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DOI 10 1515 hsz 2013 0282 Biol Chem 2014 395 6 657 665 Andr Paul Anna Trincone Sandra Siewert Georg Herrler and Christel Schwegmann We els A lysine methionine exchange in a coronavirus surface protein transforms a retention motif into an endocytosis signal Abstract Transmissible gastroenteritis virus TGEV is an enveloped RNA virus belonging to the family Corona viridae Among the viral membrane proteins the spike S protein mediates receptor recognition attachment to the host cell and fusion of viral and cellular mem branes The cytoplasmic tail of the S protein contains a tyrosine dependent sorting signal with the consensus sequence YXX In the context of the S protein of TGEV 1440YEPI1443 this motif acts as a retention signal pre venting surface expression of the protein Here we show that a chimeric S protein containing the six C terminal amino acids of the glycoprotein G of vesicular stomatitis virus VSV is no longer retained intracellularly despite the presence of the tyrosine tetrapeptide motif Following transport to the cell surface the chimeric protein was rap idly endocytosed Analysis of mutant proteins generated by site directed mutagenesis revealed that a single amino acid exchange 1445K M position 2 downstream of the tyrosine based motif was responsible for the altered sort ing behavior Keywords protein sorting protein transport TGEV S protein tyrosine based sorting signal VSV G protein Corresponding author Christel Schwegmann We els Institute for Virology Department of Infectious Diseases University of Veterinary Medicine Hannover B nteweg 17 D 30559 Hannover Germany e mail christel schwegmann tiho hannover de Andr Paul Anna Trincone Sandra Siewert and Georg Herrler Institute for Virology Department of Infectious Diseases University of Veterinary Medicine Hannover B nteweg 17 D 30559 Hannover Germany Introduction Transmissible gastroenteritis virus TGEV is a positive stranded RNA virus that belongs to the family Corona viridae genus Alphacoronavirus It causes respiratory and enteric disease in swine of all ages and is exceptionally severe in newborn animals Mortality in these animals can reach 100 Enjuanes and van der Zeijst 1995 Three membrane proteins have been described for TGEV the membrane protein M the small membrane protein E and the spike protein S The E protein is required for virus morphogenesis and maturation Fischer et al 1998 Ortego et al 2007 The M protein plays an important role during virus assembly and interacts with the E S and nucleocapsid proteins Opstelten et al 1995 Vennema et al 1996 Narayanan et al 2000 Escors et al 2001 The S protein is the major inducer of virus neutralizing antibodies and is crucial for virus entry Godet et al 1991 1994 It mediates virus attachment by interacting with porcine aminopeptidase N the cellular receptor protein for TGEV and fusion of the viral membrane with the host cell membrane Delmas et al 1992 Gallagher and Buchmeier 2001 Schwegmann Wessels et al 2002 2011 The budding event during the coronavirus repli cation cycle takes place at a pre Golgi compartment the endoplasmic reticulum Golgi intermediate compartment ERGIC Tooze et al 1984 Krijnse Locker et al 1994 Newly synthesized viral structural proteins accumulate at the site of budding and are incorporated together with the viral genome into viral particles which are then trans located to the plasma membrane and released via exocy tosis Intracellular sorting of coronavirus genomes and proteins to the site of virus budding requires strict regula tory mechanisms to achieve optimal virus production The precise regulation of viral and cellular factors involved in these processes is not fully understood Tyrosine based signals are involved at different sites in the cellular sorting machinery Previously we have demonstrated the importance of an YXX motif in the S proteins of two representatives of alpha and gammacoro naviruses the transmissible gastroenteritis coronavirus TGEV of swine and the infectious bronchitis virus IBV of chicken The TGEV S protein as well as the IBV S protein were found to be intracellularly retained because of a tyrosine based signal Schwegmann Wessels et al 2004 Winter et al 2008 This motif in the C terminal part of Brought to you by Yale University Authenticated Download Date 5 18 15 1 09 PM658 A Paul et al Role of methionine in tyrosine based sorting signals the TGEV S protein 1440YEPI1443 corresponds to YXX X can be any amino acid denotes a bulky hydrophobic amino acid motifs that have been described to be respon sible for basolateral sorting lysosomal targeting locali zation to the trans Golgi network or endocytosis in other cellular and viral proteins Howe et al 1988 Granger et al 1990 Letourneur and Klausner 1992 Thomas and Roth 1994 Rohrer et al 1996 In the case of CD3 a tyrosine containing tetrapeptide has also been described as a signal for intracellular retention Mallabiabarrena et al 1992 The glycoprotein G of vesicular