Reversible conformational change in herpes simplex virus glycoprotein B with fusion-from-without activity is triggered by mildly acidic pH
© Siekavizza-Robles et al; licensee BioMed Central Ltd. 2010
Received: 26 October 2010
Accepted: 1 December 2010
Published: 1 December 2010
The pre-fusion form of the herpes simplex virus (HSV) fusion protein gB undergoes pH-triggered conformational change in vitro and during viral entry (Dollery et al., J. Virol. 84:3759-3766, 2010). The antigenic structure of gB from the fusion-from-without (FFWO) strain of HSV-1, ANG path, resembles wild type gB that has undergone pH-triggered changes. Together, changes in the antigenic and oligomeric conformation of gB correlate with fusion activity. We tested whether the pre-fusion form of FFWO gB undergoes altered conformational change in response to low pH.
A pH of 5.5 - 6.0 altered the conformation of Domains I and V of FFWO gB, which together comprise the functional region containing the hydrophobic fusion loops. The ANG path gB oligomer was altered at a similar pH. All changes were reversible. In wild type HSV lacking the UL45 protein, which has been implicated in gB-mediated fusion, gB still underwent pH-triggered changes. ANG path entry was inactivated by pretreatment of virions with low pH.
The pre-fusion conformation of gB with enhanced fusion activity undergoes alteration in antigenic structure and oligomeric conformation in response to acidic pH. We propose that endosomal pH triggers conformational change in mutant gB with FFWO activity in a manner similar to wild type. Differences apart from this trigger may account for the increased fusion activity of FFWO gB.
Membrane fusion during enveloped virus entry is mediated by conformational change in viral fusion proteins. Herpesviruses are a paradigm for viral entry mediated by a multi-component fusion machinery. Herpesviral fusion and entry is further complicated by the likely requirement of multiple cellular cues. Herpes simplex virus (HSV) glycoproteins gB, gD, and gH-gL are necessary for entry and membrane fusion [1–3]. A cellular receptor for gD is essential for entry, but one or more additional cellular triggers is also required. There is mounting evidence for the critical, direct role of endosomal pH during HSV entry by endocytosis, which is the predominant entry pathway for HSV in many cell types including human epithelial cells [4, 5]. Lysosomotropic agents, which elevate intravesicular pH, block HSV entry by trapping virions in endocytic compartments [4, 6]. Pretreatment of isolated HSV particles with mildly acidic pH inactivates viral entry activity, which is a characteristic of viruses that are directly triggered by endosomal pH for fusion . Low pH together with soluble gD-receptor triggers association of HSV with artificial membranes .
We recently demonstrated that gB present in virions, i.e., the pre-fusion form, undergoes conformational change in direct response to mildly acidic pH of 5.5 to 6.0, both in vitro and during viral entry into cells . Low pH caused a specific change in the antigenic structure of the functional region of gB containing the hydrophobic, bipartite fusion loops. A similar range of mildly acidic pH caused a change in the oligomeric conformation of gB. Low pH triggered gB to become more hydrophobic, suggesting that membrane-interacting regions are revealed. Conformational changes in gB were reversible. Taken together, these findings support a model in which endosomal low pH serves as a cellular trigger for fusion by activating the fusion protein gB .
The product of the HSV UL45 gene is a non-glycosylated, membrane protein that is present in the virion envelope and is dispensable for viral entry via endocytic and non-endocytic cell entry pathways [9, 10]. The role of the UL45 protein in the viral envelope is not known. HSV syncytium formation mediated by a Y854K mutation in the cytoplasmic tail of gB requires wild type UL45 .
Thus, UL45 may mediate fusion events during HSV infection through a functional interaction with gB.
Fusion-from-without (FFWO) is the rapid induction of cell fusion by virions in the absence of viral protein synthesis . HSV-1 ANG path is a prototype FFWO strain. The combination of two amino acid mutations in gB, one in the ectodomain (V553A) and one in the cytoplasmic tail (A855V), confers FFWO activity to wild type HSV . Virion-cell fusion during entry has been refractory to direct study. FFWO is a surrogate assay for fusion during entry because it parallels viral entry in several respects [14–16]. Importantly, the effector and target membranes for FFWO and entry are the same. Like entry, FFWO requires an appropriate gD-receptor in the target membrane. The efficiency of gD-receptor usage for FFWO correlates with the efficiency of entry mediated by the same receptor. Lastly, antibodies to gB and gD that block FFWO also neutralize virus entry. The pre-fusion form of gB with FFWO activity has an altered antigenic conformation relative to wild type gB . Interestingly, the pre-fusion wild type gB undergoes conformational changes in these same antigenic sites upon exposure to low pH . FFWO strains of HSV require endosomal low pH for entry in a cell-specific manner, similar to wild type [4, 16]. However, FFWO itself occurs at neutral pH and is not enhanced by acidic pH (unpublished data). In this report, we investigate the relationship between pH-triggered conformation changes and fusion activity by analyzing the effect of pH on virion gB with FFWO activity.
