Tetherin Restricts Herpes Simplex Virus Type 1 and is Antagonised by Glycoprotein M

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Tetherin Restricts Herpes Simplex Virus Type 1 and is Antagonised な by Glycoprotein M に

ぬ Running Title : Restriction of HSV-1 by Tetherin ね の Caroline Blondeau1, Annegret Pelchen-Matthews2, Petra Mlcochova1,2, Mark は Marsh2, Richard S. B. Milne1ў, Greg J. Towers1ў ば 1University College London, Medical Research Council Centre for Medical ぱ Molecular Virology, Division of Infection and Immunity, University College London, ひ 90 Gower St, London WC1E 6BT, United Kingdom など 2MRC Laboratory for Molecular Cell Biology, University College London, Gower なな St., London WC1E 6BT, United Kingdom なに ўCorresponding authors なぬ Greg Towers : MRC Centre for Medical Molecular Virology, Division of Infection なね and Immunity, University College London, Cruciform Building, 90 Gower St, なの London WC1E 6BT. Phone : +44 20 3108 2112. Email : [email protected] なは Richard Milne : MRC Centre for Medical Molecular Virology, Division of Infection なば and Immunity, University College London, Cruciform Building, 90 Gower St, なぱ London WC1E 6BT. Phone : +44 20 7830 2997. Email : [email protected] なひ にど Keywords : HSV-1, tetherin, restriction, glycoprotein M, antagonism にな Abstract : 131 words. Text : 4503 words にに

JVI Accepts, published online ahead of print on 25 September 2013J. Virol. doi:10.1128/JVI.02250-13Copyright © 2013, American Society for Microbiology. All Rights Reserved.

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Abstract にぬ Tetherin is a broadly active antiviral effector that works by tethering nascent にね enveloped virions to a host cell membrane, thus preventing their release. In this にの study we demonstrate that herpes simplex virus type 1 (HSV-1) is targeted by には tetherin. We identify the viral envelope glycoprotein M (gM) as having moderate にば anti-tetherin activity. We show that gM but not gB or gD removes efficiently にぱ tetherin from the plasma membrane and can functionally substitute for the human にひ immunodeficiency virus type 1 (HIV-1) Vpu protein, the prototypic viral tetherin ぬど antagonist, in rescuing HIV-1 release from tetherin expressing cells. Our data ぬな emphasise that tetherin is a broadly active antiviral effector and contribute to the ぬに emerging hypothesis that viruses must suppress or evade an array of host cell ぬぬ countermeasures in order to establish a productive infection. ぬね ぬの ぬは ぬば ぬぱ ぬひ ねど ねな ねに ねぬ

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Introduction ねね Mammalian cells encode restriction factors that provide the host with protection ねの against virus replication. In order to establish a productive infection, a virus must ねは evade or suppress a repertoire of restriction factors directed against it by the host. ねば Restriction has been studied most extensively in the context of retrovirus ねぱ infections where numerous factors and the corresponding viral antagonists have ねひ been well characterised (24). Whilst some restriction factors, such as TRIM5g, のど appear to be specific for particular classes of virus, in this case retroviruses, のな others, such as tetherin, are broadly active against unrelated viruses. Tetherin のに (also known as BST-2, CD317, or HM1.24) was identified as the cellular factor のぬ responsible for suppression of Vpu negative human immunodeficiency virus type のね 1 (HIV-1) (30, 38) but subsequent work has shown that it is effective against a のの variety of enveloped viruses (13, 18, 22, 30, 36, 46) that use distinct mechanisms のは to antagonise its restrictive effects (5, 21, 35, 44). Tetherin is a type-2 integral のば membrane protein with a C-terminal GPI anchor. The antiviral activity of tetherin のぱ stems from this unusual double membrane linked topology that allows the のひ formation of a protein tether between the host membrane and the budding viral はど envelope, preventing release of nascent virions (32). はな はに Herpesviruses, a large family of enveloped DNA viruses, are ancient pathogens はぬ thought to have coevolved with their hosts for many generations (26). As such はね they might be expected to possess countermeasures to a variety of restriction はの factors and thus to provide a good experimental model system for studies of this はは

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aspect of the virus host interaction. To date, two members of this virus family, はば Kaposi’s sarcoma associated herpesvirus (KSHV) and human cytomegalovirus はぱ (HCMV), have been shown to interact with tetherin (25, 31, 40). Surprisingly, the はひ mode of interaction differs for these two viruses, with tetherin acting as a ばど restriction factor for KSHV but as an entry co-factor for HCMV. In this study we ばな have investigated the effect of tetherin on another human herpesvirus, herpes ばに simplex virus type 1 (HSV-1). We show that tetherin restricts the HSV-1 ばぬ replication cycle by suppressing virus release and we identify the viral envelope ばね glycoprotein M (gM) as a countermeasure contributing to antagonism of tetherin ばの restriction. ばは ばば Materials and Methods ばぱ Cell lines, plasmids, and viruses. HT1080 cells expressing internally HA-ばひ tagged human tetherin (at amino acid 154) or empty vector (LHCX) are non-ぱど clonal drug selected populations and have been described (31), as has the ぱな tetherin expression vector pCR3.1/hu-Tetherin-HA (27). The HSV-1 gM (UL10) ぱに gene was PCR amplified from HSV-1 17+ infected cell DNA and inserted into ぱぬ pCDNA3. The HSV-1 gB (UL27) and gD (US6) plasmids (pSR175 and pSC390) ぱね were gifts from from Roselyn Eisenberg and Gary Cohen (University of ぱの Pennsylvania) (6, 29). Plasmids expressing VpU, in pCDNA3 (HIV-1 release ぱは assay) or pIRESeGFP (flow cytometry) were described previously (33). HSV-1 ぱば SC16 wild-type (WT) and HSV-1 KOS K26GFP encoding a VP26-GFP fusion ぱぱ protein (8) were gifts from Gillian Elliott (Imperial College London). HSV-1 deleted ぱひ

