Structure–Function Analysis of Herpes Simplex Virus Glycoprotein B with Fusion-From-Without Activity

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Virology 382 (2008) 207–216

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Virology

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Structure–function analysis of herpes simplex virus glycoprotein B with fusion-from-without activity

Devin G. Roller, Stephen J. Dollery, James L. Doyle, Anthony V. Nicola ⁎

Department of Microbiology and Immunology, Virginia Commonwealth University School of Medicine, Richmond, Virginia, 23298-0678, USA

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Article history: Fusion-from-without (FFWO) is the rapid induction of cell fusion by virions in the absence of viral protein Received 16 July 2008 synthesis. The combination of two amino acid mutations in envelope glycoprotein B (gB), one in the Returned to author for revision ectodomain and one in the cytoplasmic tail, can confer FFWO activity to wild type herpes simplex virus 3 September 2008 (HSV). In this report, we analyzed the entry and cell fusion phenotypes of HSV that contains FFWO gB, with Accepted 18 September 2008 emphasis on the cellular receptors for HSV, nectin-1, nectin-2 and HVEM. The ability of an HSV strain with Available online 23 October 2008 FFWO gB to efficiently mediate FFWO via a specific gD-receptor correlated with its ability to mediate viral fi Keywords: entry by that receptor. A FFWO form of gB was not suf cient to switch the entry of HSV from a pH- Herpes simplex virus dependent, endocytic pathway to a direct fusion, pH-independent pathway. The conformation of gB with Glycoprotein B FFWO activity was not globally altered relative to wild type. However, distinct monoclonal antibodies had Virus–cell fusion reduced reactivity with FFWO gB, suggesting an altered antigenic structure relative to wild type. FFWO was Fusion-from-without blocked by preincubation of virions with neutralizing antibodies to gB or gD. Together with previous studies, Viral entry the results indicate that the roles of gB in FFWO and in virus–cell fusion during entry are related but not Penetration identical. This study also suggests that the FFWO function of gB is not a specific determinant for the selection Endocytosis of HSV entry pathway and that antigenic differences in FFWO gB may reflect its enhanced fusion activity. © 2008 Elsevier Inc. All rights reserved.

Introduction stomatitis virus glycoprotein G (Roche et al., 2006). Both glycoproteins are members of a new family of fusion proteins, class III (Weissenhorn Fusion of herpes simplex virus (HSV) with the host cell during viral et al., 2007). HSV-1 gB has a central, alpha-helical structure (Domain entry is thought to be initiated by interaction of viral envelope III; Fig. 4A) that is reminiscent of the coiled coil domains of class I viral glycoprotein D (gD) with a cognate receptor followed by the fusion proteins (Earp et al., 2005). The base domain (Domain I; Fig. 4A) involvement of gB and the gH–gL complex (Atanasiu et al., 2007; consists of elongated beta sheets interrupted by bipartite hydrophobic Avitabile et al., 2007; Krummenacher et al., 2007; Spear and regions that may function as internal fusion loops (Hannah et al., Longnecker, 2003; Subramanian and Geraghty, 2007). Direct study 2007). An ectodomain and a cytoplasmic tail syncytial mutation in gB of the fusion activity of herpesvirions during entry has proven are both needed for FFWO (Saharkhiz-Langroodi and Holland, 1997). difﬁcult. Fusion-from-without (FFWO) is cell fusion caused by contact The ectodomain mutation (V553A) maps to Domain III of the gB of virions with the target cell surface at high MOI in the absence of structure, which also encompasses the coiled coil. This mutation has HSV protein synthesis (Falke et al., 1985). HSV-induced FFWO shares been described as a rate-of-entry determinant (Bzik et al., 1984) and is key features with membrane fusion during viral entry. Both processes shared by the FFWO strains ANG path, HSZP, and HFEM syn. involve the interaction of the viral membrane with the host cell target The HSV-1 strains ANG (Munk and Donner, 1963) and its mouse membrane and are dependent on the presence of an appropriate gD- brain-passaged derivative, ANG path (Kaerner et al., 1983), have FFWO receptor in the target membrane (Delboy et al., 2006). Transfer of gB activity. Both strains also have unique entry phenotypes. Due to from a FFWO strain of HSV into a wild type HSV background confers mutations in the N-terminus of gD (Dean et al., 1995; Izumi and FFWO activity, suggesting that gB is a critical determinant of FFWO Stevens, 1990), ANG and ANG path have an enhanced ability to utilize activity (Saharkhiz-Langroodi and Holland, 1997). the nectin-2 receptor for entry (Delboy et al., 2006; Warner et al., The structure of an ectodomain fragment of HSV-1 gB reveals an 1998). Wild type HSV strains are readily inhibited by soluble forms of extended multi-domain architecture (Heldwein et al., 2006). gB has gD (Johnson et al., 1990; Nicola et al., 1996); however, entry of ANG or striking structural similarity to the postfusion form of vesicular ANG path, is highly resistant to inhibition by either soluble wild type gD or soluble ANG gD (Nicola et al., 1997). HSV can utilize endocytic or non-endocytic entry pathways in a ⁎ Corresponding author. 1101 East Marshall Street, Room 6-038, P.O. Box 980678, Richmond, Virginia 23298-0678, USA. Fax: +1 804 828 9946. cell-type-dependent manner (Nicola et al., 2003). The process by E-mail address: [email protected] (A.V. Nicola). which the entry pathway is selected is not clear. Viral determinants,

Table 1 specific MAbs to neutralize infection by FFWO strains of HSV. Together Composition of viruses used in this study with previous studies, the results indicate that the roles of gB in FFWO HSV-1 strain Background gB gD FFWO activitya and in virus–cell fusion during entry are related but not identical. KOS KOS KOS gB KOS gD − ANG path ANG path ANG path gB ANG path gD +b Results 27/III ANG path KOS gB ANG path gD −c d KBang KOS ANG path gB KOS gD + Utilization of entry receptors nectin-1, nectin-2 or HVEM by HSV that a Ability of virions to induce cell fusion of Vero cells in the absence of protein contains gB with FFWO activity synthesis. b Falke et al., 1985. Current models of HSV entry propose that after the binding of c Lingen et al., 1995b. d Saharkhiz-Langroodi and Holland, 1997. virion gD to one of its cognate receptors, gD interacts laterally with gB and gH–gL to trigger membrane fusion and penetration (Atanasiu et al., 2007; Avitabile et al., 2007; Krummenacher et al., 2007; Perez- cellular gD-receptors, and the background of the target cell all Romero et al., 2005; Spear and Longnecker, 2003; Subramanian and influence the entry route taken by HSV (Delboy et al., 2006; Gianni Geraghty, 2007). We first examined the receptor usage of HSVs that et al., 2004; Milne et al., 2005; Nicola et al., 2003; Nicola and Straus, contain FFWO forms of gB (Table 1). For these studies, we used the 2004; Stiles et al., 2007). The majority of HSV-1 and HSV-2 strains HSV-1 FFWO strain ANG path, its parent, ANG, and the non-FFWO, tested to date enter Chinese hamster ovary (CHO) cells expressing gD- wild type strain KOS. To evaluate specifically the role of gB, we binding receptors by a pH-dependent, endocytic mechanism (Nicola employed HSV-1 27/III which has an ANG path background but has et al., 2003). However, ANG path enters CHO-nectin-2 cells by pH- KOS gB in place of ANG path gB (Lingen et al., 1995b)(Table 1), and independent penetration at the cell surface (Delboy et al., 2006). This HSV-1 KBang which has KOS background with ANG path gB in place of led to speculation that FFWO gB, since it has enhanced fusion activity, KOS gB (Saharkhiz-Langroodi and Holland, 1997)(Table 1). KBang might function to override the requirement for endocytosis, and allow mediates FFWO of Vero cells (Saharkhiz-Langroodi and Holland, 1997) ANG path to fuse directly with the plasma membrane of CHO cells to but 27/III does not (Lingen et al., 1995b)(Table 1). accomplish entry. To determine the efficiency of receptor utilization by the panel of In this study, we analyzed the functional role of specific receptors viruses, we measured entry into receptor-negative CHO cells and into in the entry and FFWO activities of recombinant viruses that contain CHO cells expressing nectin-1, nectin-2, or HVEM (Fig. 1). HSV- chimeric gB. We also used monoclonal antibodies (MAbs) to probe the induced expression of beta-galactosidase was as an indicator of antigenic conformation of FFWO gB, and assessed the ability of gB- successful viral entry. In control experiments, we confirmed the role of

