An α-Helical Domain within the Carboxyl Terminus of Herpes Simplex Virus Type 1 (HSV-1) glycoprotein B (gB) is Associated with Cell Fusion and Resistance to Heparin Inhibition of Cell Fusion

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Virus-induced membrane fusion can be subdivided into three phases defined by studies of class I and class II fusion proteins. During Phase I, two membranes are brought into close apposition. Phase II marks the mixing of the outer membrane leaflets leading to formation of a hemifusion intermediate. A fusion pore stably forms and expands in Phase III, thereby completing the fusion process. Herpes simplex virus type 1 (HSV-1) requires four glycoproteins to complete membrane fusion, but none has been defined as class I or II. Therefore, we investigated whether HSV-1-induced membrane fusion occurred following the same general phases as those described for class I and II proteins. In this study we demonstrate that glycoprotein D (gD) and the glycoprotein H and glycoprotein L complex (gHL) mediated lipid mixing indicative of hemifusion. However, content mixing and full fusion required glycoprotein B (gB) to be present along with gD and gHL. Our results indicate that, like class I and II fusion proteins, fusion mediated by HSV-1 glycoproteins occurred through a hemifusion intermediate. In addition, both gB and gHL are probably directly involved in the fusion process. From this, we propose a sequential model for fusion via HSV-1 glycoproteins whereby gD is required for Phase I, gHL is required for Phase II, and gB is required for Phase III. We further propose that glycoprotein H and gB are likely to function sequentially to promote membrane fusion in other herpesviruses such as Epstein-Barr virus and human herpesvirus 8.lipid transfer ͉ membrane fusion ͉ fluorescence microscopy S tudies using class I and class II fusion proteins have demonstrated that virus-induced membrane fusion can be subdivided into three phases (1, 2). Phase I involves bringing opposing membranes into close proximity through a viral glycoprotein binding a cellular receptor. Phase II involves the initiation of lipid mixing between the two apposed membranes and is completed when the outer membrane leaflets are mixed to form an intermediate called hemifusion. Phase III begins when the inner membrane leaflets are mixed and continues the pore formation and expansion. The completion of Phase III signifies the completion of the fusion process. The three phases of membrane fusion (close apposition, hemifusion, and complete fusion) are useful to characterize functions of viral glycoproteins in the fusion process (1, 3, 4). Fusion proteins that have Phase I function bring membranes in close apposition and ultimately result in the initiation of the fusion process. Fusion proteins that have Phase II function are capable of mixing outer membrane leaflets leading to hemifusion. Phase III fusion proteins are capable of forming and expanding a fusion pore. For many viruses, one or two fusion proteins can carry out all phases of membrane fusion. The fusion process has yet to be characterized for viruses that require more than two fusion glycoproteins, and a major issue is whether multiple glycoproteins mediate fusion through a hemifusion intermediate.Herpes simplex ...

Section: Discussionmentioning

confidence: 99%

Herpes simplex virus type 1 mediates fusion through a hemifusion intermediate by sequential activity of glycoproteins D, H, L, and B

Subramanian

Geraghty

2007

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“…Similar to other herpesvirus gB (29,35,38), we have shown that the gB cytoplasmic tail domain is an important regulator of EBV-induced membrane fusion (8). Previously, we created truncation mutants at three amino acids: 841 (gB-841); 816 (gB-816), which removed the ER retention sequence; and 801 (gB-801), which deleted the final 46 aa of gB, including the ER retention sequence.…”

Section: The Cytoplasmic Tail Of Gb Has Different Functional Domains mentioning

confidence: 94%

“…The gB C terminus is one of the longest of the herpesvirus-encoded glycoproteins and contains important cellular-sorting signals and regulates virally induced membrane fusion (28). The role of the gB tail in regulating herpesvirus-induced membrane fusion is most apparent because many mutations that modulate fusion are located in the gB C terminus (23,24,(29)(30)(31)(32)(33)(34)(35)(36)(37)(38). For EBV, the C terminus also contains domains that are important for cell fusion and the cellular localization of gB (8,21).…”

mentioning

confidence: 99%

Cell-surface expression of a mutated Epstein–Barr virus glycoprotein B allows fusion independent of other viral proteins