Results and discussion
Together the results indicate that conformational change in HSV-1 gB with FFWO activity is induced by pH ~ 5 to 6. We have proposed that low pH triggers the pre-fusion form of wild type gB, resulting in contact of its fusion loops with the target membrane . Mildly acidic pH may have a similar effect on FFWO strains of HSV such as ANG path. The enhanced fusogenic activity of FFWO gB may manifest itself downstream of initial activation by pH, such as during refolding of gB when the two membranes are brought into apposition. Our current battery of assays likely does not detect the full range of changes that occur in gB during fusion. It is also possible that the altered pre-fusion structure of FFWO gB relative to wild type may facilitate interactions between gB and gD or gH-gL during fusion. These possibilities need to be pursued experimentally.
The pre-fusion form of HSV gB present in three different strains, HSV-1 KOS and ANG path and HSV-2 333 undergoes conformational change in response to low pH  and this study). The structure of the HSV-1 gB ectodomain truncated at residue 730 has striking structural homology to the low pH, post-fusion form of vesicular stomatitis virus (VSV) G glycoprotein [20, 21]. The available gB structure is the post-fusion form [20, 22, 23]. Whether this form is crystallized at neutral or acidic pH, the structure is essentially identical , suggesting that low pH has a negligible effect on truncated gB that already resembles an activated, post-fusion conformation. The pre-fusion x-ray structure of herpes gB is not currently known, but the pre-fusion structure of G at neutral pH has been determined . We propose that the pH-induced transition from pre- to post-fusion gB during membrane fusion is similar to G, which undergoes significant domain rearrangement. There are unique features of the regulation and execution of herpes fusion due to the multiple cellular triggers and multiple viral proteins, however. For example, low pH induces gB to become a lower-order oligomer , but acid causes a tighter, stable association of G subunits . Finally, it remains to be seen whether pH-independent entry via penetration at the plasma membrane  is accompanied by similar changes in gB conformation.
Highly fusogenic gB with FFWO activity and wild type gB undergo pH-triggered changes in antigenic conformation and oligomeric structure. The structure of gB is not globally altered. The mutant, FFWO gB may have a pre-fusion conformation that facilitates membrane fusion, but it may be triggered by low pH in a manner similar to wild type. Entry of a FFWO strain of HSV is inactivated by acid pH. Low pH-triggered changes in gB are independent of the UL45 protein. The available data support a model in which a cellular cue, such as endosomal low pH, triggers structural changes in gB that are critical for fusion and entry.
Cells and viruses
Vero cells (American Type Culture Collection [ATCC], Rockville, MD) were propagated in Dulbecco's modified Eagle's medium (DMEM; Invitrogen, Grand Island, NY) supplemented with 10% fetal bovine serum (FBS; Gemini Bio-Products, West Sacramento, CA). HSV-1 strains ANG path, KOS, and KOS UL45 D  were propagated and titered on Vero cells.
Mouse monoclonal antibodies (MAbs) to gB designated DL16, SS10 and SS106  and gB-specific rabbit polyclonal antibody R69 were kindly provided by Roselyn Eisenberg and Gary Cohen, University of Pennsylvania. The anti-gB MAbs H126  and H1817 were obtained from Virusys. Rabbit polyclonal sera to HSV-1 UL45 protein was obtained from Curtis Brandt .
Dot blot analysis
Cell-free preparations of extracellular HSV-1 ANG path or KOS strains were diluted in serum-free, bicarbonate-free DMEM with 0.2% BSA and 5 mM each of HEPES (Life Technologies), 2-(N-morpholino)ethanesulfonic acid (MES; Sigma), and sodium succinate (Sigma) to achieve final pHs ranging from 7.4 to 5.2. Samples were incubated at 37°C for 5 min. Samples were either blotted directly to nitrocellulose with a Mini Fold dot blot system (Whatman) or were first neutralized by addition of pretitrated amounts of 0.05 N NaOH. In each case, equivalent amounts of ANG path and KOS virions (106 - 107 PFU) were blotted based on reactivity of the indicated antibody with virions treated with pH 7.4. Membranes were blocked and incubated at neutral pH with anti-gB monoclonal antibody. After incubation with horseradish peroxidase-conjugated goat-anti-mouse antibody, enhanced chemiluminescent substrate (Pierce) was added, and blots were exposed to X-ray film (Kodak). To highlight reduced reactivity or the pH threshold, exposures in which gB reactivity is in the linear range of detection for a given MAb are shown. Thus, the apparent absence of reactivity does not indicate a complete failure of an antibody to bind.