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for UL10 (∆gM), and its revertant (RgM), were gifts from Dr Helena Browne ひど (University of Cambridge) and their construction has been described (34). ひな HSV-1 replication assay HT1080 cells (3x105 cells/well, 6-well plates) were ひに chilled to 4oC then incubated with HSV-1 for 1 hour. Plates were then refed and ひぬ transferred to 37oC for a further hour. The medium was then removed and ひね replaced with acid-citrate buffer (500 µl pH 3.0) to inactivate extracellular virus, ひの followed by addition of fresh media. Infected cell culture supernates were ひは recovered at various times post-infection, centrifuged to remove cellular debris ひば and virus titres determined by plaque assay on Vero cells. For cell-associated ひぱ virus titres, cells were lysed by 3 freeze thaw cycles into an equal volume of ひひ medium, cleared by centrifugation and titrated as above. The HSV-1 proteins などど ICP4 and VP5 were detected in infected cell lysates by immunoblotting using などな specific antibodies (Santa Cruz). As a loading control we detected く-actin などに (Abcam) on stripped blots. などぬ RNA interference We used lentiviral vectors encoding tetherin specific hairpins などね (shRNA1 5’-ggaguucugguguuccugauuauuucgaugaucaggagcaccagaauucc-3’, などの shRNA2 5’-gugggaaucguggauaagaaguauucguacuucuuguccgcgauucucac-3’), or などは a GFP hairpin (43) as a control. Depletion was examined by immunoblotting or by などば quantitative PCR on cDNA (see below). Cells were infected with HSV-1 96 hours などぱ post-shRNA transduction as described above. などひ Quantification of Tetherin and HSV-1 by Taqman PCR Encapsidated HSV-1 ななど genomes were quantified by extracting total DNA from DNAse-I treated ななな supernates or infected cell lysates as described (31). DNA was subjected to ななに

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quantitative Taqman PCR (Q-PCR) for HSV-1 UL27 as described (17). Absolute ななぬ copy number was determined by reference to a standard curve, plotted using ななね serial dilutions of a cloned UL27 amplicon with a detection limit of 10 UL27 ななの copies/15 µl of supernate. Copy numbers were normalized to extracted DNA ななは carrier concentration (supernates), or to quantities of extracted DNA (cells). Total ななば mRNA was extracted from transduced HeLa cells, or from HT1080 cells ななぱ expressing HA-tagged tetherin infected or not with HSV-1 SC16, and cDNA ななひ synthesised for use as template in Taqman Q-PCR reactions for tetherin and なにど GAPDH. Tetherin primers: forward, 5’-acctgcaaccacactgtgatg-3’; reverse, 5’-なにな caagctcctccactttcttttgtc-3’; tetherin probe, 5’-FAM-ccctaatggcttccctggatgcaga-なにに TAMRA-3’. Absolute copy number was determined with reference to a standard なにぬ curve derived using a tetherin encoding plasmid. Q-PCR for GAPDH was なにね performed as described (31). なにの Flow cytometry For tetherin cell surface staining, HEK293T cells in 6 well plates なには were transfected (Fugene-6, Roche) with pCR3.1/hu-Tetherin-HA and 250 ng, なにば 500 ng, or 1000 ng of plasmids expressing gM, gD or gB (33). A pIRES2eGFP なにぱ plasmid coding for VpU was used as a control. 48 hours post-transfection, cell なにひ surface tetherin expression was examined on unfixed live cells with an anti-HA なぬど monoclonal antibody (Covance) and analysed by flow cytometry. The mean なぬな fluorescence intensity of tetherin staining was measured as described (36). HSV-なぬに 1 glycoproteins were detected by immunoblotting with a rabbit anti-gM (34), なぬぬ rabbit anti-gD R8 (16), or a goat anti-gB (Santa Cruz) after membrane stripping. なぬね

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As a loading control we detected く-actin, GAPDH (Abcam) or transferrin receptor なぬの (Invitrogen). なぬは HIV-1 release assay To prepare VSV-G pseudotyped HIV-1 particles, 106 なぬば HEK293T cells were cotransfected with the gag-pol expression vector p8.91 (300 なぬぱ ng), pMDG encoding the Vesticular Stomatis Virus G glycoprotein (VSV-G) (300 なぬひ ng) and HIV-1 vector encoding YFP (450 ng) (12). 100 ng of tetherin construct なねど were co-transfected along with either 250 ng, 500 ng, or 1000 ng of HSV-1 gM, なねな gB, gD or HIV-1 Vpu. DNA dose was equalized with empty vector, pcDNA3 なねに (Invitrogen). After 48 hours, cell-associated p55-Gag and p24-Capsid, and p24-なねぬ Capsid in the supernate, were detected by immunoblotting as described (12). なねね The intensity of p55 bands in cell lysates and p24 bands in virions were analysed なねの with Image Studio 3.1.4 software (LI-COR) and ratios of p55:p24 were calculated なねは with signal intensity percentages relative to values obtained in absence of なねば tetherin. なねぱ Microscopy For electron microscopy, 1x105 HT1080 expressing HA-tagged なねひ tetherin or control cells seeded on coverslips were infected with 2x105 なのど (experiment 1) or 1x105 (experiment 2) pfu (determined on Vero cells) of HSV-1 なのな K26GFP (HT1080 are an order of magnitude less permissive than Vero to HSV-1 なのに and therefore multiplicities for these experiments can be estimated to be 0.2 and なのぬ 0.1 pfu/cell respectively). After 16 hours, the cells were fixed for 45 min in 2% なのね PFA/2% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.4), post-fixed for なのの 1 hour on ice in 1% OsO4/1.5% K3[Fe(CN)6], treated with 1.5% tannic acid (TAAB なのは Laboratories), dehydrated and embedded in Epon 812 (TAAB). Ultrathin sections なのば