McShane

Longnecker

2004

Epstein-Barr virus (EBV) infects human B lymphocytes and epithelial cells. We have compared the requirements for EBV glycoprotein-induced cell fusion between Chinese hamster ovary effecter cells and human B lymphoblasts or epithelial cells by using a virus-free cell fusion assay. EBV-encoded gB, gH, gL, and gp42 glycoproteins were required for efficient B cell fusion, whereas EBV gB, gH, and gL glycoproteins were required for Chinese hamster ovary effecter cell fusion with epithelial cell lines (AGS and SCC68) or the human embryonic kidney cell line 293-P. Fusion with human embryonic kidney 293-P cells was greater than fusion observed with B cells, indicative of an important role for cell contact. An antibody directed against the gH and gL complex inhibited epithelial cell fusion. Increased surface expression of gB alone as a result of truncations or point mutants in the carboxyl-terminal tail allowed gB-mediated fusion with epithelial cells, albeit at a lower level than with coexpression of gB, gH, and gL. Overall, gB appears to be the critical component for EBV glycoprotein-mediated cell fusion. viral entry ͉ herpesvirus T he entry process of human herpesviruses entails a two-step process: binding followed by fusion. Specific herpesvirusencoded glycoproteins and cell-surface receptors are important for both events (1-3). Entry may occur by free virus infecting a cell or by cell-cell spread (2). In either case, fusion is a prerequisite for infection. Despite herpesvirus genomes encoding many viral glycoproteins, the glycoproteins implicated in entry and fusion are limited and conserved (1). Most herpesviruses require the conserved glycoproteins gH, gL, and gB, as well as an additional, family-specific viral glycoprotein, such as gD for ␣-herpesviruses or gp42 for the ␥-herpesvirus Epstein-Barr virus (EBV), for efficient entry and fusion (1). These nonconserved glycoproteins result in cell specificity by recognizing distinct cellular receptors. For example, EBV glycoprotein 42 binds to HLA class II on B cells, an interaction essential for the infection of B cells (4, 5). Additionally, EBV gp350͞220 binds to the B cell-surface receptor CD21͞CR2, and this binding mediates the initial interaction of EBV with B cells and is thought to tether the virus to promote the subsequent binding of gp42 to HLA class II (6, 7). The gp42-class II interaction is then proposed to trigger membrane fusion, a reaction mediated by gH (EBV gp85), gL (EBV gp25), and gB (EBV gp110) (8). The exact role each of these glycoproteins plays in fusion is unclear.EBV can enter cells by means of two routes, depending on the host cell type. In primary human lymphocytes, EBV is endocytosed before fusion, whereas, in B cells in culture, the virion envelope directly fuses with the plasma membrane (9, 10). Similarly to B cells in culture, fusion of EBV with epithelial cells occurs by direct fusion of the virion envelope with the plasma membrane. Despite B lymphocytes being the primary site for latency, the tropism of EBV for epithelial cells is suppor...

“…Deletions of up to Ϸ40 aa from the C terminus of HSV-1 or HSV-2 gB either had no effect on or enhanced function in cell fusion, whereas larger deletions inhibited cell fusion (21,22). The insertion that enhanced cell fusion activity (T868) also altered epitopes in the ectodomain, suggesting both that conformation of the cytoplasmic tail can influence conformation of the ectodomain and that this altered conformation in the ectodomain may have a role in the enhanced cell fusion activity.…”

Section: Locations Of Insertions On Gb Structure In Relation To Effecmentioning

confidence: 99%

Random linker-insertion mutagenesis to identify functional domains of herpes simplex virus type 1 glycoprotein B

Lin

Spear

2007

Herpes simplex virus glycoprotein B (gB) is one of four glycoproteins essential for viral entry and cell fusion. Recently, an x-ray structure of the nearly full-length trimeric gB ectodomain was determined. Five structural domains and two linker regions were identified in what is probably a postfusion conformation. To identify functional domains of gB, we performed random linkerinsertion mutagenesis. Analyses of 81 mutants revealed that only 27 could fold to permit processing and transport of gB to the cell surface. These 27 mutants fell into three categories. Insertions into two regions excluded from the solved structure (the N terminus and the C-terminal cytoplasmic tail) had no negative effect on cell fusion and viral entry activity, identifying regions that can tolerate altered structure without loss of function. Insertions into a disordered region in domain II and the adjacent linker region also permitted partial cell fusion and viral entry activity. Insertions at 16 other positions resulted in loss of cell fusion and viral entry activity, despite detectable levels of cell surface expression. Four of these insertion sites were not included in the solved structure. Two were between residues exposed to a cavity that is too small to accommodate the 5-amino acid insertions, consistent with the solved structure being different from the native prefusion structure. Ten were between residues exposed to the surface of the trimer, identifying regions that may be critical for interactions with other viral proteins or cellular components or for transitions from the prefusion to postfusion state.cell fusion ͉ glycoprotein B ͉ viral entry ͉ mutation H erpes simplex virus (HSV) is a neurotropic virus that can cause recurrent mucocutaneous lesions of the oral or genital epithlelium, lesions on the cornea and, rarely, encephalitis. Infection of host cells occurs through virus attachment to the cell surface and subsequent membrane fusion to deliver the nucleocapsid containing the viral genome into the host cell. Virus attachment is mediated by binding of glycoproteins C (gC) or B (gB) to cell surface glycosaminoglycans, primarily heparan sulfate (1). Subsequent fusion between the virion envelope and a host cell membrane requires gB, glycoprotein D (gD), and the heterodimer glycoprotein H (gH)-glycoprotein L (gL) and one of the cellular receptors for gD. These receptors include herpesvirus entry mediator (HVEM), a member of the TNF receptor family; nectin-1 and nectin-2, cell adhesion molecules of the Ig superfamily; and specific sites in heparan sulfate generated by 3-O-sulfotransferases (2). It has been proposed that binding of gD to one of its cellular receptors induces gD to undergo a conformational change, resulting in interactions with gB and/or the gH-gL complex to trigger membrane fusion (3, 4).The exact roles for gB and gH-gL in the membrane fusion process have yet to be elucidated. Recently, it was shown (5) that gH-gL, along with gD and a gD receptor, were sufficient to induce hemifusion, mixing of the outer leafle...