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Palmitoylation of the Alphacoronavirus TGEV spike protein S is essential for incorporation into virus like particles but dispensable for S M interaction Sandra Gelhaus a Bastian Thaa b 1 Kathrin Eschke a Michael Veit b Christel Schwegmann We els a n a Institute for Virology Department of Infectious Diseases University of Veterinary Medicine Hannover Foundation B nteweg 17 30559 Hannover Germany b Institute for Virology Veterinary Faculty Free University Robert von Ostertag Str 7 13 14163 Berlin Germany article info Article history Received 17 April 2014 Returned to author for revisions 18 May 2014 Accepted 21 July 2014 Keywords Coronavirus TGEV Alphacoronavirus Spike protein Palmitoylation Cysteine rich motif Assembly abstract The spike protein S of coronaviruses contains a highly conserved cytoplasmic cysteine rich motif adjacent to the transmembrane region This motif is palmitoylated in the Betacoronaviruses MHV and SARS CoV Here we demonstrate by metabolic labeling with 3 H palmitic acid that the S protein of transmissible gastroenteritis coronavirus TGEV an Alphacoronavirus is palmitoylated as well This is relevant for TGEV replication as virus growth was compromised by the general palmitoylation inhibitor 2 bromopalmitate Mutation of individual cysteine clusters in the cysteine rich motif of S revealed that all cysteines must be replaced to abolish acylation and incorporation of S into virus like particles VLP Conversely the interaction of S with the M protein essential for VLP incorporation of S was not impaired by lack of palmitoylation Thus palmitoylation of the S protein of Alphacoronaviruses is dispensable for S M interaction but required for the generation of progeny virions Tooze et al 1984 Subcellular targeting signals in the viral proteins direct the proteins towards the budding site Corse and Machamer 2002 Lontok et al 2004 Paul et al 2014 Schwegmann Wessels et al 2004 Swift and Machamer 1991 Lateral protein interactions between M and M Locker et al 1992 M and E Lim and Liu 2001 M and N Narayanan et al 2000 as well as M and S Opstelten et al 1995 proteins play crucial roles during assembly and trigger budding This paper focuses on the S protein of TGEV This is a large type 1 membrane protein 250 kDa that is responsible for the attachment to the cellular receptor and sub sequent fusion of viral and cellular membranes The S protein is incorporated into virus particles by interaction with the M protein Nguyen and Hogue 1997 Vennema et al 1996 This is an indispensable step in the CoV replication cycle to obtain infectious virus particles Palmitoylation S acylation is a post translational modi ca tion in which a saturated fatty acid most commonly palmitic acid is linked to a cysteine residue by a thioester bond Schmidt Contents lists available at ScienceDirect journal homepage Virology http dx doi org 10 1016 j virol 2014 07 035 0042 6822 fax 49 511 9538898 E mail address christel schwegmann tiho hannover de C Schwegmann We els 1 Present address Karolinska Institutet Department of Microbiology Tumor and Cell Biology Nobels v g 16 171 77 Stockholm Sweden Virology 464 465 2014 397 405 et al 1988 Veit et al 1991 This modi cation can have effects on protein activity Huang et al 2010 transport Abrami et al 2008 2006 and stability Abrami et al 2006 Maeda et al 2010 Most of the cysteines that become acylated are located near the transmembrane region Veit 2012 Yang and Compans 1996 Yik and Weigel 2002 Palmitoylation of viral proteins has been shown to affect virus assembly or virus replication Bhattacharya et al 2004 Gaedigk Nitschko et al 1990 CoV S proteins show a conserved cysteine rich motif CRM in the cytoplasmic domain adjacent to the carboxyterminal end of the transmembrane region Fig 1 The CoV S proteins share little sequence homology at this region but the cysteine content is about 35 For the Betacoronaviruses SARS CoV and MHV it has been shown that the S proteins are palmitoylated at the CRM Petit et al 2007 Thorp et al 2006 In both viruses the palmitoylation of the S protein is implicitly necessary for the incorporation of the S protein into virus like particles VLPs or virus particles Thorp et al 2006 Ujike et al 2012 However a partial CRM can be suf cient for the incorporation of the S protein Ujike et al 2012 Yang et al 2012 In contrast to that the interaction of the S and M proteins has different requirements in both viruses Treatment with the general palmitoylation inhibitor 2 bromopalmitate dis rupted the interaction of S and M proteins in MHV Thorp et al 2006 Conversely the SARS CoV S M protein interaction is not impaired with a palmitoylation null S protein cysteine mutant McBride and Machamer 2010 These reported ndings demonstrate the different require ments in the closely related viruses MHV and SARS CoV that both belong to the genus Betacoronavirus We wanted to know whether coronaviruses from the genus Alphacoronavirus differ from these