Fig. 1. Entry of chimeric strains of HSV into CHO cells mediated by nectin-1, nectin-2, or HVEM. The indicated strain of HSV-1 was added to wild type CHO cells (A), CHO-nectin-1 cells (B), CHO-nectin-2 cells (C), or CHO-HVEM cells (D) at MOIs ranging from 0 to 10. Beta-galactosidase activity of cell lysates was quantified at 6 h post-infection as an indication of viral entry. Data are means of quadruplicate determinations. Maximal values in B, C, and D correspond to 100% of cells infected. D.G. Roller et al. / Virology 382 (2008) 207–216 209 each receptor in the entry of KOS and ANG strains (Geraghty et al., KBang utilized nectin-1 with wild type efficiency (Fig. 1B). The results 1998; Montgomery et al., 1996; Warner et al., 1998). We also are consistent with reports that nectin-1 interacts to a greater extent confirmed our previous ANG path results with nectin-1 and nectin-2 with KOS gD than with gD containing N-terminal mutations, such as (Delboy et al., 2006). ANG and ANG path gDs (Delboy et al., 2006; Krummenacher et al., The HSV chimera KBang entered wild type CHO cells with low 1998, 2004). efficiency (Fig. 1A). Interestingly, this ability is shared with KOS (Shieh HSV-1 27/III and ANG path entered CHO-nectin-2 cells efficiently et al., 1992) but not with viruses that have FFWO gB (Fig. 1A). This due to the ANG path gD allele (Fig. 1C). A similar result was obtained result suggests that a non-gB component of the KOS background with ANG (Fig. 1C). To a lesser degree, the presence of nectin-2 allows utilization of an endogenous hamster receptor. 27/III, like ANG enhanced KBang and KOS entry above the CHO cell background (Figs. path and ANG, entered CHO-nectin-1 cells ∼1 log less efficiently than 1C and A), consistent with reports that KOS gD does not effectively wild type KOS (Fig. 1B). The data also suggest that the majority of cells engage nectin-2 in CHO cells (Delboy et al., 2006; Warner et al., 1998). are not susceptible to infection by these viruses, even at high MOI. KOS and KBang which both contain wild type KOS gD, entered CHO-

Fig. 2. The pH-dependence of gD-receptor-mediated entry of HSV chimeras. CHO cells expressing nectin-1 (A), nectin-2 (B), or HVEM (C) were treated with the indicated concentrations of monensin (left) or ammonium chloride (NH4Cl; right) for 15 min. Except where indicated in the text, virus was added at an MOI of 1 for 6 h in the continued presence of inhibitor. Similar results were obtained at an MOI of 10 (data not shown). Entry was measured as the % of beta-galactosidase activity relative to that obtained in the absence of lysosomotropic agent. Data are means of quadruplicate determinations with standard error. 210 D.G. Roller et al. / Virology 382 (2008) 207–216

HVEM cells efﬁciently (Fig. 1D). At high MOIs, CHO-HVEM cells Entry pathway utilization by HSV that contains FFWO gB supported only residual entry of 27/III, ANG path and ANG, each of which bear N-terminal mutations in gD (Fig. 1D). As expected, the With the exception of ANG path (Delboy et al., 2006), all HSV form of gD present in the virus determined receptor usage. The strains tested to date enter CHO-receptor cells by a low pH, endocytic presence of FFWO gB in the viral envelope did not alter gD-receptor pathway (Nicola et al., 2003). ANG path entry into CHO-nectin-2 cells utilization by HSV. is refractory to inhibitors of endocytosis and endosomal acidiﬁcation

Fig. 3. Receptor-mediated fusion-from-without activity of chimeric strains of HSV. ANG path virions (MOI of 50 except where noted) were added to receptor-negative CHO cells (A–E) or CHO cells expressing either nectin-1 (F–J, U–V), nectin-2 (K–O), or HVEM (P–T) in the presence of 1 mM cycloheximide. Cells were incubated at 37 °C for 3 h, and were then fixed with methanol and stained with Giemsa. Magnification, 4×. (W) Fusion-from-without was quantified as indicated in Materials and methods. Means of triplicate determinations and standard deviation are shown. nd, not determined. D.G. Roller et al. / Virology 382 (2008) 207–216 211

