Conformational states of the severe acute respiratory syndrome coronavirus spike protein ectodomain

The severe acute respiratory syndrome coronavirus enters cells through the activities of a spike protein (S) which has receptor-binding (S1) and membrane fusion (S2) regions. We have characterized four sequential states of a purified recombinant S ectodomain (S-e) comprising S1 and the ectodomain of S2. They are S-e monomers, uncleaved S-e trimers, cleaved S-e trimers, and dissociated S1 monomers and S2 trimer rosettes. Lowered pH induces an irreversible transition from flexible, L-shaped S-e monomers to clove-shaped trimers. Protease cleavage of the trimer occurs at the S1-S2 boundary; an ensuing S1 dissociation leads to a major rearrangement of the trimeric S2 and to formation of rosettes likely to represent clusters of elongated, postfusion trimers of S2 associated through their fusion peptides. The states and transitions of S suggest conformational changes that mediate viral entry into cells.     

Crystal structure of severe acute respiratory syndrome coronavirus spike protein fusion core

Severe acute respiratory syndrome coronavirus is a newly emergent virus responsible for a recent outbreak of an atypical pneumonia. The coronavirus spike protein, an enveloped glycoprotein essential for viral entry, belongs to the class I fusion proteins and is characterized by the presence of two heptad repeat (HR) regions, HR1 and HR2. These two regions are understood to form a fusion-active conformation similar to those of other typical viral fusion proteins. This hairpin structure likely juxtaposes the viral and cellular membranes, thus facilitating membrane fusion and subsequent viral entry. The fusion core protein of severe acute respiratory syndrome coronavirus spike protein was crystallized, and the structure was determined at 2.8 A of resolution. The fusion core is a six-helix bundle with three HR2 helices packed against the hydrophobic grooves on the surface of central coiled coil formed by three parallel HR1 helices in an oblique antiparallel manner. This structure shares significant similarity with the fusion core structure of mouse hepatitis virus spike protein and other viral fusion proteins, suggesting a conserved mechanism of membrane fusion. Drug discovery strategies aimed at inhibiting viral entry by blocking hairpin formation, which have been successfully used in human immunodeficiency virus 1 inhibitor development, may be applicable to the inhibition of severe acute respiratory syndrome coronavirus on the basis of structural information provided here. The relatively deep grooves on the surface of the central coiled coil will be a good target site for the design of viral fusion inhibitors.     

Identification and antigenic epitope mapping of immunodominant region amino residues 510 to 672 on the spike protein of the severe acute respiratory syndrome coronavirus

The severe acute respiratory syndrome (SARS) is a newly emerging human infectious disease caused by the severe acute respiratory syndrome coronavirus (SARS-CoV). The spike (S) protein of SARS-CoV is a major virion structural protein. It plays an important role in the interaction with receptors and neutralizing antibodies. In this study, the S1 domain of the spike protein and three truncated fragments were expressed by fusion with GST in a pGEX-6p-1 vector. Western blot results demonstrated that the 510-672 fragment of the S1 domain is a linear epitope dominant region. To map the antigenic epitope of this linear epitope dominant region, a set of 16 partially overlapping fragments spanning the fragment were fused with GST and expressed. Four antigenic epitopes S1C3 (539-559), S1C4 (548-567), S1C7/8 (583-606), and S1C10/11 (607-630) were identified. Immunization of mice with each of the four antigenic epitope-fused proteins revealed that all four proteins could elicit spike protein specific antisera. All of them were able to bind to the surface domain of the whole spike protein expressed by recombinant baculovirus in insect cells. Identification of antigenic epitopes of the spike protein of SARS-CoV may provide the basis for the development of immunity-based prophylactic, therapeutic, and diagnostic clinical techniques for the severe acute respiratory syndrome.     