Betacoronaviruses with respect to their role of the S protein s CRM In our study we addressed the role of the S protein CRM of the Alphacoronavirus TGEV during assembly To this end we substi tuted cysteines in the CRM of the TGEV S protein and assessed their effect on VLP formation and on S M protein interaction We show that in contrast to the SARS CoV S protein the TGEV S protein is incorporated into VLPs irrespective of the position of its palmitoylated cysteines For an ef cient interaction between TGEV S and M proteins palmitoylation of the S protein is not necessary Our results demonstrate that despite general similarities corona viruses show differences regarding the functional role of S protein palmitoylation that may have been induced byevolutionary forces Results and discussion 2 BP treatment decreases the infectivity of TGEV The palmitate analog 2 bromopalmitate 2 BP inhibits protein palmitoylation in general Webb et al 2000 To investigate whether palmitoylation has an impact on TGEV infectivity we performed a treatment with 2 BP Fig 2A ST cells were infected with TGEV and treated with different concentrations of 2 BP 0 0 8 4 8 and 12 mM Vesicular stomatitis virus VSV grown in BHK 21 cells was employed as a control since the assembly of this virus does not depend on palmitoylation Thorp et al 2006 Whitt and Rose 1991 These 2 BP concentrations were not cytotoxic for ST cells veri ed with a WST 1 cell proliferation assay Fig 2B About 24 h after infection media were harvested and viral infectivity was determined by plaque assay The production of infectious VSV particles was not signi cantly inhibited by the 2 BP treatment Fig 2A Thorp et al 2006 Whitt and Rose 1991 In contrast to that a signi cant dose dependent decrease of TGEV infectivity was observed upon 2 BP treatment This decrease in infectivity correlated with a decrease in the amount of virus particles in the supernatant of 2 BP treated infected cells as seen by Western blot of pelleted supernatants for viral proteins Fig 2C The amount of protein expression in the cell lysates was not in uenced by 2 BP treatment Fig 2C From these results we conclude that an inhibition of palmitoylation has negative effects on the production of infectious TGEV but not VSV particles While we cannot exclude that the reduction in infectious TGEV particle numbers upon 2 BP treatment is partially caused by inhibited palmitoylation of an essential cellular factor we con sidered it most likely that the palmitoylation of a viral protein is required for the production of infectious virions This led us to assess whether the TGEV S protein is palmitoylated TGEV S is palmitoylated in virus particles Recent studies have shown that the S proteins of MHV and SARS CoV both members of the genus Betacoronavirus are palmi toylated McBride and Machamer 2010 Petit et al 2007 Shulla and Gallagher 2009 Thorp et al 2006 The cysteine rich motif CRM is highly conserved among CoV S proteins and a CRM is also found in the S protein of TGEV as a member of the genus Alphacoronavirus Fig 1 To determine whether the TGEV S protein is also palmitoylated TGEV was grown in ST cells in the presence of 3 H palmitic acid to achieve radioactive labeling followed by pelleting of the virus SDS PAGE and uorography This revealed that the S protein of TGEV is indeed S acylated visible by the band at about 250 kDa which was absent in uninfected cells Fig 2D The speci city of this labeling with 3 H palmitic acid was con rmed by hydroxylamine treatment Fig 2D Hydroxylamine cleaves thioester linkages at neutral pH and thus any palmitic acid linked to a cysteine residue by S acylation The disappearance of the TGEV S protein band after hydroxylamine treatment proves that this protein is S acylated at at least one cysteine residue Fig 1 Sequence alignment of the carboxy terminal S protein sequences from representative coronavirusspecies Underlined cysteine residues represent potential palmitoylation sites TGEV PUR 46 transmissible gastroenteritis virus strain PUR 46 FIPV feline infectious peritonitis virus HCoV NL 63 human coronavirus NL63 SARS CoV severe acute respiratory syndrome coronavirus MHV A59 mouse hepatitis virus strain A59 MERS CoV middle east respiratory syndrome coronavirus IBV Beaudette infectious bronchitis virus strain Beaudette Bul CoV bulbul coronavirus S Gelhaus et al Virology 464 465 2014 397 405398 Palmitoylation of TGEV S is necessary for incorporation into VLPs As the infectivity of TGEV was reduced after 2 BP treatment and the S protein present in TGE virions was shown to be palmitoylated we concentrated our analysis on the cysteine rich motif CRM present in the cytoplasmic domain of the TGEV S protein Fig 1 The Swt protein of the TGEV PUR MAD strain contains 10 cysteine residues within the CRM Sanchez et al 1990 The CRM of TGEV S can be split up in three cysteine clusters with three to four cysteine residues Fig 1 To test whether speci c cysteine clusters have a crucial role during virus assembly partial Cys