(Delboy et al., 2006). Thus, we used the panel of viruses to test the which has the ANG path gB allele and efficiently enters CHO-HVEM possibility that ANG path gB might be a determinant for directing cells (Fig. 1D), was inhibited by lysosomotropic agents in a manner entry by a pH-independent pathway. The receptor-expressing CHO similar to wild type (Fig. 2C). Together, the data suggest that FFWO cells were treated with monensin or ammonium chloride, which activity of gB is not sufficient to trigger direct, pH-independent entry elevate endosomal pH. For all viruses tested, entry into CHO-nectin-1 at the cell surface. cells was inhibited in a concentration-dependent manner (Fig. 2A), indicating a pH-dependent entry mechanism mediated by nectin-1 Receptor-specific fusion-from-without activity regardless of whether the FFWO allele of gB was present in the virion. 27/III entry was the least sensitive to monensin but the most sensitive We tested the ability of the panel of viruses to trigger FFWO to ammonium chloride, suggesting that something in addition to pH mediated by nectin-1, nectin-2, or HVEM. None of the viruses tested may be altered by one or both agents. induced FFWO on the receptor-negative CHO cells (Figs. 3A–E), even at Entry of 27/III into CHO-nectin-2 cells was partially resistant to an MOI of 1000 (not shown). As expected, 27/III and KOS, which have inhibition by lysosomotropic compounds in a manner similar to ANG wild type gB, did not trigger FFWO on any of the gD-receptor-bearing path (Fig. 2B). Because 27/III contains wild type KOS gB, the unique cells (Figs. 3F, G, K, L, P, and Q). Both ANG path and ANG caused FFWO mode of entry of ANG path into CHO-nectin-2 cells cannot be on CHO-nectin-2 cells (Figs. 3M–N). These two viruses caused no explained by FFWO gB alone. Another viral component of ANG path demonstrable FFWO on CHO-nectin-1 cells (Figs. 3H–I). However, likely determines the pH-independent entry phenotype. Entry of the when ANG path or ANG were added to CHO-nectin-1 cells at an MOI of parental strain ANG into CHO-nectin-2 cells was also resistant to 1000, FFWO was detected (Figs. 3U–V). Thus, FFWO correlated with inhibition by monensin and ammonium chloride (Fig. 2B). This how efficiently nectin-1 and nectin-2 receptors were utilized by ANG indicates that pH-independent entry activity was not acquired from and ANG path for entry (Figs. 1B–C). HSV-1 strain KBang mediated passage of ANG in mouse brain but was present initially in ANG. Entry FFWO of both CHO-nectin-1 and CHO-HVEM cells (Figs. 3J and T), of KBang and wild type HSV-1 strain KOS into CHO-nectin-2 cells was owing to the presence of KOS gD and ANG path gB in the virion. We tested at an MOI of 10, and was inhibited by the pH-altering agents can conclude that HVEM receptors, like nectin-1 and nectin-2 (Delboy (Fig. 2B). As an additional control, HSV-1 rid1 entry into CHO-nectin-2 et al., 2006), are capable of triggering FFWO. Addition of KBang to at an MOI of 1 or 10 was effectively inhibited by ammonium chloride CHO-nectin-2 cells caused no detectable FFWO (Fig. 3O). Quantifica- and monensin (Delboy et al., 2006) (data not shown). Entry of KBang, tion of clusters of nuclei as an indicator of FFWO (Fig. 3W) correlated

Fig. 4. Antigenic reactivity of HSV-1 ANG path gB. (A) The protein backbone of a gB monomer is shown in ribbon designation. The ectodomain mutation that is required for FFWO activity of HSV-1 ANG path (V553A) is denoted in CPK representation (pink). Other HSV-1 FFWO strains also have an alanine at this position. A mutation at aa position 303 that confers resistance to neutralization by MAb H126 is indicated. This residue is contained within the linear epitope recognized by H126. Ribbon structure was prepared with PyMOL (DeLano, 2002) and domains assigned after (Heldwein et al., 2006). (B) Detection of gB trimers in ANG path virions. HSV-1 KOS or ANG path was subjected to “native” PAGE (left) or denaturing SDS-PAGE (right) followed by Western blot with gB-specific polyclonal antiserum. gB trimers or monomers and molecular weight markers in kilodaltons are indicated. (C–F) Dot blot analysis of virion gB. Serial, two-fold dilutions of ∼5×105 PFU (left to right) of HSV-1 KOS or ANG path in PBS were blotted directly to nitrocellulose membranes. Virion gB was detected by the primary antibody (C) DL16, (D) H126, (E) R69 or (F) SS55 followed by HRP-conjugated secondary antibody. Blots were developed with ECL reagents and exposed to film. (G) HSV-1 KOS or ANG path was subjected to denaturing SDS-PAGE followed by Western blot with antibodies R69, H1817, or H126. 212 D.G. Roller et al. / Virology 382 (2008) 207–216 with the results from microscopic observations. Together with MAb H126 recognizes a linear epitope in Domain I of gB (Fig. 4A) previous work, these results underscore the importance of ANG (Heldwein et al., 2006; Kousoulas et al., 1984). Domain I amino acid path gB for receptor-mediated FFWO. For FFWO strains of HSV, a 303 is mutated in an HSV mutant that is resistant to neutralization by receptor's ability to trigger FFWO (Fig. 3) correlated with its ability to MAb H126 (Kousoulas et al., 1988). Here we show that H126 mediate entry (Fig. 1). recognized KOS gB after analysis of virions by non-denaturing approaches including dot blot, “native” PAGE, and immunoprecipita- Antigenic analysis of gB with FFWO activity tion (Fig 4D, Table 2, and not shown). Notably, H126 had greatly reduced reactivity with ANG path gB by these same measures. The presence of ANG path gB is sufficient to confer nectin-1- Additional FFWO strains of HSV including ANG, KBang, HSZP, and mediated and HVEM-mediated FFWO activity to KOS virions (Figs. 3J HFEM syn (Chapsal and Pereira, 1988) had similarly reduced reactivity and T). To address whether changes in the antigenic structure of ANG with H126 (data not shown). Importantly, when ANG path virions path gB correlate with enhanced fusogenic activity, we characterized were analyzed by denaturing SDS-PAGE, H126 and two control the reactivity of ANG path virions