Alterations in Glycoprotein gB Specified by Mutants and Their Partial Revertants in Herpes Simplex Virus Type 1 and Relationship to Other Mutant Phenotypes

Abstract

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.