stomatitis virus VSV a negative stranded ssRNA virus also harbors a carboxy terminal targeting signal of six amino acids 501 YTDIEM 506 which is important for efficient export of the G protein from the ER to the Golgi complex Sevier et al 2000 This sequence contains the above mentioned YXX consensus sequence as well as a di acidic DXE motif Nishimura and Balch 1997 Nishimura et al 1999 The aim of this study was to analyze the requirements that lead to different transport behavior of two distinct viral proteins even though they both contain tyrosine based sorting signals in similar positions of their cyto plasmic domain We focused on amino acids downstream of the tyrosine based sorting signal and their influence on intracellular sorting Chimeric proteins of S and G were constructed and were analyzed for surface trans port or intracellular retention Our findings show that a Lys Met1445 exchange in the TGEV S protein abolished intracellular retention and resulted in a protein that is transported to the plasma membrane and is subsequently endocytosed Results We were interested to know whether amino acids other than the tyrosine and the isoleucine in the 1440YEPI1443 motif of the TGEV S protein are important for its localiza tion and transport inside the cell The G protein of VSV contains a similar tyrosine based signal that is located at a similar position in the protein close to the carboxy terminus of the cytoplasmic tail Nevertheless the VSV G protein is transported to the cell surface and not retained intracellularly Wehland et al 1982 Stephens et al 1986 The tyrosine based sorting signal in the VSV G protein is described to be responsible for basolateral trans port Thomas and Roth 1994 In addition a di acidic signal nearby was shown to be important for ER export Nishimura and Balch 1997 We wondered whether the amino acids in the proximity may affect the function of the tyrosine based motif as a sorting signal We constructed different TGEV S protein mutants that contained some of the VSV G protein amino acids downstream of the tyros ine motif A schematic overview of the generated mutants is shown in Figure 1 The SG6 mutant in which the five carboxyterminal amino acids of the TGEV S protein were replaced by the six carboxyterminal amino acids of the VSV G protein differed from the parental TGEV S protein by a clear surface staining when analyzed by immuno fluorescence microscopy Figure 2A To narrow down the amino acids responsible for the differential transport behavior of the SG6 mutant the mutant SG KVH MNR was generated in which the VSV G tripeptide Met Asn Arg replaced the Lys Val His sequence in the TGEV S protein position 1445 to 1447 In addition we replaced the last two amino acids from the TGEV S protein Val His by the last three of the VSV G protein Leu Gly Lys result ing in the mutant SG VH LGK The latter mutant behaved like the parental TGEV S protein and was retained intra cellularly By contrast the mutant SG KVH MNR like the SG6 mutant was detected at the plasma mem brane Figure 2A Finally point mutants were generated Figure 1 Among them only the S K M mutant in which Figure 1 Schematic drawing of TGEV S VSV G chimeras as well as TGEV S and VSV G parental and mutant proteins The C terminal part of the cytoplasmic domain of the TGEV S and the VSV G protein sequence is shown The tyrosine based sorting signal is underlined Brought to you by Yale University Authenticated Download Date 5 18 15 1 09 PMA Paul et al Role of methionine in tyrosine based sorting signals 659 Lys1445 of the TGEV S protein was replaced by a methio nine behaved like the SG6 mutant i e it was transported to the cell surface In case of the SG6 and the S K M mutant protein we additionally generated a tyrosine to alanine mutation resulting in the mutants SG6 Y A and S YK AM These mutant proteins showed a bright expression at the cell surface when analyzed by immunofluorescence microscopy Figure 2A In this case antibody distribu tion and binding seems to be more efficient than in the other analyzed proteins The result of the immunofluo rescence assay was confirmed when the mutant proteins were surface biotinylated and subsequently analyzed by immunoprecipitation and Western blot Figure 2B upper panel All proteins containing the Lys1445Met mutation were detected on the cell surface The relative amount of surface expressed S proteins was determined using Image J Figure 2C The SG6 Y A mutant protein showed a five times higher surface expression than the SG6 protein with an intact tyrosine motif A Western blot analysis of the total amount of expressed proteins showed that all proteins were properly expressed Figure 2B lower panel The S proteins that were transported to the cell surface were expressed in two forms as indicated by an upper and a lower band black and white arrow With these proteins the upper band was most prominent whereas the lower band was predominant in intracellularly retained pro teins TGEV S SG VH LGK The upper band most likely