Biomedical subjects
Publications and source records attributed to N D Stow.
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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 herpes simplex virus type 1 origin-binding protein is encoded by gene UL9. We previously described a plasmid, pB1, which encodes a fusion protein containing only the C-terminal 317 amino acids of the UL9 polypeptide and showed that this product retains sequence-specific DNA-binding ability. Two series of pB1 mutants have now been constructed and the polypeptides were tested for origin-binding activity. Using C-terminal truncations, we show that the C-terminal 34 amino acids of UL9 are dispensable for binding and that essential residues lie between positions 801 and 818. Analysis of a series of mutants containing insertions of four amino acids at various positions identified regions of the DNA-binding domain in which alterations either abolished or had relatively little effect upon binding activity. Two mutants which were intermediate in their binding activities also exhibited temperature- or sequence-specific effects.
The origin binding protein (OBP) of herpes simplex virus type 1 is required to activate a viral origin of replication in vivo. We have used intact OBP as well as a truncated form of the protein expressed in Escherichia coli to investigate the protein-protein interactions, as well as the protein-DNA interactions involved in the formation of a nucleoprotein complex at a viral origin of replication (oriS) in vitro. The salient findings demonstrate that the N-terminal part of OBP is required for the cooperative binding of OBP to three sites (boxes I, II, and III) within oriS. A detailed model for the interaction of OBP with the viral origins of replication oriS and oriL is presented.
Cells infected with vaccinia virus strain Lister secrete a polypeptide of approximate molecular weight 35,000 (35K) into the medium. Previous studies identified a cleavable, hydrophobic region of 17 amino acids in the 35K protein which could potentially function as a signal peptide to target the protein to the secretory pathway. Here we report the use of the expression-secretion signals derived from the 35K gene to direct export and secretion of a foreign protein. Vaccinia virus recombinants carrying the bacterial chloramphenicol acetyl transferase gene (cat) immediately downstream from the promoter and the N-terminal coding sequences of the 35K gene were constructed. Our studies show that the N-terminal 22 or 42 amino acids of the 35K protein direct efficient secretion of the CAT protein. However, due to a cryptic glycosylation site within CAT, glycosylated protein was secreted, which reduced enzymatic activity. Activity was restored in the presence of tunicamycin. Removal of the glycosylation site by site-directed mutagenesis abolished glycosylation with no effect on secretion, although CAT activity was again reduced, possibly due to an effect on the active site. The results presented here demonstrate the feasibility of using the promoter and the signal sequence of the 35K gene to generate recombinant viruses for overexpression and secretion of foreign proteins.
The minimal set of seven herpes simplex virus type 1 (HSV-1) genes required for viral origin-dependent DNA synthesis was previously identified using a transient replication assay in a mammalian cell line permissive for HSV-1 growth. We have constructed recombinant baculoviruses which efficiently express the products of each of these seven genes in infected Spodoptera frugiperda (Sf) insect cells. When Sf cells were transfected with a plasmid containing a functional HSV-1 origin of replication, and subsequently superinfected with a mixture of these seven viruses, the input plasmid was amplified. This amplification exhibited properties characteristic of genuine HSV-1 DNA replication: all seven HSV-1 replication gene products were required, replicated DNA was detected as concatemers, and mutated origins were impaired to similar extents in insect cells and cells permissive for HSV-1 replication. These results demonstrate that the HSV-1 proteins expressed in Sf cells are fully competent for viral DNA synthesis, and indicate that any host function essential in mammalian cells must also be present in the infected insect cells. This system also provides a convenient method by which mutated replication proteins can be screened for function and produced in amounts sufficient for biochemical studies. Using this approach we show that the ability of the UL9 protein to bind to the viral origins of replication is not sufficient for it to facilitate DNA synthesis.