mutants were created in addition to the complete cysteine mutant where all cysteines were replaced by alanines S C 1 10 A Fig 3A As the transfection ef ciency of STcells is quite low BHK 21 cells were transfected with the plasmids encoding for the different Cys mutants The labeling with 3 H palmitic acid showed that all partial mutants were palmitoylated whereas the complete Cys mutant was not palmitoylated Fig 3B The pre sence of three cysteines in the CRM S C 1 7 A was hence suf cient for the protein to be acylated even if the degree of palmitoylation was lower for this mutant Fig 3B It is conceivable that palmitoylation of the CRM cysteines is important for fusion which is mediated by the S protein The importance of the CRM of the MHV and SARS CoV S proteins for fusion has already been proven Bos et al 1995 Chang et al 2000 McBride and Machamer 2010 Shulla and Gallagher 2009 In this study we however evaluated Fig 2 Palmitoylation of TGEV A Treatment with 2 bromopalmitate 2 BP decreases the infectivity of TGEV ST or BHK 21 cells were inoculated with TGEV or VSV respectively At 1 h p i media were replaced with growth media containing the indicated concentrations of 2 BP or ethanol as 2 BP solvent ethanol control amount of ethanol present in the highest 2 BP concentration 0 0 mM 2 BP medium without ethanol Twenty four hours later media were collected and the amount of infectious virus particles was determined by plaque assay Plaque forming units PFU ml are displayed as mean standard deviation SD n signi cantly different according to Student s t test compared to 0 0 mM 2 BP po0 05 B 2 BP is not cytotoxic to ST cells at the concentrations used ST cells were treated with the indicated concentrations of 2 BP and cell proliferation was determined by WST 1 assay The cell proliferation is indicated as mean SD in percentage C The amount of viral proteins present in viral particles is reduced after 2 BP treatment Virions from cell culture supernatants of infected cells treated with the indicated amounts of 2 BP were pelleted by ultracentrifugation 24 h p i and analyzed by SDS PAGE and Western blotting virions The indicated proteins S N and M were detected by monoclonal antibodies The corresponding cells were lysed and the respective proteins were equally analyzed cell lysates D Palmitoylation of the TGEV S protein in virus particles Infected and mock infected ST cells were labeled with 3 H palmitic acid or 35 S methionine cysteine Radiolabeled virus was analyzed by SDS PAGE and uorography A hydroxylamine treatment was performed to determine the linkage of fatty acids via a thioester bond S Gelhaus et al Virology 464 465 2014 397 405 399 whether palmitoylation is important for assembly and incorpora tion of the spike protein into newly generated virus particles To analyze this role a virus like particle assay was performed in which the incorporation of S can be analyzed separately from the in uence of the fusion capacity of the S protein The incorporation of the TGEV S protein is an essential step during assembly to form S Gelhaus et al Virology 464 465 2014 397 405400 infectious particles To determine the importance of the TGEV S protein s CRM during virus assembly we analyzed the capability of the TGEV S Cys mutants to be incorporated into virus like parti cles VLPs Fig 3D and E The co expression of the TGEV E and M proteins is the minimal requirement for VLP formation Baudoux et al 1998 If the S protein is co expressed with M and E it will be incorporated into VLPs Vennema et al 1996 In contrast to the authentic TGEV S protein the complete Cys mutant S C 1 10 Awas not incorporated into VLPs Fig 3D The analyzed partial Cys mutants S C 1 3 A S C 4 7 A S C 8 10 A and S C 1 7 A behaved like the TGEV Swt protein and were incorporated into VLPs Fig 3D To con rm these results and to account for variations in protein levels in Western blots we additionally performed immuno uorescence analysis to show the cell surface transport of S protein Fig 3E We obtained comparable intracellular expression levels of all tested TGEV S proteins and the TGEV M protein Like in the VLP assay analyzed by Western blot the S C 1 10 A mutant was the only S protein which was not detectable on the cell surface and therefore not incorporated into VLPs Fig 3E These results demonstrate that cysteines are important for the S protein incorporation into viral particles and that they are at least partially redundant In contrast to the reports for SARS CoV Ujike et al 2012 the speci c position of the cysteine residue inside the cysteine rich region seems not to be relevant for the incorporation into VLPs The presence of one cluster of 3 cysteines S C 1 7 A is enough to incorporate S into VLPs The S proteins of coronaviruses are known to form trimers Delmas and Laude 1990 To exclude that the inability of the complete Cys mutant to incorporate into VLPs is caused by a defect in oligomerization we analyzed the quaternary structure of S C 1 10 A with a sucrose density