequence to members of the protein kinase family of eukaryotes. We have investigated the possibility that these two observations are connected by preparing an antiserum to a synthetic oligopeptide corresponding to the carboxy-terminal eight amino acids of the US3 protein. This antiserum reacted on immunoblots with a polypeptide of apparent molecular weight 68,000 from extracts of cells which had been infected with HSV-1. The antiserum also reacted strongly with a 68,000 molecular weight species from a preparation of the novel HSV-1 protein kinase which had been extensively purified and resolved from other protein kinases. In addition, the purified preparation phosphorylated a protein species, also of 68,000 apparent molecular weight, when incubated with [gamma-32P]ATP. These data are consistent with gene US3 encoding the novel protein kinase induced after infection of cells with HSV-1.

The 10K virion phosphoprotein encoded by gene US9 from herpes simplex virus type 1

Gene US9 of herpes simplex virus type 1 has been predicted, from DNA sequence analysis, to encode a protein of mol wt 10,026, designated 10K (D.J. McGeoch, A. Dolan, S. Donald, and F.J. Rixon (1985). J. Mol. Biol. 181, 1-13). We have investigated this protein by using a synthetic peptide corresponding to the 11 amino acids adjacent to the amino-terminal methionine and rasing antisera in rabbits. One antiserum was able to precipitate at least 12 electrophoretically distinct polypeptide species from extracts of BHK cells infected with HSV-1. The estimated molecular weights of these polypeptides ranged from 12K to 20K and immunoblotting showed them to be related proteins. The primary translation product has an apparent mol wt of 13K. The various forms of 10K differ in their relative abundance in the infected cell and also in their degree of phosphorylation. Lower molecular weight forms of the 10K protein can be precipitated from NP-40 extracts of HSV-1 virions, suggesting that these forms of 10K are contained in the virion tegument or envelope. An association between this protein and nucleocapsids has also been observed in the nuclei of infected cells by immunoelectron microscopy. These observations imply that the product of US9 is a tegument protein which becomes associated with nucleocapsids at, or soon after, their formation in the nuclei of infected cells.

The product of gene US11 of herpes simplex virus type 1 is expressed as a true late gene

The genes of herpes simplex virus type 1 (HSV-1) can be divided into at least three temporally regulated groups termed immediate early (IE), early and late. We have studied in detail the expression of a member of the late class of genes, US11, which encodes a polypeptide of apparent molecular weight 21K. Highly specific and sensitive probes were used to monitor US11 RNA and protein synthesis during HSV-1 infection of tissue culture cells in the presence and absence of phosphonoacetic acid, an inhibitor of viral DNA replication. The results were compared with a similar study of the products of a delayed early gene, US6, encoding glycoprotein D (gD). It was found that the patterns of RNA and protein synthesis from US11 were significantly different to those of gD. US11 products appeared later and accumulated until late in infection, while gD RNA was significantly reduced at late times. In the presence of the inhibitor of DNA synthesis, US11 gene expression was reduced 50- to 100-fold while gD expression was reduced five- to tenfold. We conclude that US11 behaves as a true late gene during HSV-1 infection. However, the use of sensitive assays, which allowed the detection of very low levels of US11 gene products under conditions designed to eliminate DNA replication, brings into question the absolute requirement for DNA replication for the expression of a true late HSV-1 gene. These results are discussed in terms of current models for the regulation of late gene expression.

Novel herpes simplex virus type 1 glycoproteins identified by antiserum against a synthetic oligopeptide from the predicted product of gene US4

Gene US4 of herpes simplex virus type 1 (HSV-1) has been predicted, from DNA sequence analysis, to encode a protein of molecular weight 25237 and its properties suggest it to be a membrane-associated protein. We have investigated this protein by raising antiserum to a synthetic oligopeptide corresponding to a stretch of amino acids from an internal hydrophilic region of the predicted sequence. This antiserum immunoprecipitates three glycoprotein species of apparent mol. wt. 37 000, 48 000 and 56 000 from extracts of cells infected with HSV-1. These species are also specifically immunoprecipitated from purified virions. The in vitro translation product of gene US4 has an apparent mol. wt. of 35 000. Sequence comparisons of the short unique regions of the HSV-1 and HSV-2 genomes, in combination with published mapping data for glycoprotein G (gG) of HSV-2, has led to the conclusion that the product of gene US4 of HSV-1 is the equivalent of gG.

Purification and properties of the herpes simplex virus type 1 UL8 protein

A rapid and simple two-step scheme for the purification of herpes simplex virus type 1 UL8 protein from insect cells infected with a recombinant baculovirus has been developed. The scheme involves DEAE-Sepharose and phenyl-Sepharose chromatography and yields approximately 1.5 mg of protein from 2.4 x 10(8) infected cells. The protein remains intact during purification as judged by its reactivity with amino and carboxy termini-specific antisera. Gel filtration chromatography showed that the protein exists as a monomer in solution. No binding of the protein to ssDNA or dsDNA or to a DNA/RNA hybrid could be demonstrated using a gel mobility shift assay.

