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. 2013 Jun 4;4(3):e00332-13.
doi: 10.1128/mBio.00332-13.

Human cytomegalovirus (HCMV) glycoprotein gB promotes virus entry in trans acting as the viral fusion protein rather than as a receptor-binding protein

Paul T Wille  1 , Todd W Wisner, Brent Ryckman, David C Johnson
Affiliations

Human cytomegalovirus (HCMV) glycoprotein gB promotes virus entry in trans acting as the viral fusion protein rather than as a receptor-binding protein

Paul T Wille et al. mBio. .

Abstract

ABSTRACT Human cytomegalovirus (HCMV) glycoproteins gB and gH/gL are both necessary and sufficient for cell-cell fusion. However, it is not clear what roles these glycoproteins play in virus entry, whether acting directly in membrane fusion or in binding receptors. With other herpesviruses, it appears that gB is the fusion protein and is triggered by gH/gL, which, in some cases, binds receptors. However, for HCMV, there is published evidence that gB binds cellular ligands necessary to promote virus entry into or signaling of cells. Most mechanistic information on herpesvirus fusion proteins involves cell-cell fusion assays, which do not allow a determination of whether gB or gH/gL in the virion envelope must be oriented toward cellular membranes that contain receptors. Here, we showed that HCMV virions lacking gB were unable to enter normal cells but entered cells that expressed gB. Analyses of gB mutants lacking the cytoplasmic domain or with substitutions in putative "fusion loops" provided evidence that gB fusion activity was required for this "entry in trans." In gB-mediated entry in trans, gB is oriented toward the virion envelope that apparently lacks receptors, arguing against an essential role for gB in binding receptors or signaling molecules. In contrast, particles lacking gH/gL did not enter cells expressing gH/gL, apparently because gH/gL must be oriented toward cellular membranes (which have receptors). Coupled with our previous interference studies, in which gH/gL expressed in cells blocked HCMV entry, our findings here support the hypothesis that HCMV gH/gL binds cellular receptors before triggering gB, which acts as the fusion protein. IMPORTANCE Human cytomegalovirus (HCMV) produces major disease in neonates and immunosuppressed transplant patients. As with other herpesviruses, HCMV requires two membrane glycoproteins, gB and gH/gL, to enter host cells. However, it has not been clear how gB and gH/gL function in two steps of the HCMV entry pathway, i.e., (i) binding of cellular receptors and (ii) fusion of the virion envelope with cellular membranes. There are studies that suggest that HCMV gB is required for receptor binding and other studies suggesting that gH/gL is the receptor binding protein and gB is the fusion protein. Here, we show that HCMV virions lacking gB can enter cells that express gB in cellular membranes. In contrast, virus particles lacking gH/gL could not enter cells expressing gH/gL. Our study supports the hypothesis that gB is the fusion protein and gH/gL acts upstream of gB to bind receptors and then activate gB for fusion.

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Figures

FIG 1
FIG 1
Analyses of HCMV mutants lacking gB or gH. (A to H) Normal fibroblasts (NHDF) or fibroblasts transduced with retroviruses expressing gB (NHDF+gB) or gH (NHDF+gH) were infected with wild-type HCMV strain AD169 or TR or with HCMVΔgB or HCMVΔgH (mutant viruses derived from complementing cells). After 10 days, the cells were fixed, permeabilized, and stained for HCMV IE86. (I) Similar quantities of cell culture supernatants derived from HCMV-infected NHDF, NHDF+gB, or NHDF+gH were subjected to qPCR analyses for HCMV DNA and compared to virus genomes in HCMV BAC. (J and K) Similar quantities of virus particles were subjected to immunoblotting to detect gB, gH, the major capsid protein (MCP), or pp65.
FIG 2
FIG 2
NHDF were infected with preparations of HCMVΔgB (A and B) or HCMVΔgH (C and D) derived from NHDF (lacking gB or gH) by incubating virus with cells and then immediately centrifuging the cells at 800 × g for 30 min at 10°C. Other cells were incubated with virus, centrifuged, treated with 44% polyethylene glycol (PEG) for 30 s at 37°C, and then extensively washed. The cells were incubated for 24 h at 37°C and then stained for IE86. Numbers indicate the average number of IE86-positive cells derived from triplicate wells.
FIG 3
FIG 3
Entry in trans of HCMV particles. NHDF (fibroblasts) (A to G) or ARPE-19 epithelial cells (H to M) were transduced with nonreplicating Ad vectors to express GFP (A, B, H, and I), HCMV gB (C, D, J, and K), HCMV gH and gL (E, F, L, and M), or HSV gB (G) for 24 h. The cells were then incubated with HCMVΔgB particles lacking gB or HCMVΔgH particles lacking gH (in both cases particles derived from NHDF) and immediately centrifuged at 800 × g for 30 min at 10°C and then incubated for 4 h at 37°C. Virus was removed, and cells were incubated for an additional 20 h before being stained for HCMV IE86. Assays were done in triplicate, and the average percentages of IE86-positive cells are indicated.
FIG 4
FIG 4
Mutant forms of HCMV gB that cannot cause fusion do not promote entry in trans. NHDF were transduced for 48 h with Ad vectors that express the following wild-type and mutant forms of gB: (A) wild-type AD169 gB, sgB, and gBΔCT; (B) L241R and 882stop mutant gB; or (C) wild-type TR gB or Y157S, A154D, A154W, W240A, YIY157/GHR, or GSTW240/AAAA mutant gB, or no gB (same multiplicity of infection of AdtetTrans). The cells were then incubated with HCMVΔgB particles lacking gB, centrifuged at 800 × g for 30 min, and incubated for 4 additional hours with virus, and then the virus was removed and the cells were incubated for 20 h before being stained for HCMV IE86. Results of cell surface expression assays (Fig. S1) and cell-cell fusion assays (Fig. S2) are summarized.
FIG 5
FIG 5
Cartoon depicting HCMV particles lacking gB or gH/gL interacting with cells expressing these glycoproteins. (A) An HCMV particle lacking gB but containing gH/gL in the virion envelope can interact with cell surface receptors (green) and then activate gB (lightning bolt) that then causes fusion of the virion envelope with cell surface membranes. (B) An HCMV virion containing gB but lacking gH/gL fails to enter cells because gH/gL is oriented toward the virion envelope, which lacks gH/gL receptors, so that gH/gL does not trigger gB for fusion.
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