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(70 nm) were cut en face on a Leica EM UC7 ultramicrotome, placed on formvar-なのぱ coated slot grids and stained with lead citrate. Sections were examined with a なのひ Tecnai G2 Spirit transmission EM (FEI) and digital images were recorded with a なはど Morada 11 MegaPixel TEM camera (Olympus Soft Imaging Solutions) and the なはな ANALYSIS software. Images were adjusted for brightness and contrast and なはに figures assembled with Photoshop CS. To determine the numbers of cell surface なはぬ HSV-1 particles at least 50 consecutive /adjacent cell profiles in the section were なはね inspected for each sample. Circular profiles of HSV-1 particles, measuring なはの between 80 and 180 nm in diameter and with the morphologies indicated in Fig 3, なはは were counted. なはば For confocal microscopy, HEK293T cells were plated onto poly-L-lysine coated なはぱ coverslips in 24 well plates and transfected (Fugene-6, Roche) with plasmid なはひ encoding gM, gB or gD and/or HA-tagged tetherin. Between 20 and 48 hours なばど later cells were fixed (4% PFA), permeabilized (0.1% Triton X-100) and stained なばな using anti-HA (Covance), rabbit anti-gM, rabbit anti-gD R8, goat anti-gB or sheep なばに anti-TGN46 (Serotec) antibodies and secondary antibodies linked to Alexa-488, なばぬ 594 or 633 (Molecular Probes), or rhodamine (Pierce). Cells were observed using なばね a Leica TCS SPE, DM2500 confocal microscope (Leica Microsystems). Images なばの were adjusted for brightness and contrast with Adobe Photoshop software 10.0. なばは なばば Results なばぱ Tetherin restricts HSV-1 particle release なばひ

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To seek evidence for restriction of HSV-1 we expressed HA-tagged human なぱど tetherin in HT1080 cells. These cells were chosen because they naturally なぱな express very low levels of tetherin and are highly permissive for HSV-1 (2). As a なぱに control, we used HT1080 cells transduced with empty vector. We hypothesized なぱぬ that tetherin over-expression might saturate any anti-tetherin activities mediated なぱね by the virus and reveal tetherin sensitivity. We infected both HT1080 cell lines なぱの with HSV-1 SC16 at low multiplicity (0.01 pfu/cell) aiming to study the effect of なぱは tetherin in a multi-cycle infection by measuring the titre of virus released at なぱば various times by plaque assay on Vero cells. Tetherin expression consistently なぱぱ reduced the levels of infectious virus released, leading to a 14-fold reduction at なぱひ 48 hours post-infection (pi), as compared to controls (Fig. 1A). Quantitative PCR なひど (Q-PCR) detection of DNAse-I resistant (ie encapsidated) HSV-1 DNA in the なひな infected cell culture supernates demonstrated a comparable decrease in signal, なひに supporting the notion that tetherin suppresses virion release from infected cells なひぬ (Fig. 1B). Tetherin expression did not affect titres of cell-associated virus (Fig. なひね 1C) or levels of cell-associated viral DNA (Fig. 1D) at early time points up to 24 なひの hours pi. At later times post-infection, titres of cell-associated virus from tetherin-なひは expressing cells were 7-8 fold lower than controls (Fig. 1C) and, consistent with なひば this, levels of viral proteins detected by western blotting were also reduced (Fig. なひぱ 1E). We assume that at these later time points we see the cumulative effect of なひひ tetherin’s inhibition of viral release and the consequent reduction in number of にどど newly infected cells in subsequent rounds of infection. Importantly, in a plaque にどな assay there was no difference between tetherin expressing and control HT1080 にどに

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cells in the number, or size, of plaques obtained from a given virus dose にどぬ suggesting that tetherin had no impact on HSV-1 entry, or direct cell-to-cell にどね spread (Fig. 1F). This is consistent with the specificity of tetherin for virus release にどの over cell-to-cell spread as has been described for tetherin restriction of にどは lentiviruses HIV-1 and feline immunodeficiency virus (FIV) (9, 19). にどば にどぱ We next tested whether tetherin expression could suppress high multiplicity にどひ infection. Tetherin expressing and control HT1080 cells were infected with HSV-1 になど (input multiplicity 3 pfu/cell), and the virus yield measured at various times by になな plaque assay as before (Fig. 1G). Again, slightly less infectious virus was になに released into the supernate from tetherin expressing cells at the earliest time of になぬ 14 hours although this difference was statistically insignificant. Moreover, になね infectious titres in the supernates were equal by 24 hours after infection and up to になの 48 hours pi. Q-PCR detection of DNAse protected viral genomes confirmed this になは effect (Fig. 1H). Cell-associated virus titres at 14 hours pi were very similar and になば infected cell lysates showed similar levels of viral proteins, confirming that になぱ tetherin has no effect on viral DNA or protein synthesis (Fig. 1 I, J). These data になひ suggest that during high multiplicity infection tetherin is antagonised by HSV-1 ににど infection. To test whether tetherin protein levels were impacted by HSV-1 ににな infection we measured them by immunoblot at various time points after HSV-1 ににに infection at an input multiplicity of 2 pfu/cell (Fig. 1K). We found that tetherin ににぬ levels declined as viral protein VP5 increased. We assume that the different ににね tetherin bands observed represent differently glycosylated forms (1). Importantly, ににの