We constructed recombinant viruses based on the herpes simplex virus type 1 mutant tsK which individually were able to express the products of four viral DNA replication genes (UL5, UL8, UL9 and UL52) in the absence of any of the other proteins required for viral DNA synthesis. These viruses were used in immunofluorescence experiments to investigate the cellular localization of the four replication proteins expressed. The results demonstrated that all three components of the viral helicase-primase complex (UL5, UL8 and UL52 proteins) must be co-expressed to allow their efficient localization to the nucleus. Since the UL5 and UL52 proteins together form a complex which is enzymatically indistinguishable from a complex formed from all three proteins, a possible role of the UL8 protein may be in facilitating nuclear uptake. The UL9 protein (origin-binding protein) efficiently entered the cell nucleus when expressed alone. Both UL9 protein and the tripartite helicase-primase complex exhibited patterns of fluorescence which resembled the 'pre-replicative sites' described previously.
The herpes simplex virus type 1 helicase-primase complex consists of the products of the UL5, UL8 and UL52 genes. We have expressed these proteins in insect cells using baculovirus vectors and studied the requirements for enzymatic activities associated with the DNA unwinding function of the complex. In agreement with a recent report (Dodson, M.S., Crute, J.J., Bruckner, R.C. and Lehman, I.R. 1989, J. Biol. Chem. 264, 20835-20838) we find that DNA-dependent ATPase and DNA helicase activities are assembled in vivo in insect cells triply infected with viruses expressing the UL5, UL8 and UL52 proteins. Moreover, these activities were also detected in cells in which only the UL5 and UL52 products were expressed indicating that the presence of the UL8 protein is essential for neither the ATPase nor helicase activity of the complex.
The C-terminal 322 amino acids of the varicella-zoster virus (VZV) gene 51 product were expressed in Escherichia coli and shown to bind to specific DNA sequences within the VZV origin of DNA replication. The gene 51 product and its herpes simplex virus type 1 homolog (the UL9 protein) are capable of recognizing identical DNA sequences but the arrangement of binding sites within the origin regions of the two viruses differs. Three binding sites for the VZV gene 51 protein were identified within the VZV origin region and these lie in the same orientation and on the same side of the origin palindromic DNA sequence. DNA replication assays in transfected tissue culture cells demonstrated that the site closest to the palindrome is essential for origin activity whereas the most distal site is dispensable. The middle binding site may play an auxiliary role in DNA synthesis.
Two sites within the short region origin of DNA replication (oriS) in herpes simplex virus type 1 (HSV-1) which bind the product of the UL9 gene have previously been identified. One of these sites (site I) contains an 11 bp sequence which is also present in oriS of varicella-zoster virus, and the other (site II) includes a related element differing in two positions. A third sequence (motif III), which lies close to binding site I, differs from the site I element at only a single position. We have deleted specifically each of these three 11 bp sequences from within functional copies of HSV-1 oriS and have examined the effects on origin activity and binding of the UL9 protein. Gel retardation analyses confirmed the important roles of the regions deleted from sites I and II in interacting with the UL9 protein. In transient replication assays, copies of oriS lacking the site I or II elements exhibited undetectable or residual (4 to 8%) activity respectively. The UL9 protein did not bind to motif III even in the absence of site I sequences, although removal of the motif III sequence caused a small reduction in oriS activity. A single base change which converted the sequence within binding site I to that of motif III was sufficient to abolish both the interaction of the UL9 gene product at this locus and the replicative ability of oriS. Therefore, interaction of the UL9 protein with binding site I is essential for origin activity, but the presence of binding site II is also required for efficient replication.
Infection of tissue culture cells with vaccinia virus results in the specific secretion of several polypeptides into the medium. Previous studies identified a protein of approximate Mr 35,000 (35K) which was secreted in large amounts at both early and late times after infection with the Evans strain. We now show that a related protein is secreted by the Lister strain but not by WR, Wyeth nor Tian Tan. The gene encoding the Lister strain 35K protein was mapped within the inverted terminal repeats of the genome. The DNA sequence of this region showed that the ends of this gene are very similar to previously published sequences flanking a gene of WR which encodes a protein of approximate Mr 7,500 (7.5K). Our results suggest that the 7.5K polypeptide of WR may have arisen as a result of a deletion event and is a truncated form of the 35K Lister protein. Site-directed mutagenesis demonstrated that the 35K secreted protein encoded by Lister is not essential for growth in tissue culture.
