The Hydrophobic Domain of Infectious Bronchitis Virus E Protein ...

JOURNAL OF VIROLOGY, Jan. 2011, p. 675–685 Vol. 85, No. 20022-538X/11/$12.00 doi:10.1128/JVI.01570-10Copyright © 2011, American Society for Microbiology. All Rights Reserved.

The Hydrophobic Domain of Infectious Bronchitis Virus E ProteinAlters the Host Secretory Pathway and Is Important for

Release of Infectious Virus�

Travis R. Ruch and Carolyn E. Machamer*Department of Cell Biology, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205

Received 27 July 2010/Accepted 27 October 2010

The coronavirus (CoV) E protein plays an important role in virus assembly. The E protein is made in excessduring infection and has been shown to have ion channel activity in planar lipid bilayers. However, a role ininfection for the unincorporated E or its ion channel activity has not been described. To further investigate thefunction of the infectious bronchitis virus (IBV) E protein, we developed a recombinant version of IBV in whichthe E protein was replaced by a mutant containing a heterologous hydrophobic domain. The mutant virus,IBV-EG3, was defective in release of infectious virus particles. Further characterization of IBV-EG3 revealedthat damaged particles appeared to accumulate intracellularly. The phenotype of IBV-EG3 suggested that thehydrophobic domain of IBV E may be important for the forward trafficking of cargo, so we determined whetherIBV E facilitated the delivery of cargo to the plasma membrane. Surprisingly, we found that IBV E, but notEG3, dramatically reduced the delivery of cargo to the plasma membrane by impeding movement through theGolgi complex. Furthermore, we observed that overexpression of IBV E, but not EG3, induced the disassemblyof the Golgi complex. Finally, we determined that the delivery of IBV S to the plasma membrane was reducedin cells infected with wild-type-IBV compared to those infected with IBV-EG3. Our results indicated that thehydrophobic domain of IBV E alters the host secretory pathway to the apparent advantage of the virus.

Coronaviruses (CoVs) pose a significant threat to humanhealth. In addition to causing �20% of common cold cases,CoVs can cause deadly illness in humans, as exemplified duringthe outbreak of severe acute respiratory syndrome (SARS) in2003. Since the emergence of SARS-CoV, two other CoVs thatcause respiratory disease in humans have been identified (24),emphasizing that continued study of CoV biology has impor-tant implications for human health.

CoVs are enveloped, positive-strand RNA viruses. Unlikemany enveloped viruses that bud at the plasma membrane,CoVs assemble and bud intracellularly into the lumen of theendoplasmic reticulum-Golgi intermediate compartment(ERGIC) (7). After budding, CoVs use the host secretorypathway to exit the cell. Virions are much larger than normalprotein cargo, and it is unknown whether the virus alters thesecretory pathway to meet the demands of trafficking its viri-ons. CoVs encode four major structural proteins that facilitatehost entry and virus assembly. The membrane protein (M) hasthree transmembrane domains and forms the scaffolding forvirion assembly, the nucleocapsid protein (N) binds and pack-ages the RNA genome, the spike protein (S) is the fusionprotein that facilitates virus entry, and the envelope protein(E) plays a role in virus budding. The CoV E protein is a small(�75- to 109-amino-acid) structural protein that contains asingle hydrophobic domain (HD). CoV E was first implicatedin virion budding, where it was shown that coexpression of Ealong with M drives virus-like particle (VLP) formation (31).

Interestingly, only a fraction of the CoV E produced duringinfection is incorporated into the virion envelope. This sug-gests that additional functions for the pool of unincorporatedE protein may exist.

Recombinant CoVs lacking the E gene show decreased pro-duction of virus, indicating that CoV E is necessary for efficientvirus production (6, 9, 21). The E proteins from different CoVsare variable in both length and amino acid composition. How-ever, it has been shown that the E proteins from several dif-ferent CoVs can rescue a mutant of the mouse hepatitis virus(MHV) lacking the E gene, suggesting that the E proteins fromdifferent CoVs perform a similar function during viral infec-tion (8). Additionally, studies have demonstrated that someCoV E proteins have ion channel activity in planar lipid bilay-ers (35, 36), leading to speculation that CoV E may oligomer-ize and act as an ion channel during infection. Consistent withthis idea, computational modeling of the putative HDs of sev-eral different CoV E proteins predicts that a pentamer is astable arrangement (28). This model is supported by a recentnuclear magnetic resonance (NMR) structure of the SARS-CoV E HD (22).

Further evidence for the ion channel activity of CoV E wasobtained using the Na�/H� exchanger inhibitor hexameth-ylene amiloride (HMA), which blocks the ion channel activityof human CoV 229E E and MHV E in planar lipid bilayers.When cells infected with either virus were treated with HMA,their growth was inhibited, but HMA had no effect on thereplication of a mutant MHV lacking the E gene. (35). Ala-nine-scanning mutagenesis of the HD of MHV E showed thatdisrupting the pitch of the putative transmembrane helix re-sulted in a virus with lower titer as well as a defect in release ofinfectious virus (37). Finally, whole-cell patch clamp data sug-gest that SARS-CoV E has ion channel activity in transfected

* Corresponding author. Mailing address: Department of Cell Biol-ogy, The Johns Hopkins University School of Medicine, 725 N. WolfeSt., Baltimore, MD 21205. Phone: (410) 955-1809. Fax: (410) 955-4129.E-mail: [email protected].

� Published ahead of print on 3 November 2010.

675

on March 25, 2018 by guest

http://jvi.asm.org/

Dow

nloaded from

http://jvi.asm.org/

cells; however, it was unclear whether the expressed E proteinin these studies was present at the plasma membrane (22).These results point to an important role for the HD of CoV Ein the virus life cycle and suggest that it may act as an ionchannel during viral infection. In this study we showed that theHD of the infectious bronchitis virus (IBV) E protein is im-portant for the release of infectious virus and alters the hostsecretory pathway to the apparent advantage of the virus.

MATERIALS AND METHODS

Cell culture. HeLa and Vero cells were cultured in Dulbecco’s modified Eaglemedium (DMEM) (Invitrogen/Gibco, Grand Island, NY) with 10% or 5% fetalbovine serum (FBS) (Atlanta Biologicals, Lawrenceville, GA), respectively, and0.1 mg/ml Normocin (InvivoGen, San Diego, CA) at 37°C under 5% CO2.

Viruses and infection. The recombinant IBV Beaudette virus used as the wildtype and the generation of the IBV-EG3 mutant have been previously described(15, 40). After three rounds of plaque purification, 12 plaques were expandedand the E gene was sequenced by reverse transcription-PCR (RT-PCR). Twomutant clones carrying the mutation (clone 8 and clone 10) were chosen forfurther study. After four passages, the mutation was stable, and no other muta-tions were present in the region of the genome encoding the structural proteins.Vero cells were inoculated with virus diluted into serum-free DMEM, and viruswas adsorbed for 1 h with rocking. The inoculum was removed, and the cells wererinsed with phosphate-buffered saline (PBS) and placed in DMEM with 5% FBS.