Assay for sensitivity of oligomeric gB to detergent
The oligomeric conformation of gB from virions exposed to pH < 6.0 is sensitive to 1% SDS as assessed by "native" PAGE . HSV-1 ANG path or KOS virions (~ 105 PFU) were treated with medium adjusted to pHs ranging from 7.4 to 5.2 as described above for dot blot. Virions were adjusted to 1% SDS and were then added to polyacrylamide gel electrophoresis (PAGE) sample buffer containing 0.2% sodium dodecyl sulfate (SDS) and no reducing agent ("native" conditions). Samples were not heated and were resolved by PAGE. After transfer to nitrocellulose, membranes were blocked and incubated with rabbit polyclonal antibody specific for gB. After incubation with horseradish peroxidase-conjugated goat-anti-rabbit antibody, enhanced chemiluminescent substrate (Pierce) was added and membranes were exposed to X-ray film (Kodak).
Inactivation of virions by low pH
HSV-1 ANG path or KOS was buffered in serum-free, sodium bicarbonate-free DMEM containing 0.2% BSA with 5 mM each of HEPES, MES and succinate to achieve final pHs ranging from 7.2 to 4.8 and incubated at 37°C for 5 min. Virions were neutralized to pH 7.4 by addition of pretitrated amounts of 0.05 N NaOH. Samples were diluted in sodium bicarbonate-buffered DMEM (pH 7.6) with 10% fetal bovine serum, and added to Vero cell monolayers for 18 hr. Plaque formation was evaluated by immunoperoxidase staining. Infectivity of samples maintained at pH 7.4 was set to 100%.
This investigation was supported by Public Health Service grant AI-083850 from the National Institute of Allergy and Infectious Diseases and a grant from the Japan Health Sciences Foundation (SAA4832). We are grateful to Curtis Brandt, Gary Cohen and Roselyn Eisenberg for generous gifts of reagents. We thank Mark Delboy for critical reading of the manuscript and Abena Watson-Siriboe for technical assistance.
- Krummenacher C, Carfi A, Eisenberg RJ, Cohen GH: Herpesvirus entry into cells: The Enigma Variations. In Viral entry into host cells. Edited by: Poehlmann S. Simmons G: Landes Bioscience; 2007.
- Spear PG, Longnecker R: Herpesvirus entry: an update. J Virol 2003, 77: 10179-10185. 10.1128/JVI.77.19.10179-10185.2003PubMedPubMed CentralView Article
- Campadelli-Fiume G, Amasio M, Avitabile E, Cerretani A, Forghieri C, Gianni T, Menotti L: The multipartite system that mediates entry of herpes simplex virus into the cell. Rev Med Virol 2007, 17: 313-326. 10.1002/rmv.546PubMedView Article
- Nicola AV, McEvoy AM, Straus SE: Roles for endocytosis and low pH in herpes simplex virus entry into HeLa and Chinese hamster ovary cells. J Virol 2003, 77: 5324-5332. 10.1128/JVI.77.9.5324-5332.2003PubMedPubMed CentralView Article
- Nicola AV, Hou J, Major EO, Straus SE: Herpes simplex virus type 1 enters human epidermal keratinocytes, but not neurons, via a pH-dependent endocytic pathway. J Virol 2005, 79: 7609-7616. 10.1128/JVI.79.12.7609-7616.2005PubMedPubMed CentralView Article
- Nicola AV, Straus SE: Cellular and viral requirements for rapid endocytic entry of herpes simplex virus. J Virol 2004, 78: 7508-7517. 10.1128/JVI.78.14.7508-7517.2004PubMedPubMed CentralView Article
- Whitbeck JC, Zuo Y, Milne RS, Cohen GH, Eisenberg RJ: Stable association of herpes simplex virus with target membranes is triggered by low pH in the presence of the gD receptor, HVEM. J Virol 2006, 80: 3773-3780. 10.1128/JVI.80.8.3773-3780.2006PubMedPubMed CentralView Article
- Dollery SJ, Delboy MG, Nicola AV: Low pH-induced conformational change in herpes simplex virus glycoprotein B. J Virol 2010, 84: 3759-3766. 10.1128/JVI.02573-09PubMedPubMed CentralView Article
- Visalli RJ, Brandt CR: The HSV-1 UL45 gene is not required for growth in Vero cells. Virology 1991, 185: 419-423. 10.1016/0042-6822(91)90790-IPubMedView Article
- Dollery SJ, Lane KD, Delboy MG, Roller DG, Nicola AV: Role of the UL45 protein in herpes simplex virus entry via low pH-dependent endocytosis and its relationship to the conformation and function of glycoprotein B. Virus Res 2010, 149: 115-118. 10.1016/j.virusres.2010.01.004PubMedPubMed CentralView Article