The binding of a herpes simplex virus type 1 (HSV-1) encoded polypeptide to a viral origin of DNA replication has been studied by using a gel retardation assay. Incubation of nuclear extract from HSV-1 infected cells with a labelled origin-containing fragment resulted in the formation of a specific retarded complex, the migration of which was further reduced in the presence of an antibody reactive with the UL9 gene product. Introduction of an additional copy of the UL9 gene, under the control of an immediate early (IE) promoter, conferred the ability to express origin binding activity at the non-permissive temperature upon an HSV-1 ts mutant blocked at the IE stage of infection. Endogenous or exogenous proteolytic activity revealed the presence of a relatively protease-resistant domain which retained sequence-specific DNA binding activity. The C-terminal 317 amino acids of the UL9 gene expressed as a fusion protein in Escherichia coli also bound to the origin. Our results demonstrate that the UL9 gene product binds to a viral origin and that sequence specific recognition and binding are specified by the C-terminal 37% of the polypeptide.
The herpes simplex virus type 1 (HSV-1) mutant dl1403 contains a 2 kb deletion within the sequences encoding the immediate early polypeptide Vmw110. Previous experiments showed that although dl1403 exhibits normal patterns of gene expression following infection at an m.o.i. of 5 p.f.u./cell its growth and plaquing efficiency are impaired in low multiplicity infections, particularly in human foetal lung (HFL) cells. We have now investigated the ability of two other human herpesviruses, varicella-zoster virus (VZV) and human cytomegalovirus (HCMV), to compensate for this defect at low m.o.i. in HFL cells. Co-infection with HCMV resulted in greatly increased plaque numbers and the apparent particle/p.f.u. ratios of dl1403 stocks were reduced to values similar to those exhibited by wild-type HSV-1 stocks. Complementation of dl1403 in low multiplicity infections by HCMV and VZV was also demonstrated by an increased yield of the mutant virus and an increase in synthesis of dl1403 DNA. Ultraviolet irradiation of HCMV abolished its ability to complement dl1403 and the presence of adenovirus 5 had no stimulatory effect on dl1403 DNA replication. When HFL monolayers were infected with dilutions of dl1403 stocks such that no plaques were produced, replication of the mutant virus could be induced by superinfection with HCMV 7 days after the initial infection. These results indicate that a non-lytic interaction between dl1403 and HFL cells is a more likely consequence of a low multiplicity infection than plaque formation.
We have investigated the behaviour in mice of the herpes simplex virus type 1 (HSV-1) mutant dl1403, which contains a deletion within the gene encoding the immediate early polypeptide Vmw110. The deletion was responsible for a reduction in virulence assayed by both the intracranial and footpad routes of inoculation. Following injection into the footpad, dl1403 was able to reach the spinal cord and establish a latent infection in sensory ganglia from which virus spontaneously reactivated upon explanation. The Vmw110 polypeptide is therefore dispensable for the establishment and maintenance of latency and for reactivation from the latent state.
