Contribution of cysteine residues to the structure and function of herpes simplex virus gH/gL.
In HSV types 1 and 2, gH forms a noncovalent heterodimer with gL. Previous studies demonstrated that the first 323 amino acids of gH1 and the first 161 amino acids of gL1 are sufficient for gH/gL binding. For gL1, substitution of any of its four cysteine (C) residues (all located within the gH/gL binding region) destroyed gH binding and function. Although gH1 contains 8 cysteines in its ectodomain, gH 2 contains 7 (C3 of gH1 is replaced by arginine in gH2). We found that mutation of any of the four C-terminal cysteines led to a reduction or loss of gH/gL function. Mutation of C5 or C6 in gH1 or gH2 rendered the proteins non-functional. However, substitution of C7 and/or C8 in gH1 has a definite negative impact on cell-cell fusion, although these mutations had less effect on complementation. Remarkably, all four gH1 N-terminal cysteines could be mutated simultaneously with little effect on fusion or complementation. As gH2 already lacks C3, we constructed a triple mutant (gH2-C1/2/4) which exhibited a similar phenotype. Since gH1 is known to bind gL2 and vice versa, we wondered whether binding of gH2 to the heterologous gL1 would enhance the fusion defect seen with the gH2-C2 mutant. The combination of mutant gH2-C2 with wild-type gL1 was nonfunctional in a cell-cell fusion assay. Interestingly, the reciprocal was not true, as gH1-C2 could utilize both gL1 and gL2. These findings suggest that there is a structural difference in the gH2 N-terminus as compared to gH1. We also present genetic evidence for at least one disulfide bond within gH2, between cysteines 2 and 4.
Cairns
Tina M
TM
Department of Microbiology, School of Dental Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA. tmcairns@biochem.dental.upenn.edu
Landsburg
Daniel J
DJ
Whitbeck
J Charles
JC
Eisenberg
Roselyn J
RJ
Cohen
Gary H
GH
D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D002460:Cell Line; D006224:Cricetinae; D017931:DNA Primers; D004797:Enzyme-Linked Immunosorbent Assay; D005816:Genetic Complementation Test; D006801:Humans; D016133:Polymerase Chain Reaction; D014162:Transfection; D014759:Viral Envelope Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism
D017931:DNA Primers; D014759:Viral Envelope Proteins; C044200:glycoprotein H, herpes simplex virus type 1; C077407:glycoprotein L, Human herpesvirus 1