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all were reduced after infection. The loss of tetherin expression was somewhat にには specific as く-actin and transferrin receptor protein levels were unaffected up to ににば 16 hours after infection and GAPDH levels were only slightly reduced (Fig. 1K). ににぱ We next measured tetherin and GAPDH mRNA levels by quantitative RT-PCR in ににひ the same samples as Fig. 1K. We found that tetherin mRNA levels declined with にぬど a similar time course to protein levels suggesting a role for the virus host shutoff にぬな (VHS) function in which host mRNAs are degraded by the VHS protein encoded にぬに by the HSV-1 UL41 gene (Fig. 1L). GAPDH mRNA was also lost, although with にぬぬ less concomitant reduction in protein expression. We conclude that tetherin is にぬね partly antagonised through suppression of expression and that this likely にぬの accounts for the loss of restriction after high multiplicity infection (Fig. 1G). These にぬは data are consistent with the recently reported antagonism of tetherin by the HSV-にぬば 1 VHS response (45), but do not rule out the possibility that tetherin might be にぬぱ degraded through additional mechanisms. にぬひ にねど Tetherin depletion with shRNA increases HSV-1 release にねな To confirm that tetherin was responsible for the reduction in HSV-1 release seen にねに in Fig. 1A, we depleted endogenous levels of tetherin expression from HeLa cells にねぬ and measured release of HSV-1 into the supernate at various times after low にねね multiplicity infection (0.1 pfu/cell). HeLa cells are known to express amounts of にねの tetherin that restrict HIV-1 deleted for its tetherin antagonist Vpu (12). We used にねは two tetherin specific shRNAs and a control shRNA targeting GFP. Expression of にねば either of the anti-tetherin shRNAs improved the release of virus as indicated by にねぱ

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an increase in infectious titre in supernates particularly after 39 hours pi, にねひ compared with the titre obtained from cells expressing shGFP (Fig. 2A). Q-PCR にのど detection of DNAse protected genomes confirmed that less virus was released にのな from GFP specific shRNA expressing cells (Fig. 2B). Quantitative RT-PCR にのに showed that endogenous tetherin mRNA levels were reduced in tetherin specific にのぬ shRNA expressing HeLa cells as compared to cells expressing the hairpin にのね targeting GFP (Fig. 2C). The effect of shRNA expression was also confirmed by にのの immunoblot detecting the HA-tag in extracts of HT1080 cells expressing HA-にのは tagged tetherin (Fig. 2D) and transduced with shRNA-encoding lentivectors. にのば にのぱ Tetherin induces accumulation of HSV-1 particles at the cell surface にのひ Having established that tetherin can restrict HSV-1 release we sought to visualise にはど restricted virus on the surface of tetherin expressing cells. Thin section electron にはな microscopy revealed that in HSV-1 infected HT1080 cells that do not express にはに tetherin there were few virions associated with the cell surface. However, in にはぬ infected cells over-expressing tetherin there were areas of cell surface where にはね many virions were associated with the plasma membrane (Fig. 3 A-G). To にはの quantify this effect we counted cell surface-associated virions on control and にはは tetherin expressing cell profiles in a blinded manner. In two experiments tetherin にはば expressing cells had significantly more cell surface virions per cell profile than にはぱ control cells (Table 1, Fig. 3H). These observations are consistent with tetherin にはひ suppressing HSV-1 release from infected cells (Fig. 1). にばど にばな

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HSV-1 Glycoprotein M can Antagonise Tetherin にばに Viruses typically encode countermeasures to the repertoire of restriction factors にばぬ expressed by their natural host. Numerous viral countermeasures to tetherin にばね have been described (13, 18, 21, 22, 30, 35, 36, 46) which share one key にばの mechanistic characteristic: they remove tetherin from the location of its antiviral にばは activity. Importantly, glycoproteins from unrelated viruses including lentiviruses にばば (HIV-2, SIVtan) (9,16) and Ebola virus (14) have been shown to have anti-にばぱ tetherin activity. With this in mind, we hypothesized that the HSV-1 envelope にばひ glycoprotein M (gM) may also act as a tetherin antagonist. This protein has been にぱど shown to relocalise membrane proteins (7, 34) and a deletion mutant replicates にぱな to reduced titres in a number of cell lines (2, 23, 34). にぱに にぱぬ To investigate whether gM has a role in tetherin antagonism, we first used にぱね confocal immunofluorescence microscopy to investigate the effect of gM にぱの expression on tetherin localisation. We co-transfected HEK293T cells with にぱは plasmids encoding HSV-1 gM and HA-tagged tetherin and examined their にぱば localisation after 48 hours (Fig. 4A). In cells that were not expressing gM, or にぱぱ expressing low levels of gM (open arrowheads), tetherin was predominantly にぱひ localised to the plasma membrane, as described previously (30). However, in にひど cells staining brightly for gM, a significant proportion of the tetherin was localised にひな in the perinuclear region of the cell where it overlapped with gM labelling (solid にひに arrowheads). Further investigation indicated that the gM labelling also overlapped にひぬ with labelling for the trans-Golgi network marker TGN46 (Fig. 4B), suggesting にひね