- Haanes EJ, Nelson CM, Soule CL, Goodman JL: The UL45 gene product is required for herpes simplex virus type 1 glycoprotein B-induced fusion. J Virol 1994, 68: 5825-5834.PubMedPubMed Central
- Falke D, Knoblich A, Muller S: Fusion from without induced by herpes simplex virus type 1. Intervirology 1985, 24: 211-219. 10.1159/000149645PubMedView Article
- Saharkhiz-Langroodi A, Holland TC: Identification of the fusion-from-without determinants of herpes simplex virus type 1 glycoprotein B. Virology 1997, 227: 153-159. 10.1006/viro.1996.8327PubMedView Article
- Delboy MG, Roller DG, Nicola AV: Cellular proteasome activity facilitates herpes simplex virus entry at a postpenetration step. J Virol 2008, 82: 3381-3390. 10.1128/JVI.02296-07PubMedPubMed CentralView Article
- Delboy MG, Patterson JL, Hollander AM, Nicola AV: Nectin-2-mediated entry of a syncytial strain of herpes simplex virus via pH-independent fusion with the plasma membrane of Chinese hamster ovary cells. Virol J 2006, 3: 105. 10.1186/1743-422X-3-105PubMedPubMed CentralView Article
- Roller DG, Dollery SJ, Doyle JL, Nicola AV: Structure-function analysis of herpes simplex virus glycoprotein B with fusion-from-without activity. Virology 2008, 382: 207-216. 10.1016/j.virol.2008.09.015PubMedView Article
- Bender FC, Samanta M, Heldwein EE, de Leon MP, Bilman E, Lou H, Whitbeck JC, Eisenberg RJ, Cohen GH: Antigenic and mutational analyses of herpes simplex virus glycoprotein B reveal four functional regions. J Virol 2007, 81: 3827-3841. 10.1128/JVI.02710-06PubMedPubMed CentralView Article
- Zhou J, Blissard GW: Mapping the conformational epitope of a neutralizing antibody (AcV1) directed against the AcMNPV GP64 protein. Virology 2006, 352: 427-437. 10.1016/j.virol.2006.04.041PubMedPubMed CentralView Article
- Gaudin Y, Tuffereau C, Segretain D, Knossow M, Flamand A: Reversible conformational changes and fusion activity of rabies virus glycoprotein. J Virol 1991, 65: 4853-4859.PubMedPubMed Central
- Heldwein EE, Lou H, Bender FC, Cohen GH, Eisenberg RJ, Harrison SC: Crystal structure of glycoprotein B from herpes simplex virus 1. Science 2006, 313: 217-220. 10.1126/science.1126548PubMedView Article
- Roche S, Bressanelli S, Rey FA, Gaudin Y: Crystal structure of the low-pH form of the vesicular stomatitis virus glycoprotein G. Science 2006, 313: 187-191. 10.1126/science.1127683PubMedView Article
- Backovic M, Longnecker R, Jardetzky TS: Structure of a trimeric variant of the Epstein-Barr virus glycoprotein B. Proc Natl Acad Sci USA 2009, 106: 2880-2885. 10.1073/pnas.0810530106PubMedPubMed CentralView Article
- Lin E, Spear PG: Random linker-insertion mutagenesis to identify functional domains of herpes simplex virus type 1 glycoprotein B. Proc Natl Acad Sci USA 2007, 104: 13140-13145. 10.1073/pnas.0705926104PubMedPubMed CentralView Article
- Roche S, Rey FA, Gaudin Y, Bressanelli S: Structure of the prefusion form of the vesicular stomatitis virus glycoprotein g. Science 2007, 315: 843-848. 10.1126/science.1135710PubMedView Article
- Doms RW, Keller DS, Helenius A, Balch WE: Role for adenosine triphosphate in regulating the assembly and transport of vesicular stomatitis virus G protein trimers. J Cell Biol 1987, 105: 1957-1969. 10.1083/jcb.105.5.1957PubMedView Article
- Fuller AO, Spear PG: Anti-glycoprotein D antibodies that permit adsorption but block infection by herpes simplex virus 1 prevent virion-cell fusion at the cell surface. Proc Natl Acad Sci USA 1987, 84: 5454-5458. 10.1073/pnas.84.15.5454PubMedPubMed CentralView Article
- Kousoulas KG, Pellett PE, Pereira L, Roizman B: Mutations affecting conformation or sequence of neutralizing epitopes identified by reactivity of viable plaques segregate from syn and ts domains of HSV-1(F) gB gene. Virology 1984, 135: 379-394. 10.1016/0042-6822(84)90194-6PubMedView Article
- Visalli RJ, Brandt CR: The HSV-1 UL45 18 kDa gene product is a true late protein and a component of the virion. Virus Res 1993, 29: 167-178. 10.1016/0168-1702(93)90057-TPubMedView Article
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.