Important role for the transmembrane domain of severe acute respiratory syndrome coronavirus spike protein during entry

The spike protein (S) of severe acute respiratory syndrome coronavirus (SARS-CoV) is responsible for receptor binding and membrane fusion. It contains a highly conserved transmembrane domain that consists of three parts: an N-terminal tryptophan-rich domain, a central domain, and a cysteine-rich C-terminal domain. The cytoplasmic tail of S has previously been shown to be required for assembly. Here, the roles of the transmembrane and cytoplasmic domains of S in the infectivity and membrane fusion activity of SARS-CoV have been studied. SARS-CoV S-pseudotyped retrovirus (SARSpp) was used to measure S-mediated infectivity. In addition, the cell-cell fusion activity of S was monitored by a Renilla luciferase-based cell-cell fusion assay. S(VSV-Cyt), an S chimera with a cytoplasmic tail derived from vesicular stomatitis virus G protein (VSV-G), and S(MHV-TMDCyt), an S chimera with the cytoplasmic and transmembrane domains of mouse hepatitis virus, displayed wild-type-like activity in both assays. S(VSV-TMDCyt), a chimera with the cytoplasmic and transmembrane domains of VSV-G, was impaired in the SARSpp and cell-cell fusion assays, showing 3 to 25% activity compared to the wild type, depending on the assay and the cells used. Examination of the oligomeric state of the chimeric S proteins in SARSpp revealed that S(VSV-TMDCyt) trimers were less stable than wild-type S trimers, possibly explaining the lowered fusogenicity and infectivity.     

An exposed domain in the severe acute respiratory syndrome coronavirus spike protein induces neutralizing antibodies

Exposed epitopes of the spike protein may be recognized by neutralizing antibodies against severe acute respiratory syndrome (SARS) coronavirus (CoV). A protein fragment (S-II) containing predicted epitopes of the spike protein was expressed in Escherichia coli. The properly refolded protein fragment specifically bound to the surface of Vero cells. Monoclonal antibodies raised against this fragment recognized the native spike protein of SARS CoV in both monomeric and trimeric forms. These monoclonal antibodies were capable of blocking S-II attachment to Vero cells and exhibited in vitro antiviral activity. These neutralizing antibodies mapped to epitopes in two peptides, each comprising 20 amino acids. Thus, this region of the spike protein might be a target for generation of therapeutic neutralizing antibodies against SARS CoV and for vaccine development to elicit protective humoral immunity.     

Efficient activation of the severe acute respiratory syndrome coronavirus spike protein by the transmembrane protease TMPRSS2

The distribution of the severe acute respiratory syndrome coronavirus (SARS-CoV) receptor, an angiotensin-converting enzyme 2 (ACE2), does not strictly correlate with SARS-CoV cell tropism in lungs; therefore, other cellular factors have been predicted to be required for activation of virus infection. In the present study, we identified transmembrane protease serine 2 (TMPRSS2), whose expression does correlate with SARS-CoV infection in the upper lobe of the lung. In Vero cells expressing TMPRSS2, large syncytia were induced by SARS-CoV infection. Further, the lysosome-tropic reagents failed to inhibit, whereas the heptad repeat peptide efficiently inhibited viral entry into cells, suggesting that TMPRSS2 affects the S protein at the cell surface and induces virus-plasma membrane fusion. On the other hand, production of virus in TMPRSS2-expressing cells did not result in S-protein cleavage or increased infectivity of the resulting virus. Thus, TMPRSS2 affects the entry of virus but not other phases of virus replication. We hypothesized that the spatial orientation of TMPRSS2 vis-a-vis S protein is a key mechanism underling this phenomenon. To test this, the TMPRSS2 and S proteins were expressed in cells labeled with fluorescent probes of different colors, and the cell-cell fusion between these cells was tested. Results indicate that TMPRSS2 needs to be expressed in the opposing (target) cell membrane to activate S protein rather than in the producer cell, as found for influenza A virus and metapneumoviruses. This is the first report of TMPRSS2 being required in the target cell for activation of a viral fusion protein but not for the S protein synthesized in and transported to the surface of cells. Our findings suggest that the TMPRSS2 expressed in lung tissues may be a determinant of viral tropism and pathogenicity at the initial site of SARS-CoV infection.     