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that tetherin-gM complexes are located in the TGN. Together, these data suggest にひの that, like HIV-1 Vpu, gM can relocalise tetherin, consistent with it having a role in にひは antagonising the restriction of virus release. にひば にひぱ In order to assess the specificity of tetherin antagonism by gM we compared the にひひ localisation of tetherin after expression of gM and HSV-1 glycoproteins gD and ぬどど gB. As before, we expressed the glycoproteins transiently, together with HA-ぬどな tagged tetherin, in 293T cells and stained tetherin and each glycoprotein using ぬどに specific antibodies. We found that, as before, gM caused a relocalisation of ぬどぬ tetherin from the plasma membrane to an internal compartment (Fig. 5A). On the ぬどね other hand, gD had no such effect and tetherin mostly localised to the cell ぬどの surface as in control cells. On expression of gB we found that there was a less ぬどは extensive relocalisation of tetherin as compared to that after gM expression. We ぬどば conclude that gD has no tetherin relocalisation activity and that gB may have ぬどぱ some activity but less than that of gM. ぬどひ ぬなど To examine tetherin antagonism by HSV-1 glycoproteins further we used flow ぬなな cytometry to quantify tetherin surface expression when tetherin was co-ぬなに expressed with glycoproteins, again in 293T cells. Consistent with ぬなぬ immunofluorescence data, FACS analysis showed reduced levels of tetherin on ぬなね the surface of gM expressing cells as compared to cells expressing empty vector ぬなの (Fig. 5B). We used the HIV-1 tetherin antagonist Vpu as a positive control in ぬなは these experiments. gD expression had no effect whereas gB caused a small ぬなば

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reduction of tetherin surface staining that was less than that caused by gM (Fig. ぬなぱ 5B). All glycoproteins were expressed efficiently as measured by immunoblot (Fig. ぬなひ 5C) and there was a striking elevation in the amount of tetherin in the gM-ぬにど expressing cells, consistent with the gM-driven accumulation in the TGN (Fig. 4B). ぬにな The flow cytometry measurements (Fig. 5B) were therefore consistent with ぬにに immunofluorescent staining of tetherin expression (Fig. 5A). ぬにぬ ぬにね For a functional assessment of the antagonistic effect of HSV-1 glycoproteins on ぬにの tetherin and to provide further mechanistic insight, we asked whether they could ぬには substitute for Vpu in an HIV-1 release assay (12). HIV-1 particles released to the ぬにば supernate were detected by immunoblotting for the HIV-1 gag structural protein ぬにぱ p24 at 48 hours post-transfection. As expected, Vpu expression effectively ぬにひ antagonised tetherin restriction and HIV-1 particles (p24-CA) were detected in ぬぬど the culture supernate at levels equivalent to those obtained in the absence of ぬぬな tetherin (Fig. 5D). When we replaced Vpu with gM we saw a comparable rescue ぬぬに of HIV-1 release (intracellular-p55:supernate-p24 ratios are shown in Fig 5D). ぬぬぬ These data demonstrate that gM can rescue HIV-1 budding from tetherin ぬぬね restriction and are consistent with gM acting as an antagonist of the antiviral ぬぬの function of tetherin during HSV-1 infection. Importantly, neither gB nor gD were ぬぬは able to rescue HIV-1 release. Thus whilst gB had a weak effect on tetherin ぬぬば localisation it was unable to functionally substitute for Vpu in an assay directly ぬぬぱ measuring functional antagonism of tetherin. These observations suggest that ぬぬひ gM has specificity in antagonising tetherin. Clearly this model system depends on ぬねど

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over-expression of Vpu and gM, likely in excess of the levels achieved during an ぬねな infection; nevertheless it allows a useful functional comparison between ぬねに unrelated molecules and provides an independent demonstration that gM has ぬねぬ anti-tetherin activity. ぬねね ぬねの We next asked whether the tetherin antagonism shown by gM facilitated release ぬねは of HSV-1 from infected cells. We infected control or tetherin expressing HT1080 ぬねば cells with HSV-1 SC16 (WT), a gM deleted virus (∆gM), or a revertant virus ぬねぱ (RgM) using a low input multiplicity (0.01 pfu/cell). Titres of the three viruses ぬねひ released from control cells were indistinguishable (Fig. 6A). However, on cells ぬのど over-expressing tetherin, while all three viruses were inhibited, the ∆gM virus was ぬのな inhibited by a small but statistically significant degree over 48 hours (2-3 fold) ぬのに (Fig. 6B). We reasoned that, as in the experiments shown in Fig. 1, tetherin over-ぬのぬ expression largely saturated HSV-1 tetherin antagonism leading to inhibition of all ぬのね three viruses. Slightly stronger restriction of the ∆gM virus was likely due to its ぬのの reduced ability to antagonise tetherin. Importantly, there was no significant ぬのは difference between the HT1080 lines in the number of plaques obtained for any ぬのば of the viruses for a given dose (Fig. 6C). Absence of gM from ∆gM-infected cells ぬのぱ was confirmed by immunoblotting for gM protein in infected cell extracts (Fig. 6D). ぬのひ Finally, we infected HT1080 over-expressing HA-tagged tetherin with RgM or ぬはど ∆gM viruses and analysed cell lysates by western-blot, detecting HA-Tetherin, ぬはな the HSV-1 capsid protein VP5 or く-actin as a loading control, at various time ぬはに points pi (Fig. 6E). As early as 4-6 hours pi, we observed a reduction of tetherin ぬはぬ