Evidence was recently presented that herpes simplex virus type 1 (HSV-1) immunoglobulin G (IgG) Fc receptors are composed of a complex containing a previously described glycoprotein, gE, and a novel virus-induced polypeptide, provisionally named g70 (D. C. Johnson and V. Feenstra, J. Virol. 61:2208-2216, 1987). Using a monoclonal antibody designated 3104, which recognizes g70, in conjunction with antipeptide sera and virus mutants unable to express g70 or gE, we have mapped the gene encoding g70 to the US7 open reading frame of HSV-1 adjacent to the gE gene. Therefore, g70 appears to be identical to a recently described polypeptide which was named gI (R. Longnecker, S. Chatterjee, R. J. Whitley, and B. Roizman, Proc. Natl. Acad. Sci. USA 84:147-151, 1987). Under mildly denaturing conditions, monoclonal antibody 3104 precipitated both gI and gE from extracts of HSV-1-infected cells. In addition, rabbit IgG precipitated the gE-gI complex from extracts of cells transfected with a fragment of HSV-1 DNA containing the gI, gE, and US9 genes. Cells infected with mutant viruses which were unable to express gE or gI did not bind radiolabeled IgG; however, cells coinfected with two viruses, one unable to express gE and the other unable to express gI, bound levels of IgG approaching those observed with wild-type viruses. These results further support the hypothesis that gE and gI form a complex which binds IgG by the Fc domain and that neither polypeptide alone can bind IgG.
The properties of temperature-sensitive (ts), insertion or deletion mutants of herpes simplex virus (HSV) were investigated in an in vitro model system for latency. The studies defined virus gene products required for establishment of latency and for reactivation of latent virus. All mutants tested established latency in human foetal lung fibroblasts and could be reactivated by intertypic superinfection with HSV or with human cytomegalovirus. Two mutants of HSV type 1 used in these studies, tsK and in1411, failed to synthesize active immediate early (IE) polypeptide Vmw175 and were blocked at a very early stage of the virus replication cycle, showing that, at most, only limited gene expression is necessary for the establishment of latency. Mutant dl1403, which lacks the gene encoding IE polypeptide Vmw110, established latency as efficiently as wild-type HSV. Latent HSV type 2 was reactivated by superinfection with tsK or in1411 but not with dl1403, suggesting that polypeptide Vmw110, which is known to regulate gene expression by trans-activation, is required for reactivation in the in vitro system.
Induction of the major stress response in chick embryo fibroblasts, which follows infection at 38.5 degrees C with the herpes simplex virus mutant tsK, was investigated. Synthesis of cellular stress proteins occurred only when the mutant form of an immediate early polypeptide, Vmw175, was overproduced. Infection with mutant in 1411, which has an amber (TAG) termination signal inserted between codons 83 and 84 of the gene encoding Vmw175 and therefore specifies a truncated portion of the polypeptide, failed to stimulate stress protein synthesis. The results suggested that the presence of abnormal forms of Vmw175 at high concentrations was the signal for induction of the stress response in tsK-infected cells.
Transfection experiments with plasmids containing immediate early (IE) genes of herpes simplex virus type 1 (HSV-1) have previously demonstrated a role for the IE polypeptide Vmw110 (ICP0) in stimulating expression from plasmid-encoded early gene promoters. To gain further insights into the function of Vmw110 we isolated a deletion mutant specifying a truncated form of the polypeptide which had been shown to be inactive in transfection assays. This mutant, dl1403, contained a 2 kb deletion within both the TRL and IRL copies of the Vmw110 gene, and encoded a polypeptide consisting of the original N-terminal 105 amino acids followed by 56 amino acids specified by a reading frame not used by Vmw110. dl1403 was able to replicate and produce plaques on baby hamster kidney (BHK) cells but the yield of infectious virus was 20- to 100-fold lower than obtained with wild-type HSV-1. Surprisingly, comparison of polypeptide synthesis, DNA replication and DNA encapsidation in cells infected with 5 p.f.u./cell dl1403 or wild-type HSV-1 revealed no significant differences. In addition similar numbers of particles were produced in cells infected with the two viruses, resulting in stocks of dl1403 exhibiting significantly higher particle/p.f.u. ratios. The efficiency of plaquing of dl1403 was greatly reduced in Vero and human foetal lung cells compared with BHK cells, but following infection with 5 p.f.u./cell similar yields of infectious virus were obtained from all three cell lines. Marker rescue experiments verified that the reduced yield of dl1403 in BHK cells was a consequence of the deletion within the Vmw110 gene. The results suggest that the effect of this deletion is manifest primarily at low multiplicities of infection and can be largely overcome by increasing the virus dose.