gradient and compared it with the TGEV Swt protein The results showed that the wild type protein and the Cys mutant are both detectable in high concentration sucrose fractions indicating a comparable oligomerization poten tial Fig 3C In conclusion the palmitoylation of the TGEV S is necessary for the incorporation of the S protein into VLPs but does not affect oligomerization of S How does palmitoylation of the TGEV S protein in uence S incorporation One possible explanation might be that the conformation of the cytoplasmic tail of the S protein is crucial for the incorporation of S protein into virus particles The palmitic acids probably help in membrane attachment of the cytoplasmic tail and may thus de ne its conformation This conformation may play a minor role in the binding to the M protein but may be important for the incorpora tion of the TGEV S protein into virus like particles It is possible that the linked fatty acids determine how the protein is positioned in the ERGIC membrane and how it is inserted in the dense matrix formed by the lateral M M interactions described for coronavirus assembly de Haan et al 2000 Neuman et al 2006 The palmitoylation of proteins can concentrate them at certain membrane domains Heakal et al 2011 A lot of viruses use palmitoylation to target their proteins to lipid microdomains at the plasma membrane for assembly Bhattacharya et al 2004 Zhang et al 2000 Palmitoylated proteins concentrate with interacting proteins at intracellular microdomains as well As examples the chaperon Calnexin and the transmembrane thioredoxin protein are recruited to detergent resistant membranes of the endoplas mic reticulum by palmitoylation Hayashi and Fujimoto 2010 Lynes et al 2012 Thus a version of the TGEV S protein like the complete Cys mutant S C 1 10 A that lacks palmitic acids may interact with the M protein in an S M protein co expression experiment but may not be sorted to the membrane microdo mains at the ERGIC in which virus assembly and budding take place and may therefore not be incorporated into newly generated virions or VLPs McBride and Machamer have shown that the SARS CoV S protein is present in DRMs at the plasma membrane in single expression experiments For a complete Cys mutant of the SARS CoV S protein it was shown that it is no longer present in DRMs McBride and Machamer 2010 These results con rm the hypothesis that the palmitoylation of coronavirus S proteins directs them to membrane microdomains and that this localiza tion is indispensable for the incorporation into VLPs and virions Palmitoylation of TGEV S is dispensable for interaction with the M protein Lateral protein interactions play important roles during virus assembly The interaction of S and M proteins is essential for the incorporation of S into virus particles Thus we aimed to nd out whether the palmitoylation of S is required for the interactionwith the M protein as in the case of MHV Shulla and Gallagher 2009 Thorp et al 2006 or not as for SARS CoV McBride and Machamer 2010 Ujike et al 2012 To determine this we expressed M with HA tag together with the various cysteine mutants of S with the background of a destroyed ERGIC retention signal Y A Fig 4A With this Y A mutation S is ef ciently transported to the plasma membrane in the absence of M but is retained intracellularly by M Fig 4BandC Failure of S Y A cysteine mutants to interact with M thus leads to plasma membrane delivery of S even in the presence of M The TGEV S variants depicted in Fig 4A were expressed in BHK 21 cells by transfection in the absence or presence of HA tagged TGEV M Immuno uorescence analysis was employed to assess the localization of S and M All partial Cys mutants and even the complete Cys mutant showed a similar protein localization pattern with the M protein Fig 4B and C This pattern was identical to the one seen for the S wildtype protein in coexpression with the HA tagged M protein S wt Fig 4B and C In contrast to that the VSV G protein was not retained and not intracellularly clustered by the co expression of HA tagged TGEV M protein VSV G Fig 4B and C These results indicate that speci c interactions between the TGEV S and M proteins lead to an intracellular retention of the mutant S proteins and that the interac tion of TGEV S and M proteins is not dependent on the palmitoylation of S Palmitoylation is known to affect protein traf cking Greaves and Chamberlain 2007 Resh 1999 2006 Our results show that this is not the case for the TGEV S since the full cysteine mutant with Fig 3 Palmitoylation of S is necessary for incorporation into VLPs A Schematic illustration of TGEV S wild type and TGEV S cysteine mutants The cysteine rich motif CRM and the tyrosine based retention signal are highlighted by boxes Cysteines substituted by alanines are written in bold ED ectodomain TMD transmembrane domain CD cytoplasmic domain B The complete TGEV S cysteine mutant is not palmitoylated whereas the partial cysteine mutants are BHK 21 cells expressin