with a panel of MAbs to gB. The antibodies did detect ANG path gB (Fig. 4G). The results indicate mature ANG path gB has 9 amino acid changes relative to KOS gB that the peptide sequence to which H126 binds is present in ANG path (Saharkhiz-Langroodi and Holland, 1997). Previous studies have gB, but it may have altered accessibility. For example, the H126 indicated that both a A855V syncytial mutation in the cytoplasmic epitope may be buried in the native, trimeric structure of ANG path gB tail of gB (Lingen et al., 1995b) and a V553A mutation in the but exposed on the surface of wild type gB. In total, the analysis ectodomain (Saharkhiz-Langroodi and Holland, 1997) are required for suggests that ANG path gB has detectable differences in antigenic ANG path FFWO activity. Domain III of gB contains a central, alpha- structure relative to wild type. helical coiled coil domain (Fig. 4A; Heldwein et al., 2006). This is a common structural feature of many viral fusion proteins (Earp et al., HSV-1 ANG path has unaltered sensitivity to neutralization by 2005). The FFWO ectodomain mutation maps to Domain III, near the gB-specific monoclonal antibodies, including H126 coiled coil (Fig. 4A). The oligomeric structure of gB is disrupted by heat and high An antibody that neutralizes virus infection in the absence of concentrations of SDS (Claesson-Welsh and Spear, 1986)(Fig. 4B). complement is directed against an epitope that is important for Thus, gB oligomers can be visualized by “native” PAGE analysis when function. MAbs specific for gB were tested for their ability to neutralize samples contain 0.2% SDS and are unheated (Bender et al., 2007; HSV-1 ANG path. The dilution of antibody required to reduce plaque Cohen et al., 1986). The oligomeric form resolved under “native” numbers by 50% was determined. Since CHO-receptor cells do not conditions was revealed by the crystal structure to be a trimer support plaque formation of HSV, Vero and HaCaT cells were utilized. (Heldwein et al., 2006). After “native” PAGE, oligomers of gB were Table 2 shows that ANG path was sensitive to neutralization by MAbs detected in both ANG path and KOS virions by a polyclonal antibody to H126, SS10, SS55, and SS144. In contrast, potent neutralization of ANG gB (Fig. 4B), suggesting that gB with FFWO activity exists as a trimer. path was not detected by MAbs DL16, DL21, SS106, and SS118 at the We compared the antigenic reactivity of ANG path gB and KOS gB dilutions tested. Each of the antibodies neutralized HSV-1 KOS to a by directly blotting virions to nitrocellulose membranes and testing a similar extent. Thus, despite its unique function, ANG path gB does not panel of MAbs. Seven of nine antibodies tested displayed equivalent appear to be globally altered in structure relative to wild type. Our reactivity with gB from both viruses (Figs. 4E and F, Table 2). These results with MAb neutralization of KOS (Table 2) were consistent with MAbs detect both conformation-dependent and conformation- previous findings (Bender et al., 2005, 2007; Kousoulas et al., 1984), independent epitopes of gB (Bender et al., 2007), suggesting that with the exception of SS106 and SS118, which did not effectively FFWO gB is not globally altered with respect to wild type. neutralize HSV in our experiments. This discrepancy may be explained MAb DL16 had significantly reduced reactivity with ANG path by the use of ascites fluid instead of IgG or by different criteria for virions relative to wild type KOS virions (Fig. 4C). DL16 is conforma- neutralization. tion-dependent and specific for the oligomeric form of gB (Bender et MAb H126 effectively neutralized ANG path infection (Table 2) al., 2005), but specific residues of its epitope are not known. DL16 also despite its significantly reduced reactivity with native ANG path gB had reduced reactivity against gB from FFWO strains ANG, KBang, (Fig. 4D and Table 2). This may be explained by increased exposure of HSZP and HFEM syn (not shown). Reduced binding of DL16 may be the H126 epitope during ANG path entry. Nonetheless, these results due to altered conformation of the gB trimer of ANG path. Whether are consistent with the presence of the H126-binding sequence in one or more amino acid changes in ANG path gB contributes to DL16 ANG path gB (Fig. 4G). binding remains to be determined. HSV gB, along with gD, and gH–gL, is required for successful entry via either the endocytosis pathway or by direct penetration at the plasma membrane (Cai et al., 1988; Nicola and Straus, 2004; Spear, 1993). Vero cells support HSV entry by pH-independent fusion with Table 2 Reactivity and neutralization activity of gB-specific antibodies the plasma membrane, and HaCaT cells support entry via a pH- dependent, endocytic mechanism (Nicola et al., 2003, 2005; Wittels a b Reactivity Neutralization and Spear, 1991). All of the MAbs to gB tested exhibited similar Vero HaCaT neutralization activity whether assayed on Vero or HaCaT cells (Table MAb Notes KOS ANG path KOS ANG path KOS ANG path 2). Within the limits of these experiments, a difference in the DL16 Trimer-specific + weak −− nd nd functional role of gB in these two entry pathways was not detected. DL21 Not grouped + + −− −− H126 Domain I + − ++ ++ H1817 Domain VI + + nd nd nd nd Antibodies to gB or gD block fusion-from-without SS10 Domain IV + + + + + + SS55 Domain I + + + + + + Neutralizing antibodies specific for gB or gD can block virus entry SS106 Domain V + + −− nd nd SS118 Domain I + + −− −− at receptor-binding and/or fusion steps during viral entry (Bender et SS144 Domain V + + + + + + al., 2005; Fuller and Spear, 1987; Highlander et al., 1987, 1988; Muggeridge et al., 1990; Nicola et al., 1998; Pereira, 1994). Antibodies nd, not determined. a Measured via “native” Western blot or dot blot. to gD and gB also inhibit fusion of infected cells as measured by b +, N50% plaque reduction at MAb dilution bor =0.002; −, b50% plaque reduction. syncytium formation (or fusion-from-within) (Navarro et al., 1992; D.G. Roller et al. / Virology 382 (2008) 207–216 213