Retroviruses pseudotyped with the severe acute respiratory syndrome coronavirus spike protein efficiently infect cells expressing angiotensin-converting enzyme 2

Infection of receptor-bearing cells by coronaviruses is mediated by their spike (S) proteins. The coronavirus (SARS-CoV) that causes severe acute respiratory syndrome (SARS) infects cells expressing the receptor angiotensin-converting enzyme 2 (ACE2). Here we show that codon optimization of the SARS-CoV S-protein gene substantially enhanced S-protein expression. We also found that two retroviruses, simian immunodeficiency virus (SIV) and murine leukemia virus, both expressing green fluorescent protein and pseudotyped with SARS-CoV S protein or S-protein variants, efficiently infected HEK293T cells stably expressing ACE2. Infection mediated by an S-protein variant whose cytoplasmic domain had been truncated and altered to include a fragment of the cytoplasmic tail of the human immunodeficiency virus type 1 envelope glycoprotein was, in both cases, substantially more efficient than that mediated by wild-type S protein. Using S-protein-pseudotyped SIV, we found that the enzymatic activity of ACE2 made no contribution to S-protein-mediated infection. Finally, we show that a soluble and catalytically inactive form of ACE2 potently blocked infection by S-protein-pseudotyped retrovirus and by SARS-CoV. These results permit studies of SARS-CoV entry inhibitors without the use of live virus and suggest a candidate therapy for SARS.     

Characterization of severe acute respiratory syndrome coronavirus membrane protein

The coronavirus membrane protein (M) is the key player in the assembly of virions at intracellular membranes between endoplasmic-reticulum and Golgi-complex. Using a newly established human monoclonal anti-M antibody we detected glycosylated and nonglycosylated membrane-associated M in severe acute respiratory syndrome-associated coronavirus (SARS-CoV) infected cells and in purified virions. Further analyses revealed that M contained a single N-glycosylation site at asparagine 4. Recombinant M was transported to the plasma membrane and gained complex-type N-glycosylation. In SARS-CoV infected cells and in purified virions, however, N-glycosylation of M remained endoglycosidase H-sensitive suggesting that trimming of the N-linked sugar side chain is inhibited.     

Aromatic amino acids in the juxtamembrane domain of severe acute respiratory syndrome coronavirus spike glycoprotein are important for receptor-dependent virus entry and cell-cell fusion

The severe acute respiratory syndrome coronavirus (SARS-CoV) spike glycoprotein (S) is a class I viral fusion protein that binds to its receptor glycoprotein, human angiotensin converting enzyme 2 (hACE2), and mediates virus entry and cell-cell fusion. The juxtamembrane domain (JMD) of S is an aromatic amino acid-rich region proximal to the transmembrane domain that is highly conserved in all coronaviruses. Alanine substitutions for one or two of the six aromatic residues in the JMD did not alter the surface expression of the SARS-CoV S proteins with a deletion of the C-terminal 19 amino acids (S Delta19) or reduce binding to soluble human ACE2 (hACE2). However, hACE2-dependent entry of trypsin-treated retrovirus pseudotyped viruses expressing JMD mutant S Delta19 proteins was greatly reduced. Single alanine substitutions for aromatic residues reduced entry to 10 to 60% of the wild-type level. The greatest reduction was caused by residues nearest the transmembrane domain. Four double alanine substitutions reduced entry to 5 to 10% of the wild-type level. Rapid hACE2-dependent S-mediated cell-cell fusion was reduced to 60 to 70% of the wild-type level for all single alanine substitutions and the Y1188A/Y1191A protein. S Delta19 proteins with other double alanine substitutions reduced cell-cell fusion further, from 40% to less than 20% of wild-type levels. The aromatic amino acids in the JMD of the SARS-CoV S glycoprotein play critical roles in receptor-dependent virus-cell and cell-cell fusion. Because the JMD is so highly conserved in all coronavirus S proteins, it is a potential target for development of drugs that may inhibit virus entry and/or cell-cell fusion mediated by S proteins of all coronaviruses.     

