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Palmitoylation Strengthens Cholesterol-dependent Multimerization and Fusion Activity of Human Cytomegalovirus Glycoprotein B (gB).

ABSTRACT: Herpesviruses are a large order of animal enveloped viruses displaying a virion fusion mechanism of unusual complexity. Their multipartite machinery has a conserved core made of the gH/gL ancillary complexes and the homo-trimeric fusion protein glycoprotein B (gB). Despite its essential role in starting the viral infection, gB interaction with membrane lipids is still poorly understood. Here, evidence is provided demonstrating that human cytomegalovirus (HCMV) gB depends on the S-palmitoylation of its endodomain for an efficient interaction with cholesterol-rich membrane patches. We found that, unique among herpesviral gB proteins, the HCMV fusion factor has a Cys residue in the C-terminal region that is palmitoylated and mediates methyl-?-cyclodextrin-sensitive self-association of purified gB. A cholesterol-dependent virus-like particle trap assay, based on co-expression of the HIV Gag protein, confirmed that this post-translational modification is functional in the context of cellular membranes. Mutation of the palmitoylated Cys residue to Ala or inhibition of protein palmitoylation decreased HCMV gB export via Gag particles. Moreover, purified gBC777A showed an increased kinetic sensitivity in a cholesterol depletion test, demonstrating that palmitoyl-gB limits outward cholesterol diffusion. Finally, gB palmitoylation was required for full fusogenic activity in human epithelial cells. Altogether, these results uncover the palmitoylation of HCMV gB and its role in gB multimerization and activity.

SUBMITTER: Patrone M

PROVIDER: S-EPMC4813493 | biostudies-literature | 2016 Feb

REPOSITORIES: biostudies-literature

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Palmitoylation Strengthens Cholesterol-dependent Multimerization and Fusion Activity of Human Cytomegalovirus Glycoprotein B (gB).

Patrone Marco M Coroadinha Ana Sofia AS Teixeira Ana P AP Alves Paula M PM

The Journal of biological chemistry 20151222 9

Herpesviruses are a large order of animal enveloped viruses displaying a virion fusion mechanism of unusual complexity. Their multipartite machinery has a conserved core made of the gH/gL ancillary complexes and the homo-trimeric fusion protein glycoprotein B (gB). Despite its essential role in starting the viral infection, gB interaction with membrane lipids is still poorly understood. Here, evidence is provided demonstrating that human cytomegalovirus (HCMV) gB depends on the S-palmitoylation ...[more]

PMID: 26694613

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Project description: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.

Project description:Human cytomegalovirus (HCMV) enters host by glycoprotein B (gB)-mediated membrane fusion upon receptor-binding to gH/gL-related complexes, causing devastating diseases such as birth defects. Although an X-ray crystal structure of the recombinant gB ectodomain at postfusion conformation is available, the structures of prefusion gB and its complex with gH/gL on the viral envelope remain elusive. Here, we demonstrate the utility of cryo electron tomography (cryoET) with energy filtering and the cutting-edge technologies of Volta phase plate (VPP) and direct electron-counting detection to capture metastable prefusion viral fusion proteins and report the structures of glycoproteins in the native environment of HCMV virions. We established the validity of our approach by obtaining cryoET in situ structures of the vesicular stomatitis virus (VSV) glycoprotein G trimer (171 kD) in prefusion and postfusion conformations, which agree with the known crystal structures of purified G trimers in both conformations. The excellent contrast afforded by these technologies has enabled us to identify gB trimers (303kD) in two distinct conformations in HCMV tomograms and obtain their in situ structures at up to 21 Å resolution through subtomographic averaging. The predominant conformation (79%), which we designate as gB prefusion conformation, fashions a globular endodomain and a Christmas tree-shaped ectodomain, while the minority conformation (21%) has a columnar tree-shaped ectodomain that matches the crystal structure of the "postfusion" gB ectodomain. We also observed prefusion gB in complex with an "L"-shaped density attributed to the gH/gL complex. Integration of these structures of HCMV glycoproteins in multiple functional states and oligomeric forms with existing biochemical data and domain organization of other class III viral fusion proteins suggests that gH/gL receptor-binding triggers conformational changes of gB endodomain, which in turn triggers two essential steps to actuate virus-cell membrane fusion: exposure of gB fusion loops and unfurling of gB ectodomain.

Dataset Information

Palmitoylation Strengthens Cholesterol-dependent Multimerization and Fusion Activity of Human Cytomegalovirus Glycoprotein B (gB).

Publications

Palmitoylation Strengthens Cholesterol-dependent Multimerization and Fusion Activity of Human Cytomegalovirus Glycoprotein B (gB).

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