responsible for the enhanced fusogenicity associated with FFWO (Saharkhiz-Langroodi and Holland, 1997), we speculated that ANG path gB might allow HSV to enter by fusing directly with the plasma membrane, obviating the need for endocytosis. However, this was not the case in the model system under study (Fig. 2B). A non-gB mutation in the ANG path backbone likely affects the pH-dependence of entry. Of the three receptors tested, only nectin-2 supported pH-independent entry of ANG path-related strains into CHO cells. gD is the viral ligand for nectin-2 (Warner et al., 1998), and the three viruses that entered CHO-nectin-2 cells by a pH-independent pathway all contain ANG gD or ANG path gD. Thus, it may be the combination of this gD-nectin interaction at the surface of CHO-nectin-2 cells that triggers direct fusion with the plasma membrane during viral entry. Another possible determinant is the ANG path gH–gL complex about which little is known. These possibilities need to be tested directly. This report highlights further similarities between FFWO and – Fig. 5. Inhibition of FFWO by glycoprotein-specific antibodies. Six micrograms/milliliter virus cell fusion during entry. Like virus entry, FFWO requires an of mouse monoclonal antibody to gB (H126, H1817, or H1359), to gD (1103), or to Golgi appropriate gD-receptor in the target membrane (Delboy et al., protein p58 was mixed with HSV-1 ANG path for 1 h at 37 °C. Virus–antibody mixtures 2006). Here we show that HVEM, as well as nectin-1 and nectin-2, is were added to Vero cells (MOI of 50) for 3 h, and FFWO was detected and quantitated as capable of triggering FFWO provided that the correct partner gD is in Fig. 3. present in the virus. For any of the FFWO-inducing strains tested, if a receptor was capable of mediating entry into CHO cells, it was also fi Noble et al., 1983). Inhibition of FFWO by glycoprotein-speci c capable of mediating FFWO. Further, the efficiency of receptor usage monoclonal antibodies has not been tested. ANG path virions were for entry correlated with the efficiency of receptor usage for FFWO. preincubated with MAb and then added to Vero cells. Two MAbs to gB, Antibodies to gB and gD that neutralize virus entry also blocked H126 and H1817, both effectively blocked FFWO (Fig. 5). H126 FFWO. inhibited ANG path virion-induced fusion (Fig. 5), even though this Nectin-1 and HVEM both trigger pH-independent FFWO of CHO epitope appears to be presented differently in ANG path gB (Fig. 4 and cells, yet entry into CHO cells mediated by these receptors occurs after Table 2). MAb H1359, which recognizes gB but is non-neutralizing endocytosis and is pH-dependent. Addition of CHO cells that express fi failed to block ANG path-induced FFWO, suggesting that only speci c gB, gD, and gH–gL to target CHO cells also results in pH-independent fi regions of gB are involved in FFWO. The gD-speci c, neutralizing MAb cell fusion (Pertel et al., 2001). Both FFWO and cell–cell fusion share 1103 blocked FFWO (Fig. 5). As an additional control, the heterologous the CHO cell surface as the target membrane for fusion. Virions that mouse MAb to the Golgi protein p58 had no effect on FFWO (Fig. 5). mediate FFWO with the plasma membrane may not necessarily These results underscore the importance of functional regions on gB initiate successful infection, i.e., incoming nucleocapsids may not be and gD for HSV membrane fusion as measured by FFWO. properly directed to the nucleus. Thus, pH-dependent fusion of HSV with the endosomal membrane during viral entry may be the most Discussion physiologically relevant fusion mechanism for CHO cells because it results in infection. Nonetheless, these apparent incongruities may be Although HSV gB is required for membrane fusion (Cai et al., 1988; explained by the ability of HSV glycoproteins to mediate both pH- Hannah et al., 2007; Heldwein et al., 2006; Highlander et al., 1988; independent and pH-dependent fusion mechanisms (Nicola et al., Navarro et al., 1992; Pereira, 1994; Saharkhiz-Langroodi and Holland, 2003; Nicola and Straus, 2004). 1997; Turner et al., 1998), the exact nature of its role remains to be We show that ANG path gB, like KOS gB, is trimeric (Fig. 4B). elucidated. In the absence of a means to directly study virion–cell However, ANG path gB (Fig. 4C) and gB from other FFWO strains (data fusion during viral entry, we used FFWO as a surrogate to learn more not shown) have reduced reactivity with the trimer-specificMAb about this process. In this report, we show that relationships between DL16. Oligomeric rearrangements in viral glycoproteins can accom- gD and receptor use during FFWO are the same as those during virus pany membrane fusion activity (Allison et al., 1995; Wahlberg and entry. FFWO activity of gB is not sufficient to trigger virus entry by Garoff, 1992). DL16 may detect a difference in the arrangement of the fusion of the viral envelope with the plasma membrane. Therefore, an quaternary structure of wild type and FFWO gBs. additional property of ANG path must enable it to enter CHO-nectin-2 A linear stretch of amino acids in Domain I of ANG path gB is cells by this pH-independent pathway. Antigenic analysis suggests recognized by MAb H126. However, in the native structure of ANG that FFWO gB is conformationally distinct relative to wild type. path gB, this region has altered accessibility to H126 as measured by Altered arrangement of the putative fusion domain in ANG path gB dot blot or “native” Western blot. MAbs SS55 and SS118 recognize may account for its enhanced fusogenicity and the pH-independence Domain I of ANG path and KOS gBs to a similar extent, arguing of FFWO. against a dramatic change in the structure of Domain I. Surprisingly, HSV gD is the viral ligand for HVEM and nectin receptors H126 neutralizes ANG path and inhibits FFWO. This discrepancy (Krummenacher et al., 2007; Spear and Longnecker, 2003). PILRalpha may be explained by an unknown artifact in the blotting assays. was recently reported as a receptor for gB (Satoh et al., 2008). Despite Alternately, the H126 epitope may undergo increased exposure several distinct phenotypes associated with the entry of FFWO strains during the processes of entry and FFWO. H126 neutralizes endocytic (Delboy et al., 2006; Lingen et al., 1995a; Montgomery et al., 1996; entry of ANG path into HaCaT cells and non-endocytic entry into Nicola et al., 1997; Warner et al., 1998), the presence of FFWO gB in a Vero cells suggesting that such a change in the conformation of gB given virus had no bearing on which gD-receptor was utilized for may occur just after binding to the cell surface or just prior to entry. As expected, the allele of gD present in the virion determined fusion. the utilization of nectin-1, nectin-2, or HVEM. Domain I of gB is important for function (Bender et al., 2007; Unlike the majority of HSV-1 and HSV-2 strains tested (Nicola et al., Highlander et al., 1989; Lin and Spear, 2007; Pellett et al., 1985; Pereira 2003), ANG path can enter receptor-expressing CHO cells in a pH- et al., 1989) and is the putative fusion domain (Hannah et al., 2007). independent manner (Delboy et al., 2006). Since ANG path gB is The reduced recognition of FFWO strains by H126 is not readily 214 D.G. Roller et al. / Virology 382 (2008) 207–216 explained by differences in gB amino acid sequences relative to wild Treatments with lysosomotropic agents type. We favor the interpretation that altered antigenic reactivity is due to a distinct conformation of FFWO gB relative to wild type. The Stock solutions of ammonium chloride (1.5 M) were prepared relative orientation of Domain I of FFWO gB may alter accessibility of immediately prior to use. Monensin (75 mM; Sigma) stock solutions the H126 epitope on the trimeric structure. Interestingly, the were prepared in ethanol and stored at −20 °C. Growth medium was analogous fusion domain of VSV G is arranged differently in the removed from cells and replaced with medium containing agents or prefusion and postfusion forms, while its structure remains unaltered medium containing control concentrations of ethanol, and the (Roche et al., 2006, 2007). We propose that a change in the orientation mixture was incubated for 30 min at 37 °C. Virus was added, and of Domain I of gB contributes to the enhanced fusogenicity associated cells were incubated in the constant presence of agent for the length of with FFWO. the beta-galactosidase reporter assay. Treatments had no effect on cell viability as measured by trypan blue exclusion. Materials and methods Plaque assay Cells and viruses At 18–24 h p.i., culture medium was removed, and cells were fixed Vero cells (American Type Culture Collection; ATCC; Rockville, Md.) with ice-cold methanol-acetone solution (2:1 ratio) for 20 min at and HaCaT cells (obtained from Harvey Friedman, University of −20 °C and air-dried. Virus titers were determined by immunoperox- Pennsylvania) were propagated in Dulbecco's Modified Eagle's idase staining with anti-HSV polyclonal antibody HR50 (Fitzgerald Medium (Invitrogen, Grand Island, NY) supplemented with 10% fetal Industries, Concord, Mass.). bovine serum (FBS; Gemini Bio-Products, West Sacramento, Calif.). Chinese hamster ovary (CHO)-K1 cells stably transformed with the Fusion-from-without assay Escherichia coli lacZ gene under the control of the HSV ICP4 promoter (CHO-IEβ8 cells) (Montgomery et al., 1996). CHO-IEβ8 cells stably As described previously (Delboy et al., 2006; 2008), Vero cells were transformed to express the human gD-receptors nectin-1, nectin-2, or pretreated with growth medium containing 0.5 mM cycloheximide. HVEM (Geraghty et al., 1998; Nicola et al., 1998; Warner et al., 1998) Cell-free supernatant preparations of HSV-1 ANG path were added to (obtained from Gary Cohen and Roselyn Eisenberg, University of cells (MOI of 50) for 3 h at 37 °C in the constant presence of Pennsylvania) were propagated in Ham's F12 nutrient mixture cycloheximide. Cells were rinsed with PBS, and then fixed in 100% (Invitrogen) supplemented with 10% FBS, 150 mg/ml puromycin methanol. Monolayers were air-dried, and then nuclei were stained (Sigma, St. Louis, Mo.), and 250 mg/ml G418 sulfate (Fisher Scientific, with Giemsa. Fair Lawn, NJ). Cells were subcultured in non-selective medium prior Micrographs were taken with a Zeiss Axiovert 40C microscope to use in experiments. equipped with a Canon PowerShot G6 digital camera. Digital images HSV-1 strains KOS, ANG path (Kaerner et al., 1983), and KBang were processed with Adobe Photoshop CS2 version 9.0. To quantitate (Saharkhiz-Langroodi and Holland, 1997) were obtained from Thomas fusion, photomicrographs of random fields from triplicate wells (N500 Holland, Wayne State University. HSV-1 strain ANG (Munk and cells/well) were scored. The number of nuclei present in clusters of 4 Donner, 1963) was obtained from R. Eisenberg and G. Cohen. HSV-1 or more divided by the total number of nuclei yielded the % fusion. strains 27/III (Lingen et al., 1995b) and HSZP were obtained from This is a means of quantitation but is an underestimate of total Dietrich Falke, University of Mainz. All viruses were propagated and number of fusion events. Less than 1% FFWO was obtained in the titered on Vero cells. absence of virus.