Cleavage and activation of the severe acute respiratory syndrome coronavirus spike protein by human airway trypsin-like protease

The highly pathogenic severe acute respiratory syndrome coronavirus (SARS-CoV) poses a constant threat to human health. The viral spike protein (SARS-S) mediates host cell entry and is a potential target for antiviral intervention. Activation of SARS-S by host cell proteases is essential for SARS-CoV infectivity but remains incompletely understood. Here, we analyzed the role of the type II transmembrane serine proteases (TTSPs) human airway trypsin-like protease (HAT) and transmembrane protease, serine 2 (TMPRSS2), in SARS-S activation. We found that HAT activates SARS-S in the context of surrogate systems and authentic SARS-CoV infection and is coexpressed with the viral receptor angiotensin-converting enzyme 2 (ACE2) in bronchial epithelial cells and pneumocytes. HAT cleaved SARS-S at R667, as determined by mutagenesis and mass spectrometry, and activated SARS-S for cell-cell fusion in cis and trans, while the related pulmonary protease TMPRSS2 cleaved SARS-S at multiple sites and activated SARS-S only in trans. However, TMPRSS2 but not HAT expression rendered SARS-S-driven virus-cell fusion independent of cathepsin activity, indicating that HAT and TMPRSS2 activate SARS-S differentially. Collectively, our results show that HAT cleaves and activates SARS-S and might support viral spread in patients.     

Retroviral vectors pseudotyped with severe acute respiratory syndrome coronavirus S protein

The worldwide outbreak of severe acute respiratory syndrome (SARS) was shown to be associated with a novel coronavirus (CoV) now called SARS CoV. We report here the generation of SARS CoV S protein-pseudotyped murine leukemia virus (MLV) vector particles. The wild-type S protein pseudotyped MLV vectors, although at a low efficiency. Partial deletion of the cytoplasmic tail of S dramatically increased infectivity of pseudotypes, with titers only two- to threefold lower than those of pseudotypes generated in parallel with the vesicular stomatitis virus G protein. S-pseudotyped MLV particles were used to analyze viral tropism. MLV(SARS) pseudotypes and wild-type SARS CoV displayed similar cell types and tissue and host restrictions, indicating that the expression of a functional receptor is the major restraint in permissiveness to SARS CoV infection. Efficient gene transfer could be detected in Vero and CaCo2 cells, whereas the level of gene marking of 293T, HeLa, and HepG2 cells was only slightly above background levels. A cat cell line and a dog cell line were not susceptible. Interestingly, PK-15, a porcine kidney cell line, and primary porcine kidney cells were also highly permissive for SARS S pseudotypes and wild-type SARS CoV. This finding suggests that swine may be susceptible to SARS infection and may be a source for infection of humans. Taken together, these results indicate that MLV(SARS) pseudotypes are highly valuable for functional studies of viral tropism and entry and, in addition, can be a powerful tool for the development of therapeutic entry inhibitors without posing a biohazard to human beings.     

Sumoylation of the nucleocapsid protein of severe acute respiratory syndrome coronavirus

Severe acute respiratory syndrome coronavirus (SARS-CoV) encodes a highly basic nucleocapsid (N) protein of 422 amino acids. Similar to other coronavirus N proteins, SARS-CoV N protein is predicted to be phosphorylated and may contain nuclear localization signals, serine/arginine-rich motif, RNA binding domain and regions responsible for self-association and homo-oligomerization. In this study, we demonstrate that the protein is posttranslationally modified by covalent attachment to the small ubiquitin-like modifier. The major sumoylation site was mapped to the (62)lysine residue of the N protein. Further expression and characterization of wild type N protein and K62A mutant reveal that sumoylation of the N protein drastically promotes its homo-oligomerization, and plays certain roles in the N protein-mediated interference of host cell division. This is the first report showing that a coronavirus N protein undergoes posttranslational modification by sumoylation, and the functional implication of this modification in the formation of coronavirus ribouncleoprotein complex, virion assembly and virus-host interactions.     