represents proteins with complex N glycans whereas pro teins containing high mannose type oligosaccharides are expected in the lower band With those S protein mutants that are transported to the cell surface the mannose rich oligosaccharides that are added in the ER are converted into complex glycans during passage through the Golgi apparatus The latter type of glycosylation increases the molecular weight and results in a slower migration behav ior during SDS PAGE A similar transport behavior has been found with chimeric S proteins where the cytoplas mic tail of TGEV has been replaced by that of the F protein of Sendai virus compared to authentic TGEV S protein here the identification of the upper and lower bands as proteins with complex or high mannose glycans respec tively has been confirmed by analysis of their sensitivity to treatment with endoglycosidase H Schwegmann Wes sels et al 2004 Figure 2 Surface and total protein expression of parental TGEV S mutant S proteins and TGEV S VSV G chimeras BHK21 cells were transfected with the pCG1 expression plasmid containing the gene indicated or with no insert mock A Cells were analyzed for surface s and total t expression of the indicated proteins at 24 h post transfection by immunofluorescence microscopy B BHK21 cells were transfected with the pCG1 expression plasmid containing either no insert mock or one of the genes indicated At 24 h post transfection cells were prepared for surface biotinylation One part of the lysates was used for total protein expression analysis in a Western blot t The other part of the same samples was used for surface expression analysis s after immunoprecipitation of the proteins and detection of the biotin ligand with streptavidin peroxidase The black arrow indicates the complex glycosylated form whereas the white arrow indicates the mannose rich form of the S protein The black bar shows the position of the 250 kDa band of the prestained protein ladder C Relative amounts of surface expressed proteins shown in Figure 2B Brought to you by Yale University Authenticated Download Date 5 18 15 1 09 PM660 A Paul et al Role of methionine in tyrosine based sorting signals As the Lys1447Met exchange resulted in surface expression of this mutant protein we analyzed whether other amino acids at this position also affect the trans port of the S protein Changes of lysine to alanine hydro phobic arginine basic or glutamic acid acidic did not result in surface expression of the respective mutant pro teins They were all intracellularly retained as determined by immunofluorescence analysis as well as by surface biotinylation experiments data not shown We analyzed the intracellular localization of the S K M mutant in more detail For this purpose ER and ERGIC markers were co expressed together with the S protein Both the parental TGEV S protein as well as the K M mutant protein barely co localized with the ER marker but the parental viral protein co localized to a large extent with the ERGIC marker Figure 3A The mutant S K M showed some colocalization with the ERGIC as well but Figure 3 Colocalization analysis of TGEV S protein and S K M mutant protein with compartment markers The S protein panel A stained in red as well as the mutant protein S K M panel B were cotransfected with GFP tagged ER and GFP tagged ERGIC marker respectively Co localized proteins are shown in yellow merge the majority of the protein was distributed in a vesicle like pattern all over the cytoplasm Figure 3B This result sug gests that the tyrosine based signal in the cytoplasmic tail of TGEV S that retains the protein in the ERGIC compart ment can be suppressed by a methionine at position 1445 in such a way that the S protein is transported in the secre tory pathway beyond the ERGIC and Golgi compartments As the K M mutant was distributed in vesicular structures we investigated whether the tyrosine based signal in this mutant protein functions as an endocyto sis signal For this purpose we performed an antibody uptake assay S protein specific antibodies were bound to S K M expressing cells and incubated for 10 min at 37 C A substantial amount of the mutant protein was detected in endocytotic vesicles that could be stained after per meabilization of the cells Figure 4A second line green color in the merged image No endocytosed vesicular structures were observed when the cells were incubated at 4 C where endocytosis was prevented The punctu ated pattern on the cells at 4 C was detectable by stain ing the proteins on the cell surface and does therefore not represent endocytic vesicles In this study we included the mutant S YK AM which in addition to the K1445M mutation contained a Y1440A mutation which inacti vated the Y X X motif This mutant protein did not show any intracellular vesicles after incubation for 10 min at 37 C This result suggests that the tyrosine based reten tion signal functions as an endocytosis signal when its function as a retention signal is abolished by inserting a methionine