Chemicals Dot blot analysis of virion gB

Stocks of 0.5 M cycloheximide (Sigma) and monensin (Sigma) were Cell-free supernatants of HSV-infected Vero cells were diluted in prepared in ethanol and stored at −20 °C. 1.5 M ammonium chloride (J. PBS and blotted directly to nitrocellulose with a Mini Fold dot blot T. Baker) was prepared fresh in distilled water. system (Whatman). Strips of membrane were blocked and incubated with antibody to gB. After incubation with horseradish peroxidase- Antibodies conjugated secondary antibodies, enhanced chemiluminescent substrate (Pierce) was added, and blots were exposed to X-ray film (Kodak). Mouse monoclonal antibodies (MAbs) to gB designated DL16, DL21, SS10, SS48, SS55, SS106, SS118, and SS144 (Bender et al., 2005, 2007; SDS-PAGE and Western blotting Heldwein et al., 2006) were kindly provided by R. Eisenberg and G. Cohen. MAbs H126, H1817, and H1359 (all from Virusys) are also Cell-free supernatants of HSV-infected Vero cells were mixed with specific for gB. MAb 1103 (Goodwin Institute, Plantation, FL) is specific polyacrylamide gel electrophoresis (PAGE) sample buffer containing for gD. either no reducing agent and 0.2% sodium dodecyl sulfate (SDS) (“native” conditions) or with 200 mM dithiothreitol and 2% SDS Beta-galactosidase reporter assay for HSV entry (“denaturing” conditions). Denatured samples were boiled for 5 min at 100 °C. Proteins were resolved by PAGE. After transfer to Confluent cell monolayers grown in 96 well dishes were treated nitrocellulose, membranes were blocked and incubated with antibody with medium containing lysosomotropic compounds or vehicle to gB. After incubation with horseradish peroxidase-conjugated controls for 30 min at 37 °C. HSV was added, and cells were incubated secondary antibodies, enhanced chemiluminescent substrate (Pierce) in the constant presence of agent for 6 h. 0.5% Nonidet P-40 (Sigma) was added and membranes were exposed to X-ray film (Kodak). cell lysates were prepared, chlorophenol red-beta-D-galactopyrano- side (Roche Diagnostic, Indianapolis, In.) was added, and beta- Neutralization assay galactosidase activity was read at 595 nm with an ELx808 microtiter plate reader (BioTek Instruments, Winooski, Vt.). Beta-galactosidase Serial dilutions of MAbs were mixed with 100 PFU HSV-1 KOS or activity indicated successful entry. Mean results and standard error ANG path in DMEM supplemented with heat inactivated fetal bovine were calculated for four replicate samples. serum. The MAb-virus mixture was incubated at 37 °C for 1 h and then D.G. Roller et al. / Virology 382 (2008) 207–216 215 added to confluent monolayers of Vero or HaCaT cells in 24-well 1 glycoprotein B which alter antigenic structure and function in virus penetration. J. Virol. 63 (2), 730–738. plates. At 1 h p.i., inoculum was removed and replaced with DMEM. At Izumi, K.M., Stevens, J.G., 1990. Molecular and biological characterization of a herpes 18-24 h p.i., plaque formation of triplicate samples was determined by simplex virus type 1 (HSV-1) neuroinvasiveness gene. J. Exp. Med. 172 (2), 487–496. immunoperoxidase staining. Neutralization titer was measured as 50% Johnson, D.C., Burke, R.L., Gregory, T., 1990. Soluble forms of herpes simplex virus glycoprotein D bind to a limited number of cell surface receptors and inhibit virus plaque reduction. entry into cells. J. Virol. 64 (6), 2569–2576. Kaerner, H.C., Schroder, C.H., Ott-Hartmann, A., Kumel, G., Kirchner, H., 1983. Genetic Acknowledgments variability of herpes simplex virus: development of a pathogenic variant during passaging of a nonpathogenic herpes simplex virus type 1 virus strain in mouse brain. J. Virol. 46 (1), 83–93. This investigation was supported by Public Health Service grant Kousoulas, K.G., Pellett, P.E., Pereira, L., Roizman, B.,1984. Mutations affecting conformation AI60702 from the National Institute of Allergy and Infectious Diseases or sequence of neutralizing epitopes identified by reactivity of viable plaques and a grant from the A.D. Williams Foundation. We are grateful to segregate from syn and ts domains of HSV-1(F) gB gene. Virology 135 (2), 379–394. Kousoulas, K.G., Huo, B., Pereira, L., 1988. Antibody-resistant mutations in cross-reactive Gary Cohen, Roselyn Eisenberg, Dietrich Falke, Thomas Holland, and type-specific epitopes of herpes simplex virus 1 glycoprotein B map in separate Juergen Podlech, and Stephen Straus for the kind gifts of reagents. domains. Virology 166 (2), 423–431. We thank Mark Delboy for critical reading of the manuscript. Krummenacher, C., Nicola, A.V., Whitbeck, J.C., Lou, H., Hou, W., Lambris, J.D., Geraghty, R.J., Spear, P.G., Cohen, G.H., Eisenberg, R.J., 1998. Herpes simplex virus glycoprotein D can bind to poliovirus receptor-related protein 1 or herpesvirus entry mediator, two structurally unrelated mediators of virus entry. J. Virol. 72 (9), 7064–7074. References Krummenacher, C., Baribaud, F., Ponce de Leon, M., Baribaud, I., Whitbeck, J.C., Xu, R., Cohen, G.H., Eisenberg, R.J., 2004. Comparative usage of herpesvirus entry mediator Allison, S.L., Schalich, J., Stiasny, K., Mandl, C.W., Kunz, C., Heinz, F.X., 1995. Oligomeric A and nectin-1 by laboratory strains and clinical isolates of herpes simplex virus. rearrangement of tick-borne encephalitis virus envelope proteins induced by an Virology 322 (2), 286–299. acidic pH. J. Virol. 69 (2), 695–700. Krummenacher, C., Carfi, A., Eisenberg, R.J., Cohen, G.H., 2007. Herpesvirus entry into Atanasiu, D., Whitbeck, J.C., Cairns, T.M., Reilly, B., Cohen, G.H., Eisenberg, R.J., 2007. cells: the enigma variations. In: Poehlmann, S., Simmons, G. (Eds.), “Viral Entry into Bimolecular complementation reveals that glycoproteins gB and gH/gL of herpes Host Cells”. Landes Bioscience. simplex virus interact with each other during cell fusion. Proc. Natl. Acad. Sci. U. S. A. Lin, E., Spear, P.G., 2007. Random linker-insertion mutagenesis to identify functional 104 (47), 18718–18723. domains of herpes simplex virus type 1 glycoprotein B. Proc. Natl. Acad. Sci. U. S. A. Avitabile, E., Forghieri, C., Campadelli-Fiume, G., 2007. Complexes between herpes 104 (32), 13140–13145. simplex virus glycoproteins gD, gB, and gH detected in cells by complementation of Lingen, M., Seck, T., Dehoust, U., Weise, K., Falke, D., 1995a. Amino acid substitutions in split enhanced green fluorescent protein. J. Virol. 81 (20), 11532–11537. glycoprotein D mediate the ability of fusion from without-positive herpes simplex Bender, F.C., Whitbeck, J.C., Lou, H., Cohen, G.H., Eisenberg, R.J., 2005. Herpes simplex virus type 1 strains to penetrate at 4 degrees and in the presence of soluble virus glycoprotein B binds to cell surfaces independently of heparan sulfate and glycoprotein D. Intervirology 38 (5), 283–289. blocks virus entry. J. Virol. 79 (18), 11588–11597. Lingen, M., Seck, T., Weise, K., Falke, D., 1995b. Single amino acid substitutions in the Bender, F.C., Samanta, M., Heldwein, E.E., de Leon, M.P., Bilman, E., Lou, H., Whitbeck, J.C., glycoprotein B carboxy terminus influence the fusion from without property of Eisenberg, R.J., Cohen, G.H., 2007. Antigenic and mutational analyses of herpes simplex herpes simplex virus type 1. J. Gen. Virol. 