The unique N terminus of the herpes simplex virus type 1 large subunit is not required for ribonucleotide reductase activity

Using purified bacterially expressed herpes simplex virus type 1 ribonucleotide reductase large subunit (R1) and the proteolytic enzymes chymotrypsin and trypsin, we have generated stable N-terminal truncations. Chymotrypsin removes 246 amino acids from the amino terminus to produce a fragment (dN246R1) which retains full enzymic activity and affinity for the small subunit (R2). Treatment of R1 with trypsin produces a 120K protein and a cleavage at amino acid residue 305 to produce a fragment (dN305R1) which remains associated with a 33K N-terminal polypeptide. Although this 33K-dN305R1 complex retains full binding affinity for R2 its reductase activity is reduced by approximately 50%. Increasing the concentration of trypsin removes the 33K N-terminal polypeptide resulting in dN305R1 which, when bound to R2, has full ribonucleotide reductase activity. Like R1, dN246R1 and dN305R1 each exist as dimers showing that the first 305 amino acids of R1 are not necessary for dimer formation. These results indicate that, in structural studies of subunit interaction, dN246R1 or dN305R1 can be considered as suitable replacements for intact R1.

Identification of Two Herpes Simplex Virus Type 1-induced Proteins (21K and 22K) which Interact Specifically with the a Sequence of Herpes Simplex Virus DNA

We have used a DNA competition binding assay to search for herpes simplex virus (HSV) proteins which are able to bind to specific sequences of the genome of HSV. Cloned DNAs from different regions of the virus genome were tested. Two late polypeptides, one major of apparent molecular weight 21 000 and one minor of 22 000, were preferentially bound by a variety of fragments containing the HSV-1 400 bp a sequence (a direct repeat present at the ends of the molecule and in inverted orientation between the long and short regions of the genome) but not by other competing DNAs including ones containing an origin of replication. We interpret our result as evidence that the HSV type 1-induced 21K and 22K polypeptides interact specifically with DNA sequences within this 400 bp HSV-1 a sequence.

Identification of structural domains within the large subunit of herpes simplex virus ribonucleotide reductase

The large subunit (R1) of herpes simplex virus (HSV) ribonucleotide reductase is a bifunctional protein consisting of a unique N-terminal protein kinase domain and a ribonucleotide reductase domain. Previous studies showed that the two functional domains are linked by a protease sensitive site. Here we provide evidence for two subdomains, of 30K and 53K, within the reductase domain. The two fragments, which were produced by limited proteolysis and were resistant to further degradation, remained tightly associated in a complex containing two molecules of each. They were capable of binding the R2 subunit of HSV ribonucleotide reductase with approximately the same affinity as the intact protein but the complex did not complement the small subunit (R2) to give an active enzyme. At low concentrations (0.4 micrograms/ml) of trypsin or V8 protease, cleavage between the subdomains was prevented by the presence of the N-terminal protein kinase domain. At higher protease concentrations (1 micrograms/ml) the N-terminal domain is extensively proteolysed and the 30K and 53K domains were generated. Identical results were obtained using purified R1 isolated from infected cell extracts or following expression in Escherichia coli. The origin of the two domains was investigated by N-terminal sequencing of the 53K fragment and by examining their reactivity with a panel of R1-specific monoclonal antibodies which we isolated and epitope mapped for that purpose. The trypsin cleavage site was found to lie between arginine 575 and asparagine 576, and proteolysis in this region was not prevented by the presence of R2 or the nonapeptide YAGAVVNDL. We propose that the ribonucleotide reductase region of HSV R1 exists in a two domain structure, and that the interdomain linking region is protected by the unique N terminus.

Epitope mapping identifies an exposed loop between the unique amino- and conserved carboxy-domains of the large subunit of herpes simplex virus type 1 ribonucleotide reductase

The large subunits of herpes simplex virus types 1 and 2 ribonucleotide reductases contain unique amino-terminal regions comprising 311 and 318 residues respectively, which are not found in ribonucleotide reductases from other sources. We report the mapping of the epitope recognized by monoclonal antibody 1026, which is specific for the large subunit (R1) of HSV-1, and then deduce the structural relationship of the amino-terminal region of R1 with the rest of the protein. A panel of 10 fusion proteins containing sequences spanning the entire R1 subunit were constructed. They were used together with proteolytic fragments of R1 and several synthetic peptides to show that the epitope is discontinuous and appears to be a loop structure centered on a previously located trypsin-sensitive site at residue 305. The existence of the loop was suggested by the observation that reactivity of the antibody with R1 could be blocked by peptides corresponding to residues 289 to 303 and 308 to 313 which flank the trypsin-sensitive site. Our results suggest that the unique amino-terminal region of R1 consists of a structurally distinct domain which is linked to the conserved carboxy region by an exposed loop.