For the one-step growth curve, triplicate wells of Vero cells were infected withwild-type IBV (IBV-wt), IBV-EG3-8, or IBV-EG3-10 at a multiplicity of infec-tion (MOI) of 1. The supernatants and cells were collected separately every 4 hfor 24 h. The supernatants were clarified and frozen at �80°C. The cells werewashed with PBS, covered with 0.5 ml of DMEM with 5% FBS, and subjected tothree freeze-thaw cycles. The virus titer was determined by plaque assay on Verocells. Each sample was plaqued in triplicate, and the plaques were stained byoverlay of 0.8% agarose in DMEM with 0.015% neutral red.

Plasmids. cDNAs encoding IBV E, EG3, and IBV M have been describedpreviously (3, 5). The pCAGGS expression vectors for each were constructed byexcising the coding sequence from pBluescript (Stratagene, La Jolla, CA) andsubcloning into pCAGGS-MCS (20) using SacI and EcoRI restriction sites. ThecDNA for a soluble version of the membrane protease dipeptidyl peptidase IV(solDPPIV) (32) was excised from pMB88 (a gift from A. Hubbard, JohnsHopkins University School of Medicine), cloned into pBluescript using SacI andMfeI restriction sites, and subsequently moved into pCAGGS using KpnI andSacI restriction sites (32). pCAGGS-Tac-S and pCAGGS VSV G have beendescribed previously (17, 18). The pcDNA-mCherry �-tubulin was a kind giftfrom Roger Tsien (University of California at San Diego) (26).

Transient transfection. Fugene6 (Roche, Indianapolis, IN) was used to tran-siently transfect cells according to the manufacturer’s protocol. HeLa cells wereplated in 35-mm dishes and transfected with the following amounts of plasmiddiluted into Opti-MEM (Invitrogen/Gibco, Grand Island, NY) with Fugene6: 0.5�g pCAGGS IBV E, 0.5 �g pCAGGS EG3, 0.5 �g pCAGGS IBV M, 1 �gpCAGGS VSV G, 1 �g pCAGGS solDPPIV, 1 �g pCAGGS Tac-S, and 1 �gpcDNA-mCherry-�-tubulin. The cells were used in experiments at 18 to 22 hafter transfection.

Antibodies. Rabbit antibodies recognizing the C termini of IBV E, IBV M, andIBV S have been described previously (5, 12, 14). The mouse anti-IBV S mono-clonal antibody was a kind gift from Ellen Collisson (Western University Collegeof Veterinary Medicine). The rat anti-IBV E polyclonal tail antibody has beendescribed previously (5). The rabbit anti-vesicular stomatitis virus (anti-VSV)polyclonal antibody used for immunoprecipitation has been described previously(33). Mouse anti-p230 and mouse anti-GM130 were from BD Biosciences (SanDiego, CA). Rabbit anti-giantin antibody was from Covance (Emeryville, CA).Mouse anti-LAMP1 (developed by J. T. August and J. E. K. Hildreth) wasobtained from the Developmental Studies Hybridoma Bank (developed underthe auspices of the NICHD and maintained by the University of Iowa, Depart-ment of Biological Sciences, Iowa City, IA). Mouse anti-calnexin antibody wasfrom StressGen (Victoria, British Columbia, Canada). The rabbit anti-dsRed wasfrom Clontech (Palo Alto, CA). The mouse anti-c-Myc used to immunoprecipi-tate solDPPIV was from Roche (Indianapolis, IN). The horseradish peroxidase-conjugated donkey anti-rabbit antibody was from Amersham/GE Healthcare(Piscataway, NJ). The Alexa Fluor 488-conjugated donkey anti-rabbit IgG, AlexaFluor 488-conjugated donkey anti-mouse IgG, Alexa Fluor 568-conjugated don-key anti-rabbit IgG, and Alexa Fluor 568-conjugated anti-mouse IgG were from

Invitrogen/Molecular Probes (Eugene, OR). The Alexa Fluor 594-conjugatedtransferrin was from Invitrogen/Molecular Probes. The Texas Red-conjugateddonkey anti-rat and fluorescein-conjugated donkey anti-rat IgGs were from Jack-son ImmunoResearch Laboratories, Inc. (West Grove, PA).

Electron microscopy. For analysis of infected cells by transmission electronmicroscopy (TEM), Vero cells were infected with IBV-wt or IBV-EG3 at anMOI of 0.1. At 14 h postinfection (p.i.), cells were fixed and embedded essen-tially as described previously (2). Briefly, cells were fixed in 1.5% glutaraldehydein 0.1 M sodium cacodylate buffer, treated with 1% OsO4 in 0.1 M sodiumcacodylate with 1% potassium ferricyanide, dehydrated, and embedded in Epon.For quantitation, carriers were defined as membrane-bound structures contain-ing three or more virions. Aberrant material was defined as structures other thanvirions present within the limiting membranes of carriers. For IBV-wt-infectedcells, 16 cells and 54 carriers were counted, and for IBV-EG3-infected cells, 10cells and 83 carriers were counted. Thin sections were viewed at 80 kV. Imageswere collected on a Hitachi 7600 microscope using an AMT charge-coupled-device (CCD) camera.

Coimmunoprecipitation. Vero cells were infected with IBV-wt or IBV-EG3 atan MOI of 0.1. At 20 h p.i. cells were starved in cysteine-methionine-free DMEMfor 15 min, labeled with 150 �Ci of Expre35S35S [35S]methionine-cysteine (Per-kin-Elmer, Waltham, MA) in cysteine-methionine-free DMEM for 90 min andchased for 30 min in normal growth medium. Labeled cells were washed withPBS, cross-linked with 1 mM dithiobis(succinimidyl propionate) (DSP) for 10min, and quenched in 40 mM glycine in PBS. After cross-linking, cells were lysedin detergent solution (62.5 mM EDTA, 1% NP-40, 0.4% deoxycholic acid, 50mM Tris-HCl [pH 8]) with protease inhibitor cocktail (Sigma). Samples wereclarified, and SDS was added to 0.2%. All samples were precleared with Staph-ylococcus aureus Pansorbin cells (Calbiochem, San Diego, CA). IBV E wasimmunoprecipitated with the rabbit anti-IBV E antibody. Immune complexeswere collected with 20 �l of washed Staphylococcus aureus Pansorbin cells andwashed two times in radioimmunoprecipitation assay (RIPA) buffer (10 mM Tris[pH 7.4], 0.1% SDS, 1% deoxycholic acid, 1% NP-40, 150 mM NaCl). Thecross-linking was reversed prior to gel electrophoresis with 5% �-mercaptoeth-anol. Samples were separated by 15% SDS-PAGE, and labeled proteins werevisualized by using a Molecular Imager FX phosphorimager (Bio-Rad) usingQuantity One software.

