CROSS-LINKING OF GLYCOPROTEIN OLIGOMERS DURING HERPES-SIMPLEX VIRUS TYPE-1 ENTRY

Herpes simplex virus (HSV) has 10 glycoproteins in its envelope. Glyco protein B (gB), gC, go, gH, and gL have been implicated in virus entry . We previously used chemical cross-linking to show that these five gl ycoproteins were close enough to each other to be cross-linked into ho modimeric and hetero-oligomeric forms; hetero-oligomers of gB-gC, gC-g D, gD-gB, gH-gL, gC-gL, and gD-gL were found in purified virions. To b etter understand the roles of these glycoproteins in viral entry, we h ave modified a standard HSV penetration assay to include cross-linkers . This allowed us to examine changes in associations of viral glycopro teins during the entry process. HSV-1(KOS) was adsorbed at 4 degrees C to human neuroblastoma cells (SY5Y). The temperature was raised to 37 degrees C and cells were treated,vith cross-linker at various times a fter the temperature shift. Cytoplasmic extracts were examined by West ern blotting (immunoblotting) for viral glycoproteins. We found that ( i) as in virus alone, the length and concentration of the cross-linkin g agent affected the number of specific complexes isolated; (ii) the s ame glycoprotein patterns found in purified virions were also present after attachment of virions to cells; and (iii) the ability to cross-l ink HSV glycoproteins changed as virus penetration proceeded, e.g., gB and go complexes which were present during attachment disappeared wit h increasing time, and their disappearance paralleled the kinetics of penetration. However, this phenomenon appeared to be selective since i t was not observed with gC oligomers. In addition, we examined the cro ss-linking patterns of gB and go in null viruses K082 and KOSgD beta. Neither of these mutants, which attach but cannot penetrate, showed ch anges in glycoprotein cross-linking over time. We speculate that these changes are due to conformational changes which preclude cross-linkin g or spatial alterations which dissociate the glycoprotein interaction s during the penetration events.