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. 2013 Nov;87(21):11332-45.
doi: 10.1128/JVI.01700-13. Epub 2013 Aug 14.

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

Doina Atanasiu  1 , Wan Ting Saw, John R Gallagher, Brian P Hannah, Zene Matsuda, J Charles Whitbeck, Gary H Cohen, Roselyn J Eisenberg
Affiliations

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

Doina Atanasiu et al. J Virol. 2013 Nov.

Abstract

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.

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Figures

Fig 1
Fig 1
Method and validation. (A) Schematic representation of the dual split-luciferase assay (DSP assay). B78 cells were transfected with the four essential glycoproteins and a plasmid that encodes the inactive N-terminal half of luciferase coupled to the N-terminal half of GFP. Nectin-1-expressing cells were transfected with the other half of the luciferase-GFP fusion construct. The major differences between the two versions are the amino acid sequence and the split point in Renilla luciferase (RL). The terms “1–7” and “8–11” refer to the names of the beta-strands of GFP included in each DSP plasmid. Fusion (content mixing) occurs upon coculture of the two cell populations, leading to the restoration of functional luciferase and intact GFP. The enzyme catalyzes the conversion of the membrane-permeant substrate EnduRen to a luminescent product. (B) Typical fusion time course using DSP plasmids. (C) To determine rates, luminescence values were normalized by considering the luminescence units at 7 h to be 100% and calculating the slope of the line between 3 and 7 h. (D) For GFP fluorescence, coverslips of target and effector cells were fixed and examined by confocal microscopy. Fusion events were calculated by multiplying the number of green syncytia by the number of nuclei per syncytium. Values were normalized by considering the 7th-hour counts to be 100%.
Fig 2
Fig 2
gB is the rate-limiting protein. (A) B78 cells were transfected with varying amounts of DNA corresponding to one viral glycoprotein, while the other three were maintained at 125 ng along with one of the DSP plasmids. Fusion was triggered with receptor-bearing target cells carrying the second DSP half as shown in Fig. 1A. Data were normalized to the rates observed for cells transfected with 125 ng of each glycoprotein (filled bars). To determine rates, luminescence values were normalized by considering the luminescence units at 7 h to be 100% and calculating the slope of the line between 3 and 7 h. (B) B78 cells were transfected the same way as for panel A, and surface expression of gB, gD, and gH/gL was measured using CELISA. Data were normalized by considering the absorbance value for cells transfected with 125 ng of each glycoprotein (filled bars) to be 100%. Data from 3 independent experiments were averaged, and the curve was considered linear when the R2 value was equal to or higher than 0.96.
Fig 3
Fig 3
The rate of fusion with the WT HSV glycoproteins is constant over a wide time frame. Two sets of plasmids (DSP or RLuc8) were used to measure fusion rates over a short and a long time course using the WT versions of each HSV glycoprotein. (A and B) The rates measured over the same period for cells transfected with DSP plasmids (A) and cells transfected with RLuc8 plasmids (B) are similar. The sensitivity is markedly enhanced with RLuc8 plasmids. (B and C and D) Raw data are shown for comparison. RLuc8 plasmids yield robust luminescence readings taken every 5 min for 2 h (C) or every minute for 30 min (D).
Fig 4
Fig 4
Functional region 1 (FR1) with fusion loops. (A) Ribbon representation of a gB protomer. The amino acid sequences of fusion loop 1 (FL1) (blue) and FL2 (cyan) are shown. The color scheme for the gB protomer is as published previously (4). The fusion loops are enlarged (box) to highlight individual amino acids. The colors of these residues correspond to the colors of the curves in panels B to E. (B) The DSP plasmids were used to measure the rates of fusion for FL1 mutants over 8 h. (C) The RLuc8 plasmids were used to measure the rates of fusion for FL1 mutants over 120 min. The same phenotypes as those for the 8-h time course are seen over 2 h. (D) The DSP plasmids were used to measure the rates of fusion for FL2 mutants over 8 h. (E) The RLuc8 plasmids were used to measure the rates of fusion for FL2 mutants over 120 min. For panels B and D, luminescence values we normalized to the 8th-hour reading for the WT. For panels C and E, luminescence values were normalized to the 2-h reading for the WT.
Fig 5
Fig 5
Functional region 3 (FR3) and low-rate-of-fusion mutations. (A) (Left) Ribbon structure of gB with hypothetical membrane. The site of the LL871/872AA mutation in the cytoplasmic tail is shown in a cartoon representation. (Right) Enlarged gB crown with color-coded residues. (B) Ribbon representation of a gB crown protomer showing residues altered in low-rate-of-fusion mutants and a low-rate-of-virus-entry mutant. (C) Expression of low-rate-of-fusion mutants on the cell surface. Data are normalized to WT levels. (D and E) All mutants show slower kinetics than the WT in either the long (D) or the short (E) time course.
Fig 6
Fig 6
FR3 and high-rate-of-entry mutants. (A) Ribbon structure of the gB crown. Residues altered in fast-entry mutants are depicted as spheres. (B) All mutants were expressed at or near WT levels. (C and D) The kinetics of fusion were either WT (C) or enhanced over WT kinetics (D) and were maintained over a long period. (E) Both hyperfusogenic mutants exhibited a fast phenotype right from the initial stages of fusion.
Fig 7
Fig 7
A mutant with alterations in the cytoplasmic tail of gB exhibits a superhigh initial fusion rate. (A) Surface expression of the LL871/872AA cytoplasmic tail mutant. (B and C) Kinetics of fusion of the LL871/872AA mutant over the first 20 min (B) or over 8 h (C) in comparison to those of WT gB and gB H657R.
Fig 8
Fig 8
Proposed model of prefusion gB. The prefusion gB structure was generated using the MatchMaker program (UCSF Chimera) by aligning structural domains of postfusion HSV gB to the known structural domains of the prefusion form of VSV G. H657 (black spheres) is shown. This residue is located in the center of the prefusion model (front view), covered by FR2 (green), and on the outside of each protomer in the postfusion form. The relationships of H657 with other residues in the crown (brown) and stalk (yellow) change as gB transitions from a pre- to a postfusion structure; this is more evident in the top views, where only domains III (yellow) and IV (brown) are shown.
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References

    1. Eisenberg RJ, Atanasiu D, Cairns TM, Gallagher JR, Krummenacher C, Cohen GH. 2012. Herpes virus fusion and entry: a story with many characters. Viruses 4:800–832 - PMC - PubMed
    1. Spear PG, Eisenberg RJ, Cohen GH. 2000. Three classes of cell surface receptors for alphaherpesvirus entry. Virology 275:1–8 - PubMed
    1. Backovic M, Longnecker R, Jardetzky TS. 2009. Structure of a trimeric variant of the Epstein-Barr virus glycoprotein B. Proc. Natl. Acad. Sci. U. S. A. 106:2880–2885 - PMC - PubMed
    1. Heldwein EE, Lou H, Bender FC, Cohen GH, Eisenberg RJ, Harrison SC. 2006. Crystal structure of glycoprotein B from herpes simplex virus 1. Science 313:217–220 - PubMed
    1. Kadlec J, Loureiro S, Abrescia NG, Stuart DI, Jones IM. 2008. The postfusion structure of baculovirus gp64 supports a unified view of viral fusion machines. Nat. Struct. Mol. Biol. 15:1024–1030 - PubMed

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