Trafficking assays. In all trafficking assays, HeLa cells were used at 18 to 22 hposttransfection. Cells were starved in cysteine-methionine-free DMEM for 15min, labeled with 50 to 100 �Ci of Expre35S35S [35S]methionine-cysteine (Perkin-Elmer, Waltham, MA) per dish in cysteine-methionine-free DMEM for 20 min,and chased for various times in normal growth medium. Labeled cells werewashed with PBS and lysed in detergent solution with protease inhibitor cocktail.Samples were clarified, and SDS was added to 0.2%. All samples were preclearedwith Staphylococcus aureus Pansorbin cells. After incubation with the appropri-ate antibody, immune complexes were collected with 20 �l of washed Staphylo-coccus aureus Pansorbin cells and washed two times in RIPA buffer. For the VSVG surface immunoprecipitation assay, HeLa cells expressing VSV G along withIBV M, IBV E, or EG3 were pulse-labeled as described above. At each timepoint, labeled dishes were washed two times with cold PBS-BSA (1% BSA and0.02% NaN3 in PBS) and then incubated on ice with rabbit anti-VSV diluted into400 �l PBS-BSA for 4 h. Cells were lysed in detergent solution, and immunecomplexes were isolated as described above. This pool of protein was counted as“surface.” The internal pool of VSV G was determined by reimmunoprecipitat-ing the samples with rabbit anti-VSV. Immune complexes were eluted in loadingsample buffer and separated by 10% SDS-PAGE. For the solDPPIV secretionassay, HeLa cells coexpressing solDPPIV along with IBV E, IBV M, or EG3were pulse-labeled as described above. The supernatants were taken from eachsample and clarified, 5� detergent solution was added to 1�, and SDS wasadded to 0.2%. solDPPIV was immunoprecipitated from the cell and superna-tant fractions with mouse anti-c-Myc antibody. For the VSV G endo-�-N-acetyl-glucosaminidase H (endo H) assay, HeLa cells coexpressing VSV G along withIBV E, IBV M, or EG3 were pulse-labeled and collected as described above.VSV G was immunoprecipitated using rabbit anti-VSV antibody. Immune com-plexes were eluted in 1% SDS (pH 6.8) at 100°C and digested in 75 mMNa-citrate (pH 5.5) with 0.2 �l endo H (100 units) (New England Biolabs,Beverly, MA) at 37°C overnight. For Tac-S endo H assays, HeLa cells coexpress-ing Tac-S along with IBV E, IBV M, or EG3 were pulse-labeled and collected asdescribed above. Tac-S was immunoprecipitated using rabbit anti-SARS-CoV Santibody (17). Immune complexes were eluted and digested with endo H. Con-centrated sample buffer (200 mM Tris-HCl [pH 6.8], 8% SDS, 60% glycerol,0.2% bromophenol blue) was added to each sample, and the proteins were runon 10% SDS-PAGE. Labeled proteins were visualized by using a Molecular

676 RUCH AND MACHAMER J. VIROL.


http://jvi.asm.org/

Dow

nloaded from

http://jvi.asm.org/

Imager FX phosphorimager (Bio-Rad) and quantified using Quantity One soft-ware.

[3H]palmitate incorporation. Vero cells were infected with either IBV-wt orIBV-EG3 at an MOI of 0.1. At 12.5 h p.i., cells were labeled with either 25 �Ciof [35S]methionine-cysteine ([35S]-Promix; Amersham) or 500 �Ci of [3H]palmi-tate for 3 h as previously described (3). Cells were lysed in detergent solution andclarified, and IBV E was immunoprecipitated with the rabbit anti-IBV E anti-body as described above. Samples were subjected to SDS-PAGE, and gels wereimpregnated with 2,5-diphenyloxazole (PPO) and processed by fluorography at�80°C.

Indirect-immunofluorescence microscopy. HeLa cells plated on glass cover-slips were processed for immunofluorescence at 18 to 20 h after transfection. Forstaining p230, GM130, and giantin, cells were washed with PBS and fixed in 3%paraformaldehyde. The fixative was quenched in PBS containing 10 mM glycine(PBS-Gly), and the cells were permeabilized in 0.5% Triton X-100 (TX-100) for3 min. For staining calnexin and LAMP1, cells were fixed in 3% paraformalde-hyde and permeabilized in 0.05% saponin for 10 min. Saponin was included in allantibody dilutions for the latter samples. For staining tubulin, cells were fixedand permeabilized in methanol for 20 min at �20°C. Coverslips were incubatedin primary antibody with 1% BSA for 18 min at room temperature and washedtwo times with PBS-Gly. Primary antibodies were used at the following dilutions:rabbit anti-IBV E, 1:800; mouse anti-p230, 1:200; mouse anti-GM130, 1:800;mouse anti-LAMP1, 1:200; rat anti-IBV E, 1:500; rabbit anti-giantin, 1:800;rabbit anti-dsRed, 1:500; and rabbit anti-calnexin, 1:100. After being washed twotimes with PBS-Gly, cells were incubated in secondary antibody with 1% BSA for18 min. Secondary antibodies were used at the following dilutions: Alexa Fluor488-conjugated anti-rabbit IgG, 1:1,000; Alexa Fluor 568-conjugated anti-mouseIgG, 1:1,000; Texas Red-conjugated anti-rat IgG, 1:500; and fluorescein isothio-cyanate (FITC)-conjugated anti-rat IgG, 1:200. Coverslips were washed twotimes with PBS-Gly and mounted on microscope slides in glycerol containing 0.1M N-propylgallate. All images and enlargements were adjusted independently.The surface staining for IBV S was carried out as follows. Vero cells were in-fected with IBV-wt or IBV-EG3 at an MOI of 0.1. At 9 to 10 h p.i., cells wereincubated with mouse anti-IBV S antibody (1:4) for 12 min at 37°C. Cells werethen fixed with 3% paraformaldehyde and permeabilized with 0.5% TX-100 for3 min. Rabbit anti-IBV S antibody (1:800) was used to stain for total IBV S.Images were collected using an Axioskop microscope (Zeiss, Thornwood, NJ)equipped for epifluorescence using an ORCA-03G charge-coupled-device cam-era (Hamamatsu, Japan) and IP Lab software (Scanalytics, Vienna, VA). ImageJ (National Institutes of Health) was used to quantify the images. Cells wereoutlined, and the mean fluorescence intensity was calculated for the same areaon the surface and total images. The percent surface was calculated by dividingthe surface signal by the total signal.

Syncytium size. Vero cells were infected with either IBV-wt or IBV-EG3 at anMOI of 0.1. The size of syncytia was determined at 10 to 13 h p.i. after fixationand permeabilization by costaining with rabbit anti-IBV E (1:800) as describedabove and for nuclei with Hoechst 33285 (0.1 �g/ml). Syncytia were classified ascells with three or more nuclei. Syncytia from four independent infections werecounted at between 10.5 and 14 h p.i. For IBV-wt, 131 syncytia were scored, andfor IBV-EG3, 136 syncytia were scored.

RESULTS

The HD of IBV E is important for efficient viral replication.To better understand the role of the HD of IBV E, we createda chimeric version of IBV E in which the HD (amino acids 12to 32) was replaced with the HD from the vesicular stomatitisvirus glycoprotein (VSV G) (amino acids 463 to 482) (Fig. 1A).The HD of VSV G was chosen because it is the same length asthe HD of IBV E and does not contain any known localizationor oligomerization signals. We then made a recombinant ver-sion of IBV wherein EG3 was inserted in place of E in the virusgenome. The recombinant virus (IBV-EG3) was plaque puri-fied, and several clones were sequenced by RT-PCR to confirmthe mutation. Selected clones carrying EG3 in place of E weregrown and passaged in Vero cells. After expansion and passageof several clones, RT-PCR sequencing showed that the muta-tion was stable and no additional mutations were presentwithin the structural proteins.