at postion 1445 To confirm these results endocytosis was also analyzed by a biotin internalization assay The SG6 Y A mutant served as a negative control in this assay Figure 1 It behaves like the S YK AM mutant as far as the transport to the cell surface is concerned Figure 2A Figure 4B shows that all three mutants were expressed at the plasma membrane 4 C MESNA and that SG6 and S K M were efficiently endocytosed after incubation at 37 C for 10 min 37 C MESNA Endocy tosis of S K M was already observed at the earliest after 2 min at 37 C and increased over time data not shown In contrast the SG6 Y A mutant was not endocytosed as it lacks a tyrosine based internalization motif Figure 4B 37 C The endocytosis rate for SG6 and S K M was determined from three independent experiments The amount of endocytosed protein varied between the differ ent experiments with a peak at an endocytosis time of 10 min Both proteins showed an average endocytosis rate of about 7 min SG6 7 2 min S K M 7 6 min This indicated that other VSV G derived amino acids in the SG6 protein compared to the S K M did not have an enhanc ing effect on endocytosis Brought to you by Yale University Authenticated Download Date 5 18 15 1 09 PMA Paul et al Role of methionine in tyrosine based sorting signals 661 These results demonstrate that a single amino acid exchange outside the Y X X motif may result in a com pletely different transport behavior Whereas the authen tic TGEV S protein is retained in the ERGIC compartment the mutant protein S K M is transported to the cell surface and subsequently endocytosed Taken together our results indicate that the function of a tyrosine based sorting motif as a retention signal can be affected by a Figure 4 Internalization analysis of TGEV S proteins with mutations in or near the potential endocytosis signal A Surface and intracellular staining of S K M and S YK AM in an antibody uptake assay After surface binding of the anti S protein antibody cells were incubated at 4 C or 37 C respectively Surface protein antibody complexes as well as endocytosed protein antibody complexes could be stained by secondary antibodies conjugated with two different fluorescent dyes red surface green surface and endocytosed In the overlays merge endocytosed protein is shown in green whereas surface protein is visible in red or yellow B Biotin internalization assay of SG6 SG6 Y A and S K M BHK21 cells were transfected with the pCG1 expression plasmid containing the gene indicated Cells were analyzed at 24 h post transfection for endocytosis as described in the experimen tal procedures section In order to determine the total amount of surface biotinylated proteins the samples were compared with those that were neither warmed to 37 C nor treated with MESNA To analyze the cleavage efficiency of the biotin disulfide bond under reducing conditions one part of the cells was kept at 4 C to inhibit endocytosis and then treated with MESNA After incubation at 37 C the endocytosed protein is seen as a band at 220 kDa single amino acid downstream of the respective tetrapep tide motif As the initial point of our analysis was the comparison of the TGEV S tyrosine based sorting signal with the VSV G tyrosine based sorting signal we proceeded to introduce a lysine in the original VSV G protein at position 6 instead of the methionine Additionally we constructed a double mutant with a tyrosine to alanine mutation in addition to the methionine to lysine mutation In the Western blot analysis of lysates from transfected cells the parental G proteins as well as both mutant proteins were expressed in comparable amounts Figure 5A Both the G M K and the G YM AK proteins were expressed at the cell surface but in substantially lower amounts when compared to the parental G protein Figure 5A upper panel The surface biotinylated samples of the VSV G proteins were taken from the same well as the lysates i e these two results can be directly compared In an immunofluorescence assay the mutant proteins were expressed in a similar surface distributed pattern as the parental VSV G data Figure 5 Protein expression and internalization analysis of VSV G parental and mutant proteins A Surface and total protein expression of VSV G parental and mutant proteins VSV G protein G M K mutant protein and G YM AK mutant protein were expressed in BHK21 cells Cells were surface biotinylated at 24 h post transfection After lysis one part of the samples was used for total G protein analysis in a Western blot t The other part was immunoprecipitated with monoclonal anti VSV G antibody and surface expression of the proteins was analyzed after SDS PAGE and Western blot with streptavidin peroxidase B Intra cellular distribution of VSV G parental and mutant proteins Cells were analyzed for total expression of the indicated proteins at 24 h post transfection by immunofluorescence microscopy 1000 origi nal magnification C Internalization of VSV G proteins BHK21 cells were transfected with the pcDNA3 1 ex