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protein in infected cells compared to non-infected cells, as previously shown (Fig. ぬはね 1K). Tetherin was almost completely lost by 10 hours pi. gM expression was not ぬはの responsible for loss of tetherin as indicated by the observation that the ∆gM virus ぬはは infection also led to tetherin loss. These data indicate that whilst HSV-1 gM can ぬはば act as a tetherin antagonist, the virus has at least one other anti-tetherin activity ぬはぱ responsible for loss of tetherin protein. ぬはひ ぬばど Discussion ぬばな Here we extend the repertoire of tetherin restricted viruses to HSV-1 and the list ぬばに of tetherin antagonists to include the HSV-1 glycoprotein gM. It is striking that ぬばぬ glycoproteins are common as tetherin antagonists, with anti-tetherin activity being ぬばね described for glycoproteins from the lentiviruses SIVtan and HIV-2 as well as the ぬばの filovirus Ebola (13, 21, 22). Our data suggest a complex relationship between ぬばは tetherin and HSV-1. Tetherin is clearly antagonised by HSV-1 in at least two ぬばば independent ways, by gM mediated relocalisation and through a gM independent ぬばぱ suppression of tetherin mRNA (Fig. 1) likely through the VHS response (45). ぬばひ ぬぱど The modest effect of tetherin on HSV-1 replication in our experiments may be ぬぱな because HSV-1 is largely insensitive to tetherin restriction. However, we ぬぱに speculate that gM’s role as a tetherin antagonist may not solely be to improve ぬぱぬ HSV-1 budding. Rather its presence within incoming virions might be important ぬぱね for suppressing tetherin innate signalling. Tetherin activates innate immune ぬぱの signalling cascades via NFκB inducing an innate immune response on ぬぱは

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engagement with virus (11). As a pattern recognition receptor tetherin may be a ぬぱば particularly important target for early antagonism before VHS takes effect. The ぬぱぱ ability of gM to rescue HIV-1 from tetherin restriction provides good evidence for ぬぱひ functional tetherin antagonism by gM. Rescue of HIV-1 from tetherin restriction ぬひど by gM appears to be more potent than rescue of HSV-1 from tetherin (compare ぬひな Fig. 5, HIV-1, with Fig. 6, HSV-1). We assume that this is in part because, in the ぬひに case of HIV-1, all tetherin antagonism is abrogated by deletion of Vpu and thus ぬひぬ tetherin restriction is maximal and entirely rescued by gM expression. However, ぬひね in the case of HSV-1, in the absence of gM, tetherin is still antagonised by VHS ぬひの (45) and potentially other as yet uncharacterised viral functions. Indeed, gB had a ぬひは minor effect on tetherin localisation measured by immunofluorescent staining and ぬひば flow cytometry although no effect in an HIV-1 tethering assay. gD had no ぬひぱ measurable effect in any of our assays. ぬひひ ねどど How gM achieves ligand specificity, remains unknown but we note that HIV-1 ねどな Vpu is also promiscuous, removing multiple proteins from the cell surface (4, 15, ねどに 28, 42). It is unclear whether gM actively removes tetherin, or whether like HIV-1 ねどぬ Vpu, gM prevents tetherin from reaching the cell surface (10, 37). Regardless, ねどね the end result is accumulation of tetherin, detected by immunoblot (Fig. 5C) and ねどの the presence of tetherin in a compartment positive for TGN46 staining (Fig. 4B). ねどは Notably, HIV-2 Env also relocalises tetherin to a TGN46 positive compartment ねどば without degrading it (22). Thus, it appears that, in common with other viral ねどぱ tetherin countermeasures, gM acts by removing tetherin from the site of virus ねどひ

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budding, in this case TGN46-negative endocytic tubules (14). We envisage the ねなど interaction between tetherin and outgoing HSV-1 virions occurring in this ねなな compartment and the relocalisation of tetherin we observe by ねなに immunofluorescence (Fig. 4B) provides a plausible basis for the anti-tetherin ねなぬ activity of gM. We did not see any chains of tethered virions similar to those ねなね formed by tetherin-restricted lentiviruses (30). This may reflect spatial constraints ねなの imposed by budding into tubules. Indeed we assume that newly budded tethered ねなは virions would not be easily apparent by electron microscopy because they would ねなば simply be restrained at the location in which they normally reside. However, ねなぱ following release, virions would remain tethered to the cell surface, as we ねなひ observe (Fig. 3). This phenomenon may provide some explanation as to how ねにど tetherin restricts release of HSV-1 and not direct cell-to-cell spread, reported by ねにな the lack of impact on plaque size. We assume that surface tethered virions may ねにに be able to interact with target cell receptors to initiate infection despite being ねにぬ tethered to the infected cell membrane. Such a process has been described for ねにね lentiviruses HIV-1 and FIV (9, 19). ねにの ねには Redundant herpesvirus-encoded tetherin antagonists are also found in KSHV (3, ねにば 25, 31). KSHV encodes the E3 ubiquitin ligase K5 that recruits tetherin to cause ねにぱ its degradation, yet can still antagonise tetherin after K5 depletion with RNAi (31). ねにひ Thus large viruses, such as herpesviruses, may encode several partially ねぬど redundant tetherin antagonists that have subtly different roles in restriction factor ねぬな antagonism. The complex relationship between herpesviruses and tetherin may ねぬに