One-step growth curves were performed for IBV-wt andIBV-EG3. Since the whole genome of the recombinant viruswas not sequenced, we used two different clones of IBV-EG3to control for any clonal variation. At each time point, thereleased and cell-associated viruses were collected separately.When we compared the production of total infectious virus(released plus cell associated), we found that both clones ofIBV-EG3 grew to a titer that was approximately 10-fold lowerthan that of IBV-wt (Fig. 1B). More strikingly, we found thatover 80% of the infectivity of IBV-wt was present in the re-leased virions, while in both clones of IBV-EG3 the majority ofthe infectious virus was present within the cells (Fig. 1C).

FIG. 1. Defect in growth and release of IBV-EG3 infectious par-ticles. (A) The HD of IBV E was precisely replaced with the HD ofVSV G. (B) Vero cells were infected with IBV-wt or two clones ofIBV-EG3 (8 and 10) at an MOI of 1, and a one-step growth curve wasperformed. At each time point, the released and cell-associated viruseswere collected separately. Infectivity was determined by triplicateplaque assay, and the total infectivity (the sum of the released andcell-associated titers) was plotted. Error bars indicate the standarddeviation for each time point. (C) Percentage of infectious virus in thesupernatant at 12, 16, 20, and 24 h p.i. Error bars indicate the standarddeviation for each time point.

VOL. 85, 2011 IBV E ALTERS THE HOST SECRETORY PATHWAY 677


http://jvi.asm.org/

Dow

nloaded from

http://jvi.asm.org/

These results indicate that IBV-EG3 is defective in the pro-duction of infectious virus (reduced 10-fold) and has an evenlarger defect in the release of infectious virus (reduced �44-fold).

Damaged IBV-EG3 virions accumulate intracellularly. Pre-liminary characterization of IBV-EG3 demonstrated that mostparticles purified from infected cells lacked surface spikes andthat the majority of IBV S was cleaved near the virion envelope(15). Additionally, the total protein content of purified virionsindicated that IBV-EG3 particles were released at 2- to 3-fold-lower levels than IBV-wt (data not shown), even though re-leased infectious particles were reduced �44-fold.

To address whether IBV-EG3 virions were accumulatingintracellularly, we performed transmission electron microscopy(TEM) on Vero cells infected with either IBV-wt or IBV-EG3.In cells infected with IBV-wt, virions were present in pleomor-phic structures packed with virions (Fig. 2A, upper panels,arrows). However, in IBV-EG3-infected cells, virions accumu-lated in vacuole-shaped carriers along with aberrant structuresthat may be degraded material (Fig. 2B, lower panels, arrow-heads). We next quantified how often virion carriers containedaberrant material, and we found that IBV-EG3 virions weremuch more likely to have aberrant material than IBV-wt. It ispossible that IBV-EG3 virions are trafficked incorrectly andend up in degradative compartments, resulting in the release ofdamaged virions.

The HD of IBV E is not necessary for interaction with IBVM or palmitoylation of E. We have previously shown that Eand EG3 can both drive VLP formation when coexpressed withIBV M, suggesting that both proteins are competent for virusassembly (4). Here we tested how well M interacts with EG3 inthe context of an infection. We radiolabeled infected cells with[35S]methionine-cysteine and immunoprecipitated IBV E aftercross-linking with dithiobis(succinimidyl propionate) (DSP) topreserve the interaction after detergent solubilization. The re-sults showed that both E and EG3 coimmunoprecipitated M(E/M signal ratio of �1.2 for both), suggesting that altering theHD of IBV E does not interfere with E-M interaction (Fig.3A). This is consistent with our previous observation that bud-

FIG. 2. IBV-EG3 virions accumulate intracellularly. (A) Vero cells were infected with IBV-wt or IBV-EG3 and subjected to TEM at 14 h p.i.The upper panels are representative images from cells infected with IBV-wt; arrows indicate virion carriers. The lower panels are representativeimages from cells infected with IBV-EG3; arrowheads indicate aberrant material within IBV-EG3 carriers. Scale bars are 500 nm. (B) Quantitationof the percentage of carriers containing aberrant material from IBV-wt- and IBV-EG3-infected cells. Error bars indicate standard errors of themeans. For IBV-wt, 54 carriers were examined from 16 cells, and for IBV-EG3, 83 carriers were examined from 10 cells.

FIG. 3. The HD of IBV E is not required for interaction with IBVM or for IBV E palmitoylation. (A) Vero cells were infected withIBV-wt or IBV-EG3 at an MOI of 0.1. At 20 h p.i., cells were labeledwith [35S]cysteine-methionine for 90 min and cross-linked with DSP,and IBV E was immunoprecipitated (IP) as described in Materials andMethods. The immune complexes were subjected to SDS-PAGE andautoradiography after the cross-linking was reversed. The signal ratioof E to M is �1.2 for both samples. (B) Vero cells were infected witheither IBV-wt or IBV-EG3 at an MOI of 1. At 12.5 h p.i., cells werepulse-labeled with either [35S]cysteine-methionine or [3H]palmitate for180 min. After labeling, IBV E was immunoprecipitated from eachsample and subjected to SDS-PAGE, and labeled proteins were de-tected by fluorography.



http://jvi.asm.org/

Dow

nloaded from

http://jvi.asm.org/

ded IBV-EG3 virions were present in Golgi stacks as deter-mined by TEM (15).

It has been shown that palmitoylation of MHV E is impor-tant for virus production (1, 13). Therefore, we determinedwhether replacing the HD of IBV E affected its palmitoylation.We radiolabeled infected cells with either [35S]methionine-cysteine or [3H]palmitate and compared the amount of palmi-tate incorporated to total protein using fluorography. The re-sults showed that E and EG3 were palmitoylated to similarextents (Fig. 3B). Thus, the heterologous HD of IBV-EG3does not disrupt the interaction with IBV M or affect proteinpalmitoylation.

IBV E, but not EG3, affects delivery of cargo to the plasmamembrane. Our data demonstrate that the HD of IBV E isimportant for the efficient release of infectious virus but doesnot appear to be critical for particle assembly. We hypothe-sized that the HD of IBV E facilitates the forward trafficking ofvirions through the host secretory pathway and that rapidmovement through the secretory pathway helps virions avoidcellular degradation pathways. To this end, we tested whetherIBV E enhances the movement of protein cargo toward theplasma membrane. We coexpressed IBV E or EG3 along withthe model cargo glycoprotein VSV G in HeLa cells. As a

control, we coexpressed VSV G with IBV M, a membraneprotein that is also targeted to the Golgi region. Transfectedcells were pulse-labeled with [35S]methionine-cysteine, and thedelivery of VSV G to the plasma membrane was determined bysurface immunoprecipitation at various times postchase. Toour surprise, the overexpression of IBV E reduced delivery ofVSV G to the plasma membrane, while overexpression of EG3had no effect (Fig. 4A). This result was the opposite of what weexpected; instead of facilitating the movement of VSV G to theplasma membrane, IBV E inhibited the trafficking of VSV G.To further characterize how IBV E affected protein trafficking,we examined other cargo molecules. Since VSV G is a mem-brane-bound protein and virions are soluble cargo, we nextassayed the effect of overexpressing IBV E and EG3 on thesecretion of a soluble version of the membrane protease dipep-tidyl peptidase IV (solDPPIV). solDPPIV is a secreted versionof DPPIV that was created by inserting a signal sequencecleavage site at the end of the transmembrane domain (32).Cells coexpressing solDPPIV along with either IBV E or EG3were pulse-labeled with [35S]methionine-cysteine, the superna-tants were collected at various times postchase, and theamount of solDPPIV secreted was determined by immunopre-cipitation followed by SDS-PAGE and phosphorimaging. We