76 (Pt 7), 1843–1849. virus glycoprotein B reveal four functional regions. J. Virol. 81 (8), 3827–3841. Milne, R.S., Nicola, A.V., Whitbeck, J.C., Eisenberg, R.J., Cohen, G.H., 2005. Glycoprotein D Bzik, D.J., Fox, B.A., DeLuca, N.A., Person, S., 1984. Nucleotide sequence specifying the receptor-dependent, low-pH-independent endocytic entry of herpes simplex virus glycoprotein gene, gB, of herpes simplex virus type 1. Virology 133 (2), 301–314. type 1. J. Virol. 79 (11), 6655–6663. Cai, W.H., Gu, B., Person, S., 1988. Role of glycoprotein B of herpes simplex virus type 1 in Montgomery, R.I., Warner, M.S., Lum, B.J., Spear, P.G.,1996. Herpes simplex virus-1 entry viral entry and cell fusion. J. Virol. 62 (8), 2596–2604. into cells mediated by a novel member of the TNF/NGF receptor family. Cell 87 (3), Chapsal, J.M., Pereira, L., 1988. Characterization of epitopes on native and denatured 427–436. forms of herpes simplex virus glycoprotein B. Virology 164 (2), 427–434. Muggeridge, M.I., Roberts, S.R., Isola, V.J., Cohen, G.H., Eisenberg, R.J., 1990. Herpes Claesson-Welsh, L., Spear, P.G., 1986. Oligomerization of herpes simplex virus simplex virus. In: Van Regenmortel, M.H.V., Neurath, A.R. (Eds.), “Immunochem- glycoprotein B. J. Virol. 60 (2), 803–806. istry of Viruses. The Basis for Serodiagnosis and Vaccines.”, Vol. 2. Elsevier Cohen, G.H., Isola, V.J., Kuhns, J., Berman, P.W., Eisenberg, R.J., 1986. Localization of Biochemical Press, Amsterdam, pp. 459–481. discontinuous epitopes of herpes simplex virus glycoprotein D: use of a Munk, K., Donner, D., 1963. [Cytopathic effect and plaque morphology of various herpes nondenaturing (“native” gel) system of polyacrylamide gel electrophoresis coupled simplex virus strains.]. Arch. Gesamte Virusforsch. 13, 529–540. with Western blotting. J. Virol. 60 (1), 157–166. Navarro, D., Paz, P., Pereira, L., 1992. Domains of herpes simplex virus I glycoprotein B Dean, H.J., Warner, M.S., Terhune, S.S., Johnson, R.M., Spear, P.G., 1995. Viral that function in virus penetration, cell-to-cell spread, and cell fusion. Virology 186 determinants of the variable sensitivity of herpes simplex virus strains to gD- (1), 99–112. mediated interference. J. Virol. 69 (8), 5171–5176. Nicola, A.V., Straus, S.E., 2004. Cellular and viral requirements for rapid endocytic entry Delboy, M.G., Patterson, J.L., Hollander, A.M., Nicola, A.V., 2006. Nectin-2-mediated of herpes simplex virus. J. Virol. 78 (14), 7508–7517. entry of a syncytial strain of herpes simplex virus via pH-independent fusion with Nicola, A.V., Willis, S.H., Naidoo, N.N., Eisenberg, R.J., Cohen, G.H., 1996. Structure– the plasma membrane of Chinese hamster ovary cells. Virol. J. 3 (1), 105. function analysis of soluble forms of herpes simplex virus glycoprotein D. J. Virol. 70 Delboy, M.G., Roller, D.G., Nicola, A.V., 2008. Cellular proteasome activity facilitates (6), 3815–3822. herpes simplex virus entry at a postpenetration step. J. Virol. 82 (7), 3381–3390. Nicola, A.V., Peng, C., Lou, H., Cohen, G.H., Eisenberg, R.J., 1997. Antigenic structure of Earp, L.J., Delos, S.E., Park, H.E., White, J.M., 2005. The many mechanisms of viral soluble herpes simplex virus (HSV) glycoprotein D correlates with inhibition of HSV membrane fusion proteins. Curr. Top. Microbiol. Immunol. 285, 25–66. infection. J. Virol. 71 (4), 2940–2946. Falke, D., Knoblich, A., Muller, S., 1985. Fusion from without induced by herpes simplex Nicola, A.V., Ponce de Leon, M., Xu, R., Hou, W., Whitbeck, J.C., Krummenacher, C., virus type 1. Intervirology 24 (4), 211–219. Montgomery, R.I., Spear, P.G., Eisenberg, R.J., Cohen, G.H., 1998. Monoclonal Fuller, A.O., Spear, P.G., 1987. Anti-glycoprotein D antibodies that permit adsorption but antibodies to distinct sites on herpes simplex virus (HSV) glycoprotein D block block infection by herpes simplex virus 1 prevent virion–cell fusion at the cell HSV binding to HVEM. J. Virol. 72 (5), 3595–3601. surface. Proc. Natl. Acad. Sci. U. S. A. 84 (15), 5454–5458. Nicola, A.V., McEvoy, A.M., Straus, S.E., 2003. Roles for endocytosis and low pH in herpes Geraghty, R.J., Krummenacher, C., Cohen, G.H., Eisenberg, R.J., Spear, P.G., 1998. Entry of simplex virus entry into HeLa and Chinese hamster ovary cells. J. Virol. 77 (9), alphaherpesviruses mediated by poliovirus receptor-related protein 1 and 5324–5332. poliovirus receptor. Science 280 (5369), 1618–1620. Nicola, A.V., Hou, J., Major, E.O., Straus, S.E., 2005. Herpes simplex virus type 1 enters Gianni, T., Campadelli-Fiume, G., Menotti, L., 2004. Entry of herpes simplex virus human epidermal keratinocytes, but not neurons, via a pH-dependent endocytic mediated by chimeric forms of nectin1 retargeted to endosomes or to lipid rafts pathway. J. Virol. 79 (12), 7609–7616. occurs through acidic endosomes. J. Virol. 78 (22), 12268–12276. Noble, A.G., Lee, G.T., Sprague, R., Parish, M.L., Spear, P.G., 1983. Anti-gD monoclonal Hannah, B.P., Heldwein, E.E., Bender, F.C., Cohen, G.H., Eisenberg, R.J., 2007. Mutational antibodies inhibit cell fusion induced by herpes simplex virus type 1. Virology 129 evidence of internal fusion loops in herpes simplex virus glycoprotein B. J. Virol. 81 (1), 218–224. (9), 4858–4865. Pellett, P.E., Kousoulas, K.G., Pereira, L., Roizman, B., 1985. Anatomy of the herpes Heldwein, E.E., Lou, H., Bender, F.C., Cohen, G.H., Eisenberg, R.J., Harrison, S.C., 2006. simplex virus 1 strain F glycoprotein B gene: primary sequence and predicted Crystal structure of glycoprotein B from herpes simplex virus 1. Science 313 (5784), protein structure of the wild type and of monoclonal antibody-resistant mutants. 217–220. J. Virol. 53 (1), 243–253. Highlander, S.L., Sutherland, S.L., Gage, P.J., Johnson, D.C., Levine, M., Glorioso, J.C., 1987. Pereira, L., 1994. Function of glycoprotein B homologues of the family herpesviridae. Neutralizing monoclonal antibodies specific for herpes simplex virus glycoprotein Infect. Agents Dis. 3 (1), 9–28. D inhibit virus penetration. J. Virol. 61 (11), 3356–3364. Pereira, L., Ali, M., Kousoulas, K., Huo, B., Banks, T., 1989. Domain structure of herpes Highlander, S.L., Cai, W.H., Person, S., Levine, M., Glorioso, J.C., 1988. Monoclonal simplex virus 1 glycoprotein B: neutralizing epitopes map in regions of continuous antibodies define a domain on herpes simplex virus glycoprotein B involved in virus and discontinuous residues. Virology 172 (1), 11–24. penetration. J. Virol. 62 (6), 1881–1888. Perez-Romero, P., Perez, A., Capul, A., Montgomery, R., Fuller, A.O., 2005. Herpes simplex Highlander, S.L., Dorney, D.J., Gage, P.J., Holland, T.C., Cai, W., Person, S., Levine, M., virus entry mediator associates in infected cells in a complex with viral proteins gD Glorioso, J.C., 1989. Identification of mar mutations in herpes simplex virus type and at least gH. J. Virol. 79 (7), 4540–4544. 216 D.G. Roller et al. / Virology 382 (2008) 207–216

