Experiments done with a temperature-sensitive mutant of herpes simplex virus type 1 (HSV-1) have revealed that one of the virion glycoproteins, designated VP7(B2), is apparently not required for the production of enveloped virus particles, whereas it does play a critical role in virion infectivity. The mutant, designated HSV-l[HFEM]tsB5, fails to accumulate VP7(B2) at nonpermissive temperature and produces virions that lack detectable quantities of this glycoprotein and that have very low specific infectivity. The poor infectivity of the virions is most readily explained by failure of penetration into the host cell rather than by failure of adsorption to cells because it was shown that the VP7(B2)-deficient virions can bind to cells and that polyethylene glycol, an agent known to promote membrane fusion, can significantly enhance infectivity of the adsorbed virions.
The herpes simplex virus type 1 (HSV-1) (strain 17) DNA polymerase gene has been cloned into an Escherichia coli-yeast shuttle vector fused to the galactokinase gene (GAL-I) promoter. Genes controlled by the GAL-I promoter are induced by galactose, uninduced by raffinose, and repressed by glucose. Cell extracts from a strain of Saccharomyces cerevisiae harboring this vector (Y-MH202, expresser cells) grown in the presence of galactose and assayed in high salt (100 mM ammonium sulfate) contained a novel DNA polymerase activity. No significant high-salt DNA polymerase activity was detected in extracts from expresser cells grown in the presence of raffinose or in extracts from control cells containing the E. coli-yeast shuttle vector without the HSV-1 DNA polymerase gene grown in the presence of raffinose or galactose. Immunoblot analysis of the cell extracts by using a polyclonal rabbit antiserum prepared against a highly purified HSV-1 DNA polymerase preparation revealed the specific induction of the HSV-1-140-kilodalton DNA polymerase polypeptide in expresser cells grown in galactose. Extracts from the same cells grown in raffinose or control cells grown in either raffinose or galactose did not contain this immunoreactive polypeptide. The high-salt DNA polymerase activity in the extracts from expresser cells grown in galactose was inhibited >90% by either acyclovir triphosphate or aphidicolin, as expected for HSV-1 DNA polymerase. In addition, the high-salt polymerase enzyme activity could be depleted from extracts by immunoprecipitation by using purified immunoglobulin G from this same polyclonal rabbit antiserum. These results demonstrate the successful expression of functional HSV-1 DNA polymerase enzyme in S. cerevisiae.
The ts B5 mutant of herpes simplex virus type 1 (HSV-1) strain HFEM was shown previously to be temperature sensitive for accumulation of the mature form of glycoprotein gB, for production or activity of a factor required in virus-induced cell fusion, and for production of virions with normal levels of infectivity. In addition, a previous study showed that virions produced by ts B5 at permissive temperature were more thermolabile than HFEM virions and contained altered gB that did not assume the dimeric conformation characteristic of HFEM. Results presented here demonstrate that, at permissive temperature, ts B5 differs from HFEM in another respect: plaques formed by ts B5 are syncytial on Vero cells (but not on HEp-2 cells), whereas plaques formed by HFEM are nonsyncytial on both cell types. In addition, our results indicate that ts B5 produces an oligomeric form of gB, but that it differs in electrophoretic mobility and stability from the gB dimers of HFEM. The major purpose of this study was to investigate the dependence of the various ts B5 mutant phenotypes on the temperature sensitivity of gB accumulation and on the alterations in oligomeric conformation of gB produced at permissive temperature. For this work the following HSV-1 strains related to ts B5 or HFEM were analyzed: (i) phenotypic revertants selected from ts B5 stocks for nonsyncytial plaque morphology on Vero cells or for ability to form plaques at restrictive temperature (38.5°C); (ii) a plaque morphology variant of HFEM selected for its syncytial phenotype on Vero cells; (iii) temperature-sensitive recombinants previously isolated from a cross between ts B5 and the non-temperature-sensitive syncytial strain HSV-1(MP); and (iv) a phenotypic revertant selected from one of the recombinant stocks for its ability to form plaques at 39°C. These strains were all compared with ts B5 and HFEM at three different temperatures in two different cell lines with respect to plaque formation, yield of infectious progeny, virus-induced cell fusion, and accumulation of gB. The results of our analyses on all the strains tested revealed the following correlations between mutant phenotypes and the accumulation and oligomeric conformation of gB. (i) There was a direct and quantitative relationship between the accumulation in infected cells of infectious progeny and of the mature form of gB, providing strong support for the hypothesis that this form of gB is necessary to the production of infectious virions. The oligomeric conformation of gB characteristic of HFEM is apparently not required for virion infectivity; nor was virion thermostability necessarily related to the presence of the HFEM-like oligomeric form of gB. (ii) The previously reported correlation between temperature sensitivity of gB accumulation and virus-induced cell fusion was confirmed for ts B5 and extended to other virus strains, and coordinate reversion of these traits was also demonstrated, providing support for the hypothesis that gB has a role in virus-induced cell fusion. At 37°C, intermediate between permissive and restrictive temperatures, some of the mutants and partial revertants induced cell fusion despite reduced accumulations of the mature form of gB, suggesting that the amount of mature gB present did not determine the extent of fusion and that other forms of gB as well as other factors should be investigated with regard to the process of cell fusion. (iii) Some of the mutants and partial revertants could form plaques at 38.5°C despite reduced accumulations of gB and infectious progeny, indicating that the cell-to-cell transmission of viral infection may be at least in part independent of these factors.
The herpes simplex virus type 1 ICP4 and ICPO polypeptides are immediate-early proteins that positively and negatively regulate expression of other viral genes in trans. ICP4 has recently been shown to bind DNA bearing the consensus sequence 5'-ATCGTCNNNN(T/C)CG(A/G)C-3', present upstream of a number of viral genes. To test the hypothesis that this DNA-binding activity is involved in ICP4-mediated gene regulation, site-specific mutagenesis was employed to mutate the version of this sequence in the promoter of the ICPO gene. The mutation eliminated detectable binding of ICP4 to the promoter as measured in vitro by a gel electrophoresis band shift assay. The ability of the mutated ICPO promoter to direct synthesis of a reporter gene was also investigated in a transient transfection assay. Whereas ICP4 was found to transactivate the wild-type ICPO promoter two-to threefold, the mutated promoter was transactivated seven-to ninefold. In assays containing the ICPO transactivator gene, ICP4 down regulated the wild-type promoter far more efficiently than the mutated promoter. Finally, both the wild-type and mutated ICPO promoters exhibited a similar response to ICP4 in transfections that included a vector expressing the viral transactivator protein VP16. These experiments suggest that the sequence-specific DNA-binding activity of ICP4 is an essential element of its role as a negative regulator of gene expression.
A mutation (asparagine 815 to serine 815) was introduced into the herpes simplex virus type 1 (HSV-1) DNA polymerase (pol). The HSV-1 pol enzyme in lysates of Saccharomyces cerevisiae cells expressing the mutant protein showed increased resistance to acyclovir triphosphate and increased sensitivity to phosphonoacetate but was not substantially altered with respect to sensitivity to phosphonoformate or aphidicolin. These results directly demonstrate that both resistance to acyclovir triphosphate and sensitivity to phosphonoacetate can be conferred by this mutation in the absence of other viral factors and that the yeast expression system can be used for structure-function studies on HSV-1 pol.
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