FIG. 4. Overexpression of IBV E reduces the delivery of cargo to the plasma membrane. (A) HeLa cells were transfected with plasmidsencoding VSV-G along with IBV M, IBV E, or EG3 as indicated. The transfected cells were pulse-labeled with [35S]cysteine-methionine, and thearrival of VSV G at the plasma membrane was determined by surface immunoprecipitation as described in Materials and Methods. The gels showthe amount of newly made VSV G surface (S) and internal material (I) for each time point. The graph indicates the percentage of total VSV Gat the cell surface. Error bars represent standard errors of the means from at least two experiments. (B) HeLa cells were transfected with plasmidsencoding solDPPIV along with IBV M, IBV E, or EG3 as indicated, pulse-labeled with [35S]cysteine-methionine, and chased in nonradioactivemedium, and the amount secreted was determined by immunoprecipitating solDPPIV from the supernatant and cellular fractions of each sample.The gels on the left show the secreted and cellular pools of solDPPIV at each time point. The graph on the right was generated by calculating thepercentage of total solDPPIV in the supernatant. Error bars represent standard errors of the means from three experiments.



http://jvi.asm.org/

Dow

nloaded from

http://jvi.asm.org/

observed that overexpression of IBV E severely inhibited thesecretion of solDPPIV, while overexpression of EG3 had noeffect (Fig. 4B). Taken together, these results show that IBV Einhibits the delivery of protein cargo to the cell surface.

IBV E delays movement of cargo through the Golgi complex.To determine if the decrease in protein trafficking caused byIBV E was occurring at the Golgi complex or at a post-Golgistep, we analyzed the processing of N-linked oligosaccharideson cargo proteins. Measuring the acquisition of resistance toendoglycosidase H (endo H), which occurs in the medial Golgicompartment, is a common method for determining the rate ofglycoprotein movement through the Golgi complex. Cells co-expressing VSV G along with IBV E, EG3, or IBV M werepulse-labeled and chased for various times. After collection ofthe cell lysates at each time point, VSV G was immunopre-cipitated and digested with endo H. The percentage of VSV Gthat was resistant to endo H at each time point was determinedafter SDS-PAGE and phosphorimaging. The results showedthat IBV E, but not EG3 or IBV M, dramatically reduced the

amount of processed VSV G (Fig. 5A). This suggests that IBVE impedes trafficking at or prior to the medial Golgi compart-ment. We next assayed the effect of IBV E on a different cargoprotein, Tac-S. This protein contains the ectodomain of inter-leukin-2� and the endodomain of SARS-CoV S, which con-tains a weak dibasic ER retrieval signal (17). IBV E alsodelayed the movement of Tac-S through the medial Golgicompartment (Fig. 5B). The results of these experiments showthat IBV E, but not EG3, decreases the anterograde move-ment of several cargo proteins through the Golgi complex. Thisresult is consistent with the observation that IBV E reduces thedelivery of cargo proteins to the cell surface.

Overexpression of IBV E causes the Golgi complex to dis-assemble. Given the trafficking defect observed in cells ex-pressing IBV E, we determined whether the overexpression ofIBV E had any effect on the on the morphology of secretoryorganelles. Using indirect-immunofluorescence microscopy ofHeLa cells overexpressing either IBV E or EG3, we examinedthe staining pattern for the cis-Golgi protein GM130. GM130

FIG. 5. IBV E reduces protein traffic through the Golgi complex. (A) HeLa cells were transfected with plasmids encoding VSV-G along withIBV M, IBV E, or EG3. The transfected cells were pulse-labeled with [35S]cysteine-methionine, and VSV G was immunoprecipitated at varioustimes of chase and digested with endo H. The gels on the left show the mature (**) and immature (*) bands for VSV G in each sample. The middleband represents VSV G with one of its two oligosaccharides processed and was included in the mature fraction. The graph on the right shows thepercentage of endo H-resistant VSV G over time. Error bars represent standard errors of the means from at least two experiments. (B) HeLa cellswere transfected with a plasmids encoding Tac-S along with IBV M, IBV E, or EG3 as indicated. The transfected cells were pulse-labeled with[35S]cysteine-methionine, and Tac-S was immunoprecipitated at various times of chase and digested with endo H. The gels on the left show themature (**) and immature (*) bands for Tac-S in each sample. The percentage of endo H-resistant Tac-S is shown in the graph on the right. Errorbars represent standard errors of the means from at least two experiments.



http://jvi.asm.org/

Dow

nloaded from

http://jvi.asm.org/

staining was dispersed in cells overexpressing IBV E but not incells overexpressing EG3 (Fig. 6A). However, lysosomes(LAMP1), early endosomes (transferrin receptor), and the ER(calnexin) were all unaffected by the overexpression of IBV E(data not shown). We also examined the distributions of actinand tubulin to determine if IBV E induced any rearrangementof the cytoskeleton, which could disrupt the Golgi structure.However, both actin filaments and microtubules appeared tobe normal in cells overexpressing IBV E (data not shown).

We next tested several different Golgi markers to see if theirlocalization was affected by IBV E. Both the trans-Golgi pro-

tein p230 and the membrane-bound cis/medial Golgi proteingiantin were dispersed in cells overexpressing IBV E (Fig. 6B).In addition, the localization of the ERGIC marker ERGIC-53was also disrupted (data not shown). This suggests that theoverexpression of IBV E alters the structure of the entire Golgicomplex. Closer examination of the staining patterns forGM130, p230, and giantin showed that IBV E only partiallycolocalized with each marker (Fig. 6). Additionally, we ob-served that both p230 and GM130 only partially colocalizedwith giantin in cells with disrupted Golgi complexes (Fig. 7).Taken together, the results suggest that the Golgi complex is

FIG. 6. IBV E disrupts the morphology of the Golgi complex. (A) HeLa cells overexpressing IBV E or EG3 were double labeled with anti-IBVE and anti-GM130 antibodies. The same field is shown for each pair of samples. (B) HeLa cells overexpressing IBV E were double labeled withrat anti-IBV E and rabbit antigiantin (cis/medial Golgi), or rabbit anti-IBV E and mouse anti-p230 (trans-Golgi). Boxed areas in each field areenlarged at the right; arrows indicate areas of overlap, and arrowheads indicate areas where only one marker is present. (C) Color merges of theenlargements shown in panels A and B. IBV E is shown in green, and the respective marker is shown in red.



http://jvi.asm.org/

Dow

nloaded from

http://jvi.asm.org/

not simply disassembling into ministacks, where the Golgi res-ident proteins should mostly colocalize (29). Instead, moreextensive disassembly must be occurring, which could also ex-plain the reduction in cargo trafficking.