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also explain the puzzling result that whilst HSV-1 and KSHV are restricted by ねぬぬ tetherin, this protein has been reported to act as a cofactor for HCMV replication ねぬね (25, 31, 40). Tetherin expression improves HCMV infectivity by a few fold, ねぬの perhaps related to its ability to stabilise lipid rafts, which play a key role in HCMV ねぬは entry (20, 41). We did not observe an equivalent enhancement with HSV-1 (Fig. ねぬば 1-2) and it remains unclear whether HCMV also encodes proteins that ねぬぱ manipulate tetherin to prevent it from restricting the virus. ねぬひ ねねど The increasing variety of viruses that are restricted by tetherin illustrates the ねねな power of a restriction factor that targets the fundamental processes of viral ねねに budding and release. The diversity of tetherin antagonists that viruses have ねねぬ evolved also emphasises the importance of overcoming this system of host ねねね defence. It is likely that the study of the ongoing evolutionary conflict between ねねの viruses and tetherin, that is suggested by the Red Queen hypothesis (39), will ねねは lead to significant enhancements to our understanding of the cell biology of both ねねば viruses and their hosts and the relationships between them. ねねぱ ねねひ Acknowledgements ねのど We are grateful to Helena Browne, Gary Cohen, Roselyn Eisenberg, Gillian Elliott, ねのな Roger Everett, Stuart Neil, Claire Pardieu, Sam Wilson and Ben Webb for ねのに reagents and Mahdad Noursadeghi for help with statistical analyses. This work ねのぬ was funded by Wellcome Trust Senior Fellowship 090940 to GJT, the UK ねのね

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はにに Figure legends はにぬ Figure 1:Human tetherin restricts HSV-1 particle release. HT1080 cells はにね expressing HA-tagged tetherin (THN) or empty vector (Vector) were infected with はにの HSV-1 SC16 at 0.01 (A-E) or 3 pfu/cell (G-J). Supernates were harvested at the はには indicated times and titrated for HSV-1 infectivity by plaque assay (A, G) or はにば subjected to DNase-I treatment followed by Q-PCR for the gB gene UL27 (B, H). はにぱ At the same time, cells were harvested, and frozen/thawed 3 times before はにひ titration for HSV-1 infectivity on Vero cells (C, I), or extracted DNA was subjected はぬど to Q-PCR for the gB gene UL27 (D), or lysates were analysed by immunoblot (E, はぬな J). Statistical significance as p value, was determined by 2 way ANOVA (** はぬに p<0.01, *** p<0.001). Tetherin had no effect on DNA replication up to 22h pi (D) はぬぬ (2 way ANOVA, p>0.05). After high MOI infection (3 pfu/cell), tetherin had no はぬね

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effect on HSV-1 sup titres (G) (2 way ANOVA, p>0.05), or cell-associated virus はぬの titres (I) (t-test, p>0.05). (F) HT1080 cells expressing HA tagged tetherin or empty はぬは vector were infected with HSV-1 SC16, washed with acid citrate buffer 1h later はぬば before addition of overlay media for plaque assay. Plaques were counted 48h はぬぱ later after crystal violet staining or immune-alkaline phosphatase staining (for the はぬひ plaques shown). Results +/-SEM are from 3 independent experiments. (K, L) はねど HT1080 cells expressing HA-tagged tetherin or empty vector were infected with はねな HSV-1 SC16 (2 pfu/cell) and cell lysates were prepared at the indicated times はねに and analysed by immunoblot detecting VP5 expression and HA-tag (tetherin), はねぬ transferrin receptor (TfR),く-actin and GAPDH after membrane stripping (K); or はねね tetherin and GAPDH mRNA level were assessed in non-infected and infected はねの cells by Q-PCR. Under these conditions tetherin mRNA was not detectable in the はねは empty vector HT1080 cells. HSV-1 infection decreased tetherin mRNA copy はねば number (L) (2 way ANOVA, p<0.01). Results +/-SD are representative of at least はねぱ 2 separate experiments, except when specified. はねひ Figure 2: shRNA-mediated depletion of tetherin increases HSV-1 release. はのど HeLa cells were transduced with HIV-1 vector encoding either of 2 tetherin-はのな specific shRNAs or a shRNA targeting GFP (A-C). Cells were then infected with はのに HSV-1 SC16 (0.1 pfu/cell). HSV-1 infectivity in the supernates was measured by はのぬ plaque assay (A) and DNAse protected genomes by Q-PCR (B). Tetherin mRNA はのね depletion was assessed in HeLa by Q-PCR normalized to GAPDH (C). The はのの increase in HSV titres after tetherin depletion in (A) was significant (2 way はのは ANOVA, p<0.05). Tetherin reduction was also assessed by immunoblotting HA-はのば

