Dual split protein-based fusion assay reveals that mutations to herpes simplex virus (HSV) glycoprotein gB alter the kinetics of cell-cell fusion induced by HSV entry glycoproteins.
Herpes simplex virus (HSV) entry and cell-cell fusion require glycoproteins gD, gH/gL, and gB. We propose that receptor-activated changes to gD cause it to activate gH/gL, which then triggers gB into an active form. We employed a dual split-protein (DSP) assay to monitor the kinetics of HSV glycoprotein-induced cell-cell fusion. This assay measures content mixing between two cells, i.e., fusion, within the same cell population in real time (minutes to hours). Titration experiments suggest that both gD and gH/gL act in a catalytic fashion to trigger gB. In fact, fusion rates are governed by the amount of gB on the cell surface. We then used the DSP assay to focus on mutants in two functional regions (FRs) of gB, FR1 and FR3. FR1 contains the fusion loops (FL1 and FL2), and FR3 encompasses the crown at the trimer top. All FL mutants initiated fusion very slowly, if at all. However, the fusion rates caused by some FL2 mutants increased over time, so that total fusion by 8 h looked much like that of the WT. Two distinct kinetic patterns, "slow and fast," emerged for mutants in the crown of gB (FR3), again showing differences in initiation and ongoing fusion. Of note are the fusion kinetics of the gB syn mutant (LL871/872AA). Although this mutant was originally included as an ongoing high-rate-of-fusion control, its initiation of fusion is so rapid that it appears to be on a "hair trigger." Thus, the DSP assay affords a unique way to examine the dynamics of HSV glycoprotein-induced cell fusion.
Department of Microbiology, School of Dental Medicine.
Doina
D
Atanasiu
Wan Ting
WT
Saw
John R
JR
Gallagher
Brian P
BP
Hannah
Zene
Z
Matsuda
J Charles
JC
Whitbeck
Gary H
GH
Cohen
Roselyn J
RJ
Eisenberg
D000818:Animals; D002459:Cell Fusion; D045744:Cell Line, Tumor; D004252:DNA Mutational Analysis; D051379:Mice; D050505:Mutant Proteins / Q000235:genetics / Q000378:metabolism; D014759:Viral Envelope Proteins / Q000235:genetics* / Q000378:metabolism*
D050505:Mutant Proteins; D014759:Viral Envelope Proteins; C562160:glycoprotein B, human herpesvirus 1