To compare the level of overexpression of IBV E in trans-fected cells to that during infection, we used immunoblotting

and normalized the signal to the number of E-expressing cellsdetermined by indirect-immunofluorescence microscopy. Wefound that the level of E expressed per cell at 18 h posttrans-fection was similar to the level present at 16 h p.i. at an MOIof 0.2 (data not shown). Thus, transfected cells express anamount of IBV E per cell similar to that expressed by infectedcells at a late time postinfection.

The HD of IBV E reduces accumulation of IBV S on thesurface of infected cells. Because IBV E reduces protein trafficthrough the Golgi complex in transfected cells, we assayedwhether IBV E could also affect trafficking in infected cells.We measured the accumulation of IBV S on the surface ofcells infected with IBV-wt or IBV-EG3 using a surface immu-nofluorescence assay. We labeled the surface of cells infectedwith IBV-wt or IBV-EG3 with mouse anti-IBV S antibodies.We then fixed and permeabilized the cells and labeled the totalamount of IBV S by costaining with a rabbit anti-IBV S anti-body. When we compared the signal from the surface to thetotal for each sample, we found that cells infected with IBV-EG3 had a greater proportion of surface IBV S (Fig. 8A),although the total signal for IBV S was similar. This result isconsistent with our observation of reduced cargo trafficking.Since more IBV S was at the surface of cells infected withIBV-EG3, we reasoned that cells infected with IBV-EG3should also produce larger syncytia. We examined the size ofsyncytia in cells infected with either IBV-wt or IBV-EG3 byphase microscopy and found that syncytia were larger andmore prevalent in IBV-EG3-infected cells (Fig. 8B). We quan-tified the difference by counting the number of nuclei persyncytia in IBV-wt- or IBV-EG3-infected cells at 11.5 h p.i.Over 80% of IBV-wt syncytia had three to five nuclei, whileover 80% of IBV-EG3 syncytia contained more than five nu-clei. Thus, IBV E, but not EG3, decreases the accumulation ofIBV S on the surface of infected cells and reduces the size ofsyncytia in IBV-wt-infected cells.

DISCUSSION

We have shown that the HD of IBV E plays an importantrole in the release of infectious IBV particles from Vero cells.A recombinant virus with IBV E containing a heterologousHD (EG3) was competent for virus assembly but showed adefect in the release of infectious particles. The finding that theHD was not required for assembly is consistent with our earlierobservation that the cytoplasmic tail of IBV E is sufficient forinteraction with IBV M (4). Further characterization of themutant virus showed that it accumulated intracellularly in vac-uole-like structures along with aberrant material. We hypoth-esized that the mutant virions were accumulating intracellu-larly and becoming damaged and were subsequently releasedas noninfectious particles. Thus, we initially thought that theHD of IBV E might alter the secretory pathway to promoteanterograde trafficking. However, in overexpression experi-ments IBV E, but not EG3, caused a dramatic reduction inprotein trafficking to the plasma membrane by impeding cargotrafficking through the Golgi complex. We also observed thatoverexpression of IBV E disrupted Golgi morphology but didnot affect the ER or endosomal compartments. Finally, weobserved that cells infected with IBV-EG3 had increased sur-face levels of IBV S, leading to larger syncytia.

FIG. 7. Golgi markers do not colocalize in IBV E-expressing cells.(A) HeLa cells overexpressing IBV E were double labeled for twoGolgi markers: rabbit antigiantin was used along with either mouseanti-p230 or mouse anti-GM130 as indicated. Boxed areas in each fieldare enlarged at the right; arrows indicate areas of overlap, and arrow-heads indicate areas where only one marker is present. (B) Colormerges of the enlargements shown in panel A. Giantin is shown ingreen, and p230 and GM130 are shown in red.



http://jvi.asm.org/

Dow

nloaded from

http://jvi.asm.org/

Previously our lab reported that expression of IBV E usinga recombinant vaccinia virus in BHK-21 cells did not disruptGolgi structure (5). In the current study we expressed IBV Efrom a plasmid using lipid-based transient transfection ofHeLa cells. One possible reason for the difference we observedin the previous study could be the different cell types used. Wetested this by overexpressing IBV E via lipid-based plasmidtransfection in several different cell lines, including BKH-21. Inall cases, IBV E disrupted the Golgi morphology (data notshown). We next tested whether the lipid-based transfectionmethod was responsible for the effects that we observed. Weused a CaPO4-based transfection method as well as nucleofec-tion and observed the same effects on the Golgi complex as wesaw using lipid-based methods (data not shown). Thus, thediscrepancy in the data is likely due to the method used toexpress IBV E. Vaccinia virus expression produces a largeamount of protein very rapidly, whereas transient transfectiontends to produce a more modest amount of protein over alonger period of time.

Our lab previously reported that IBV does not replicateefficiently when IBV S accumulates on the surface of cells earlyin infection (39). The ability of IBV E to reduce protein traf-

ficking is likely beneficial to the virus because it prevents theaccumulation of IBV S on the surface of infected cells, therebyreducing syncytium size and number. Large syncytia may dieprematurely, which prevents robust virus replication. Increasedsyncytium size may also make virion trafficking more challeng-ing due to the intracellular rearrangements caused by cell-cellfusion. Furthermore, reducing protein trafficking during hostinfection may have other positive effects for the virus, such asreducing the amount of antigen on the cell surface or prevent-ing antigen display to the immune system by the major histo-compatibility complex I.

The importance of the E protein in the release of infectiousparticles has been observed for other CoVs. Mutations intro-duced into the HD of MHV E via alanine scanning producedmutant viruses which, among other defects, were compromisedin release of infectious virus (37). Studies investigating the roleof transmissible gastroenteritis coronavirus (TGEV) E showedthat when E protein was deleted from the virus, virions accu-mulated intracellularly, and infectious virus could not be re-covered unless E was provided in trans (21). These results,combined with our data showing that IBV E alters the secre-

FIG. 8. Mutation of the HD of IBV E leads to increased accumulation of IBV S on the surface of infected cells. (A) Vero cells were infectedwith either IBV-wt or IBV-EG3 at an MOI of 0.1. At 9 to 10 h p.i., cells were subjected to surface staining for IBV S, followed by fixation andstaining for total S. Quantification of the surface labeling was performed using Image J as described in Materials and Methods. The graphrepresents data from at least 15 cells from two independent infections; error bars represent standard errors of the means (P � 0.03). (B) Vero cellswere infected with either IBV-wt or IBV-EG3 at an MOI of 0.1 and imaged under phase microscopy at 11.5 h p.i. Syncytium size was quantifiedafter labeling with rabbit anti-IBV E to mark infected cells and Hoechst 33258 to label nuclei (graph on right). Syncytia were classified as cells withthree or more nuclei. Syncytia from four independent infections were counted at between 10.5 and 14 h p.i. For IBV-wt 131, syncytia were scored,and for IBV-EG3, 136 syncytia were scored.



http://jvi.asm.org/

Dow

nloaded from

http://jvi.asm.org/

tory pathway, suggest that the CoV E protein supports therelease of infectious particles.