Pertel, P.E., Fridberg, A., Parish, M.L., Spear, P.G., 2001. Cell fusion induced by herpes Stiles, K.M., Milne, R.S., Cohen, G.H., Eisenberg, R.J., Krummenacher, C., 2008. The herpes simplex virus glycoproteins gB, gD, and gH–gL requires a gD receptor but not simplex virus receptor nectin-1 is down-regulated after trans-interaction with necessarily heparan sulfate. Virology 279 (1), 313–324. glycoprotein D. Virology 373 (1), 98–111. Roche, S., Bressanelli, S., Rey, F.A., Gaudin, Y., 2006. Crystal structure of the low-pH form Subramanian, R.P., Geraghty, R.J., 2007. Herpes simplex virus type 1 mediates fusion of the vesicular stomatitis virus glycoprotein G. Science 313 (5784), 187–191. through a hemifusion intermediate by sequential activity of glycoproteins D, H, L, Roche, S., Rey, F.A., Gaudin, Y., Bressanelli, S., 2007. Structure of the prefusion form of the and B. Proc. Natl. Acad. Sci. U. S. A. 104 (8), 2903–2908. vesicular stomatitis virus glycoprotein g. Science 315 (5813), 843–848. Turner, A., Bruun, B., Minson, T., Browne, H., 1998. Glycoproteins gB, gD, and gHgL of Saharkhiz-Langroodi, A., Holland, T.C., 1997. Identiﬁcation of the fusion-from-without herpes simplex virus type 1 are necessary and sufﬁcient to mediate membrane determinants of herpes simplex virus type 1 glycoprotein B. Virology 227 (1), fusion in a Cos cell transfection system. J. Virol. 72 (1), 873–875. 153–159. Wahlberg, J.M., Garoff, H., 1992. Membrane fusion process of Semliki Forest virus. I: low Satoh, T., Arii, J., Suenaga, T., Wang, J., Kogure, A., Uehori, J., Arase, N., Shiratori, I., Tanaka, pH-induced rearrangement in spike protein quaternary structure precedes virus S., Kawaguchi, Y., Spear, P.G., Lanier, L.L., Arase, H., 2008. PILRalpha is a herpes penetration into cells. J. Cell. Biol. 116 (2), 339–348. simplex virus-1 entry coreceptor that associates with glycoprotein B. Cell 132 (6), Warner, M.S., Geraghty, R.J., Martinez, W.M., Montgomery, R.I., Whitbeck, J.C., Xu, R., 935–944. Eisenberg, R.J., Cohen, G.H., Spear, P.G., 1998. A cell surface protein with herpesvirus Shieh, M.T., WuDunn, D., Montgomery, R.I., Esko, J.D., Spear, P.G., 1992. Cell surface entry activity (HveB) confers susceptibility to infection by mutants of herpes receptors for herpes simplex virus are heparan sulfate proteoglycans. J. Cell. Biol. simplex virus type 1, herpes simplex virus type 2, and pseudorabies virus. Virology 116 (5), 1273–1281. 246 (1), 179–189. Spear, P.G., 1993. Membrane fusion induced by herpes simplex virus. In: Bentz, J. (Ed.), Weissenhorn, W., Hinz, A., Gaudin, Y., 2007. Virus membrane fusion. FEBS Lett. 581 (11), “Viral Fusion Mechanisms”. CRC Press, Inc., Boca Raton, FL, pp. 201–232. 2150–2155. Spear, P.G., Longnecker, R., 2003. Herpesvirus entry: an update. J. Virol. 77 (19), Wittels, M., Spear, P.G., 1991. Penetration of cells by herpes simplex virus does not 10179–10185. require a low pH-dependent endocytic pathway. Virus Res. 18 (2–3), 271–290.