An analysis of protein-protein interactions of herpes simplex virus glycoproteins involved in viral entry

Herpes simplex virus (HSV) envelope glycoproteins gB, gC, gE/gI and gH/gL are found in oligomeric states. Glycoproteins gB, gC, gD, gH and gL have been implicated in virus entry. In this project, I sought to explore the organization of glycoproteins in the virion envelope, and examine glycoprotein associations during viral entry. Using purified glycoproteins and virions, I quantitated glycoprotein amounts, and used chemical crosslinkers to detect oligomers. Glycoproteins in purified virus preparations were detected with polyclonal antibodies, and molar ratios of 1: 2: 11: 16 and 1: 1: 14: 9 were calculated for gB: gC: gD: gH in two strains. gL was detected, but could not be quantitated. To identify complexes of these glycoproteins, HSV-1(KOS) was incubated with crosslinkers of various sizes, solubilized, and examined by Western blotting. Bis(sulfosuccinimidyl) suberate, an 11.4 A crosslinker, gave optimal results, detecting homodimers of gB, gC and gD. Immunoprecipitation of extracts and Western blotting with heterologous antibodies identified oligomers containing gB-gC, gC-gD, gD-gB, gC-gL, gD-gL and gH-gL, suggesting that these glycoproteins are capable of forming associations. To examine the roles of oligomers in entry, I modified the standard HSV penetration assay, including crosslinkers to examine changes in glycoprotein associations. Virus was adsorbed at 4$\sp\circ$C to human neuroblastoma cells (SY5Y), the temperature was raised to 37$\sp\circ$C, crosslinker was added at varying times, and cytoplasmic extracts were examined for oligomers. Again, the length and concentration of the crosslinker affected the complexes detected. The same glycoprotein patterns found in purified virus were also present after attachment of virus to cell. The ability to crosslink HSV glycoproteins changed as penetration proceeded; gB, gD and gH-gL complexes formed at zero time disappeared with increasing time, paralleling the kinetics of penetration. This phenomenon was selective, since it was not observed with gC. Finally, I examined the crosslinking of null viruses K082 and KOSgD$\beta$, and found that these mutants, which cannot penetrate, did not demonstrate any change in glycoprotein crosslinking. These results suggest associations of gB, gD and gH-gL may be functionally important in entry