The apparent disassembly of the Golgi complex in responseto expression of IBV E raises some interesting questions. Pre-viously it has been reported that MHV infection drives therearrangement of the Golgi complex in a two-step process,where initially the Golgi complex is dispersed from its jux-tanuclear position by an unknown mechanism, followed by thecondensation of the Golgi complex in the centers of syncytia,seemingly driven by cell-cell fusion (10). More recent work byUlasli et al. has greatly expanded our understanding of themembrane rearrangements caused by coronaviruses. These au-thors describe the formation of large virion-containing vacu-oles from ERGIC/Golgi membranes concurrent with the scat-tering of the Golgi complex (30). These results suggest that therearrangement of the Golgi complex may be important forforming virion carriers. It is interesting to speculate on why thevirus would need to alter the secretory pathway in order toproperly traffic its virions. Virions are much larger than normalcargo and may traverse the secretory pathway using a differentroute, or they may require different machinery than conven-tional cargo. The morphological changes in the Golgi complexthat we observed in the presence of IBV E may help to createan environment that promotes virion trafficking.

The mechanism by which IBV E modifies the secretory path-way is not understood. One possibility is that IBV E oligomer-izes and forms an ion channel at the Golgi complex. Otherviruses encode small hydrophobic proteins that oligomerizeand form ion channels, with the best characterized being in-fluenza virus M2 (23). The proton channel activity of influenzavirus M2 plays an important role during the entry of influenzavirus by acidifying the lumen of the virion following endocyto-sis, which allows for uncoating of the virus (34). For somestrains of influenza virus, M2 also acts at the trans-Golgi net-work (TGN), where it raises the pH of the TGN lumen toprevent the premature activation of the fusion protein, hem-agglutinin (27). A consequence of increasing the TGN pH isthat normal protein trafficking is slowed both through theGolgi complex and to the plasma membrane (25). It is possiblethat IBV E follows a similar paradigm for ion channel activityin the secretory pathway. In vitro data suggest that an IBV Echannel would likely conduct Na� or K� ions (35). If IBV Edisrupts the Na�/K� gradient at the Golgi complex, it couldhave a deleterious impact on the Na�/H� exchangers at theGolgi complex that are critical for maintaining the properlumenal pH of the Golgi complex (19). It is also conceivablethat the presence of an IBV E ion channel at the Golgi com-plex could affect the homeostasis of other ions critical to Golgifunction, such as Ca2� or Cl� (11, 16). In addition to the effecton protein trafficking, changing the ion balance within theGolgi complex could inactivate proteases that damage virions.This would explain why IBV S is damaged in cells infected withIBV-EG3 and not in those infected with IBV-wt. Additionally,a change in the lumenal ion concentration could have an effecton the structure of the Golgi complex. It is clear that morework will be required to understand the putative ion channelactivity of IBV E. This information will be essential for under-standing the function of IBV E at the Golgi complex.

While there are considerable in vitro data focused on the ionchannel activity of CoV E, it has not yet been demonstrated

that the CoV E protein possesses ion channel activity in in-fected cells. Thus, it is important to consider other possibilitiesfor the mechanism of IBV E function. It may be that the HDof IBV E facilitates a protein-protein interaction that affectsprotein trafficking. One possibility is that the HD of IBV Einteracts with the membrane domain of SNARES that regulatevesicle fusion at the Golgi complex. It is also possible that IBVE may interact with cellular ion channels present in Golgimembranes and modulate their activity. For example, if theHD of IBV E was able to bind tightly to the V0 subunit of theV-ATPase, it could prevent assembly of the active pump andrender it unable to acidify the Golgi lumen. This would lead totrafficking and morphological changes similar to those causedby overexpression of IBV E (38). Finally, by mutating the HDof IBV E, we may have disrupted the formation of IBV Eoligomers. While oligomerization would be required for ionchannel activity, it may also be important for the types ofprotein-protein interactions described above.

While the CoV E proteins are small, they appear to havemultiple functions. Previous characterization of IBV E showedthat the C-terminal tail of the protein contains targeting infor-mation and facilitates interaction with IBV M (3, 4). Here, wetook advantage of a mutant version of IBV E that was com-petent for assembly but defective in release of infectious par-ticles. We showed that the HD of IBV E alters the cellularsecretory pathway. This indicates that multiple domains of IBVE are important for its proper function, and this is possibly truefor all CoV E proteins. Future studies will examine whichresidues within the HD of IBV E are critical for its effect onthe secretory pathway. Additionally, it will be important todetermine how IBV alters the secretory pathway through di-rect ion channel activity, protein-protein interaction, or someother mechanism.

ACKNOWLEDGMENTS

This work was supported by National Institutes of Health grant R21A1072312.

We thank Soonjeon Youn for the initial isolation of the recombinantIBV and IBV-EG3. We also thank members of the Machamer lab aswell as Jeremy Rotty, Corrin McBride, and David Zuckerman forhelpful discussions and critical reading of the manuscript.

REFERENCES

1. Boscarino, J. A., H. L. Logan, J. J. Lacny, and T. M. Gallagher. 2008.Envelope protein palmitoylations are crucial for murine coronavirus assem-bly. J. Virol. 82:2989–2999.

2. Cluett, E. B., and C. E. Machamer. 1996. The envelope of vaccinia virusreveals an unusual phospholipid in Golgi complex membranes. J. Cell Sci.109:2121–2131.

3. Corse, E., and C. E. Machamer. 2002. The cytoplasmic tail of infectiousbronchitis virus E protein directs Golgi targeting. J. Virol. 76:1273–1284.

4. Corse, E., and C. E. Machamer. 2003. The cytoplasmic tails of infectiousbronchitis virus E and M proteins mediate their interaction. Virology 312:25–34.

5. Corse, E., and C. E. Machamer. 2000. Infectious bronchitis virus E proteinis targeted to the Golgi complex and directs release of virus-like particles.J. Virol. 74:4319–4326.

6. DeDiego, M. L., E. Alvarez, F. Almazan, M. T. Rejas, E. Lamirande, A.Roberts, W. J. Shieh, S. R. Zaki, K. Subbarao, and L. Enjuanes. 2007. Asevere acute respiratory syndrome coronavirus that lacks the E gene isattenuated in vitro and in vivo. J. Virol. 81:1701–1713.

7. Klumperman, J., J. K. Locker, A. Meijer, M. C. Horzinek, H. J. Geuze, andP. J. Rottier. 1994. Coronavirus M proteins accumulate in the Golgi complexbeyond the site of virion budding. J. Virol. 68:6523–6534.

8. Kuo, L., K. R. Hurst, and P. S. Masters. 2007. Exceptional flexibility in thesequence requirements for coronavirus small envelope protein function.J. Virol. 81:2249–2262.



http://jvi.asm.org/

Dow

nloaded from

http://jvi.asm.org/

9. Kuo, L., and P. S. Masters. 2003. The small envelope protein E is notessential for murine coronavirus replication. J. Virol. 77:4597–4608.

10. Lavi, E., Q. Wang, S. R. Weiss, and N. K. Gonatas. 1996. Syncytia formationinduced by coronavirus infection is associated with fragmentation and rear-rangement of the Golgi apparatus. Virology 221:325–334.