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tag (tetherin) or く-actin (after membrane stripping) in shRNA transduced HA-はのぱ tagged tetherin expressing HT1080 cells (D). Results +/-SD are representative of はのひ 3 separate experiments. ははど Figure 3: Tetherin retains HSV-1 particles at the cell surface. Electron ははな microscopy of HT1080 cells expressing HA-tagged tetherin (A - E) or empty ははに vector (F and G) infected with HSV-1 K26GFP. The boxed areas in A, B, C and F ははぬ are shown at higher magnification in C, D E and G, as indicated. N, nucleus; the ははね asterisks mark accumulations of viral nucleocapsids. In D, E and G, selected ははの HSV-1 particles with typical morphology are indicated by the black arrows, while ははは some of the more tangentially cut electron-dense viral profiles are marked with ははば black arrowheads. Scale bars are 10 µm in A, B and E, or 1 µm in C, D, E and G. ははぱ (H) Counts of cell surface virus particles from both experiments (Exp) are plotted. ははひ Particle counts from cells expressing vector are shown as open symbols, counts はばど from HA-tetherin expressing cells as filled symbols. The number of cells はばな analysed is indicated below the figure. Bars show mean ± standard deviation, p はばに values were determined using an unpaired t-test with Welch's correction on cells はばぬ having at least one viral particle on the cell surface. (Panels A - G show data from はばね experiment 1). はばの Figure 4: gM relocalises tetherin to a TGN46 positive compartment. はばは HEK293T cells expressing HA-tetherin alone or with HSV-1 gM were stained with はばば anti-HA (red), and anti-gM (A) or TGN46 (B) (green), and DAPI (blue, nuclei). The はばぱ last panel in A is a zoom of the area marked on the Overlay+DAPI panel. In (A) はばひ Open arrowheads indicate tetherin predominantly localised to the plasma はぱど

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membrane in cells not expressing, or expressing low levels of gM. Solid はぱな arrowheads (in cells staining brightly for gM) indicate tetherin localised to はぱに perinuclear regions and colocalised with gM. Images are representative of at はぱぬ least 2 separate experiments. Scale bar = 20 µm. はぱね Figure 5: Ability of HSV-1 glycoproteins to remove tetherin from the cell はぱの surface and rescue HIV from restriction. HEK293T cells expressing HA-はぱは tetherin alone or with HSV-1 gM, gD, or gB were stained with anti-HA (red), and はぱば anti-gM, anti-gD or anti-gB (green) respectively, and DAPI (blue, nuclei). Images はぱぱ are representative of 2 separate experiments. Scale bar = 20 µm. (B) Flow はぱひ cytometric detection of cell surface tetherin on HEK293T cells expressing HA-はひど tetherin alone (EV) or in combination with 3 different amounts (250, 500 and はひな 1000 ng) of gM, gD or gB plasmids. Cells transfected with 1000 ng of a VpU はひに plasmid were used as a control. Mean fluorescence intensities for tetherin are はひぬ plotted as a percentage of the EV value. Data shown are mean of 3 or 4 はひね experiments ± SEM. Statistical significance was determined using a t-test はひの comparing MFI from cells transfected with 1000 ng of plasmids (* p<0.05, *** はひは p<0.001, NS non significant). (C) Cells transfected for the flow cytometric assay はひば in (B) were used for immunoblotting gM, gD, gB, HA-tetherin and く-actin, はひぱ sequentially on the same membrane after stripping. (D) HEK293T cells were はひひ cotransfected with 3 HIV-1 vector plasmids and, in addition, an empty (no THN) ばどど or tetherin expression vector (EV), or a tetherin expression vector plus 3 different ばどな amounts (as in B and C) of gM, gD, gB or VpU plasmids. p24 capsid (CA) in ばどに supernates (virions) and p55-Gag expression in cell lysates, as well as ばどぬ

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glycoprotein expression, were analysed 48h later by immunoblotting, with く-actin ばどね used as a loading control after membrane stripping. Intracellular-p55 : supernate-ばどの p24 ratios are indicated below p24-stained membranes and are relative to the ばどは ratio obtained with no tetherin. ND indicates that rations could not be determined ばどば due to absence of any p24 band. The image is representative of at least 3 ばどぱ experiments. ばどひ Figure 6: gM antagonises tetherin during HSV-1 infection. Control (A) and ばなど HA-tagged tetherin expressing (B) HT1080 cells were infected with HSV-1 SC16 ばなな (WT), a gM-deleted virus (∆gM), or a revertant virus (RgM), at 0.01 pfu/cell, and ばなに supernates harvested at the indicated times post infection were titrated for HSV-1 ばなぬ infectivity by plaque assay. 2 way ANOVA of 3 separate tetherin expression ばなね experiments confirms statistically significant differences attributable to time and ばなの the presence of gM (p<0.01). (C) HSV-1 SC16 WT, ∆gM, or RgM, were plaque ばなは assayed on cell lines used in A and B. Results +/-SEM are from 2 independent ばなば experiments. (D) Immunoblot detection of ICP4, gM and く-actin in lysates of ばなぱ HT1080 cells infected with the 3 viruses or uninfected (NI). (E) Immunoblot ばなひ detection of VP5 and HA-tag (tetherin), or く-actin after membrane stripping, in ばにど lysates of HA-tagged tetherin-expressing HT1080 cells infected with ∆gM (∆) or ばにな RgM (R) virus at 2 pfu/cell or uninfected (-). Results are representative of 3 ばにに separate experiments. ばにぬ ばにね ばにの ばには

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Table 1 HSV-1 particles counting at the cell surface ばにば ばにぱ Experiment 1 Experiment 2

Vector THN Vector THN

No of cell profiles examined 60 51 62 70

No of infected cells* 32 33 20 27

No of HSV-1 profiles at the cell surface

419

1258

389

886

Cell surface HSV-1 particles per infected cell profile

13.09

38.12

19.45

32.81

ばにひ * Cell profiles showing at least one viral particle at the cell surface ばぬど ばぬな ばぬに ばぬぬ ばぬね ばぬの

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