11. Lissandron, V., P. Podini, P. Pizzo, and T. Pozzan. Unique characteristics ofCa2� homeostasis of the trans-Golgi compartment. 2010. Proc. Natl. Acad.Sci. U. S. A. 107:9198–9203.

12. Lontok, E., E. Corse, and C. E. Machamer. 2004. Intracellular targetingsignals contribute to localization of coronavirus spike proteins near the virusassembly site. J. Virol. 78:5913–5922.

13. Lopez, L. A., A. J. Riffle, S. L. Pike, D. Gardner, and B. G. Hogue. 2008.Importance of conserved cysteine residues in the coronavirus envelope pro-tein. J. Virol. 82:3000–3010.

14. Machamer, C. E., and J. K. Rose. 1987. A specific transmembrane domain ofa coronavirus E1 glycoprotein is required for its retention in the Golgiregion. J. Cell Biol. 105:1205–1214.

15. Machamer, C. E., and S. Youn. 2006. The transmembrane domain of theinfectious bronchitis virus E protein is required for efficient virus release.Adv. Exp. Med. Biol. 581:193–198.

16. Maeda, Y., T. Ide, M. Koike, Y. Uchiyama, and T. Kinoshita. 2008. GPHRis a novel anion channel critical for acidification and functions of the Golgiapparatus. Nat. Cell Biol. 10:1135–1145.

17. McBride, C. E., J. Li, and C. E. Machamer. 2007. The cytoplasmic tail of thesevere acute respiratory syndrome coronavirus spike protein contains a novelendoplasmic reticulum retrieval signal that binds COPI and promotes inter-action with membrane protein. J. Virol. 81:2418–2428.

18. McBride, C. E., and C. E. Machamer. 2010. A single tyrosine in the severeacute respiratory syndrome coronavirus membrane protein cytoplasmic tailis important for efficient interaction with spike protein. J. Virol. 84:1891–1901.

19. Nakamura, N., S. Tanaka, Y. Teko, K. Mitsui, and H. Kanazawa. 2005. FourNa�/H� exchanger isoforms are distributed to Golgi and post-Golgi com-partments and are involved in organelle pH regulation. J. Biol. Chem. 280:1561–1572.

20. Niwa, H., K. Yamamura, and J. Miyazaki. 1991. Efficient selection forhigh-expression transfectants with a novel eukaryotic vector. Gene 108:193–199.

21. Ortego, J., J. E. Ceriani, C. Patino, J. Plana, and L. Enjuanes. 2007. Absenceof E protein arrests transmissible gastroenteritis coronavirus maturation inthe secretory pathway. Virology 368:296–308.

22. Pervushin, K., E. Tan, K. Parthasarathy, X. Lin, F. L. Jiang, D. Yu, A.Vararattanavech, T. W. Soong, D. X. Liu, and J. Torres. 2009. Structure andinhibition of the SARS coronavirus envelope protein ion channel. PLoSPathog. 5:e1000511.

23. Pinto, L. H., L. J. Holsinger, and R. A. Lamb. 1992. Influenza virus M2protein has ion channel activity. Cell 69:517–528.

24. Pyrc, K., B. Berkhout, and L. van der Hoek. 2007. The novel human coro-naviruses NL63 and HKU1. J. Virol. 81:3051–3057.

25. Sakaguchi, T., G. P. Leser, and R. A. Lamb. 1996. The ion channel activityof the influenza virus M2 protein affects transport through the Golgi appa-ratus. J. Cell Biol. 133:733–747.

26. Shaner, N. C., R. E. Campbell, P. A. Steinbach, B. N. Giepmans, A. E.Palmer, and R. Y. Tsien. 2004. Improved monomeric red, orange and yellowfluorescent proteins derived from Discosoma sp. red fluorescent protein.Nat. Biotechnol. 22:1567–1572.

27. Sugrue, R. J., G. Bahadur, M. C. Zambon, M. Hall-Smith, A. R. Douglas,and A. J. Hay. 1990. Specific structural alteration of the influenza haemag-glutinin by amantadine. EMBO J. 9:3469–3476.

28. Torres, J., J. Wang, K. Parthasarathy, and D. X. Liu. 2005. The transmem-brane oligomers of coronavirus protein E. Biophys. J. 88:1283–1290.

29. Turner, J. R., and A. M. Tartakoff. 1989. The response of the Golgi complexto microtubule alterations: the roles of metabolic energy and membranetraffic in Golgi complex organization. J. Cell Biol. 109:2081–2088.

30. Ulasli, M., M. H. Verheije, C. A. de Haan, and F. Reggiori. 2010. Qualitativeand quantitative ultrastructural analysis of the membrane rearrangementsinduced by coronavirus. Cell. Microbiol. 12:844–861.

31. Vennema, H., G. J. Godeke, J. W. Rossen, W. F. Voorhout, M. C. Horzinek,D. J. Opstelten, and P. J. Rottier. 1996. Nucleocapsid-independent assemblyof coronavirus-like particles by co-expression of viral envelope protein genes.EMBO J. 15:2020–2028.

32. Weisz, O. A., C. E. Machamer, and A. L. Hubbard. 1992. Rat liver dipepti-dylpeptidase IV contains competing apical and basolateral targeting infor-mation. J. Biol. Chem. 267:22282–22288.

33. Weisz, O. A., A. M. Swift, and C. E. Machamer. 1993. Oligomerization of amembrane protein correlates with its retention in the Golgi complex. J. CellBiol. 122:1185–1196.

34. Wharton, S. A., R. B. Belshe, J. J. Skehel, and A. J. Hay. 1994. Role of virionM2 protein in influenza virus uncoating: specific reduction in the rate ofmembrane fusion between virus and liposomes by amantadine. J. Gen. Virol.75:945–948.

35. Wilson, L., P. Gage, and G. Ewart. 2006. Hexamethylene amiloride blocks Eprotein ion channels and inhibits coronavirus replication. Virology 353:294–306.

36. Wilson, L., C. McKinlay, P. Gage, and G. Ewart. 2004. SARS coronavirus Eprotein forms cation-selective ion channels. Virology 330:322–331.

37. Ye, Y., and B. G. Hogue. 2007. Role of the coronavirus E viroporin proteintransmembrane domain in virus assembly. J. Virol. 81:3597–3607.

38. Yilla, M., A. Tan, K. Ito, K. Miwa, and H. L. Ploegh. 1993. Involvement ofthe vacuolar H(�)-ATPases in the secretory pathway of HepG2 cells. J. Biol.Chem. 268:19092–19100.

39. Youn, S., E. W. Collisson, and C. E. Machamer. 2005. Contribution oftrafficking signals in the cytoplasmic tail of the infectious bronchitis virusspike protein to virus infection. J. Virol. 79:13209–13217.

40. Youn, S., J. L. Leibowitz, and E. W. Collisson. 2005. In vitro assembled,recombinant infectious bronchitis viruses demonstrate that the 5a open read-ing frame is not essential for replication. Virology 332:206–215.



http://jvi.asm.org/

Dow

nloaded from

http://jvi.asm.org/

The Hydrophobic Domain of Infectious Bronchitis Virus E Protein ...

Documents

Transcript of The Hydrophobic Domain of Infectious Bronchitis Virus E Protein ...