Hosting the severe acute respiratory syndrome coronavirus: specific cell factors required for infection

As with all viruses, the severe acute respiratory syndrome coronavirus (SARS-CoV) utilizes specific host cell factors during its infection cycle. Some of these factors have been identified and are now increasingly scrutinized as targets to intervene with infection. In this brief review, we describe the current understanding of how the SARS-CoV is able to use the cellular machinery for its replication.     

Calreticulin as a hydrophilic chimeric molecular adjuvant enhances IgG responses to the spike protein of severe acute respiratory syndrome coronavirus

Fragment 450-650 of the spike (S) protein (S450-650) of severe acute respiratory syndrome-associated coronavirus (SARS-CoV) contains epitopes capable of being recognized by convalescent sera of SARS patients. Vaccination of mice with recombinant S450-650 (rS450-650) can induce Abs against SARS-CoV, although the titer is relatively low. In the present study, a fusion protein linking a fragment (residues 39-272) of murine calreticulin (CRT) to S450-650 in a prokaryotic expression system was created. Compared with target antigen alone, the recombinant fusion product (rS450-650-CRT) has much improved hydrophilicity and immunogenicity. The S450-650-specific IgG Abs of BALB/c mice subcutaneously immunized with rS450-650-CRT were in substantially higher titer (approximately fivefold more). Furthermore, the fusion protein, but not rS450-650 alone, was able to elicit S450-650-specific IgG responses in T cell deficient nude mice. Given that rCRT/39-272 can drive the maturation of bone-marrow-derived dendritic cells, directly activate macrophages and B cells, and also elicit helper T cell responses in vivo, we propose that fragment 39-272 of CRT is an effective molecular adjuvant capable of enhancing target Ag-specific humoral responses in both a T cell-dependent and independent manner. Fusion protein rS450-650-CRT is a potential candidate vaccine against SARS-CoV infection.     

Mucosal immunization with surface-displayed severe acute respiratory syndrome coronavirus spike protein on Lactobacillus casei induces neutralizing antibodies in mice

Induction of mucosal immunity may be important for preventing SARS-CoV infections. For safe and effective delivery of viral antigens to the mucosal immune system, we have developed a novel surface antigen display system for lactic acid bacteria using the poly-gamma-glutamic acid synthetase A protein (PgsA) of Bacillus subtilis as an anchoring matrix. Recombinant fusion proteins comprised of PgsA and the Spike (S) protein segments SA (residues 2 to 114) and SB (residues 264 to 596) were stably expressed in Lactobacillus casei. Surface localization of the fusion protein was verified by cellular fractionation analyses, immunofluorescence microscopy, and flow cytometry. Oral and nasal inoculations of recombinant L. casei into mice resulted in high levels of serum immunoglobulin G (IgG) and mucosal IgA, as demonstrated by enzyme-linked immunosorbent assays using S protein peptides. More importantly, these antibodies exhibited potent neutralizing activities against severe acute respiratory syndrome (SARS) pseudoviruses. Orally immunized mice mounted a greater neutralizing-antibody response than those immunized intranasally. Three new neutralizing epitopes were identified on the S protein using a peptide neutralization interference assay (residues 291 to 308, 520 to 529, and 564 to 581). These results indicate that mucosal immunization with recombinant L. casei expressing SARS-associated coronavirus S protein on its surface provides an effective means for eliciting protective immune response against the virus.     

Antigenic and immunogenic characterization of recombinant baculovirus-expressed severe acute respiratory syndrome coronavirus spike protein: implication for vaccine design

The spike (S) glycoprotein of severe acute respiratory syndrome coronavirus (SARS-CoV) mediates the receptor interaction and immune recognition and is considered a major target for vaccine design. However, its antigenic and immunogenic properties remain to be elucidated. In this study, we immunized mice with full-length S protein (FL-S) or its extracellular domain (EC-S) expressed by recombinant baculoviruses in insect cells. We found that the immunized mice developed high titers of anti-S antibodies with potent neutralizing activities against SARS pseudoviruses constructed with the S proteins of Tor2, GD03T13, and SZ3, the representative strains of 2002 to 2003 and 2003 to 2004 human SARS-CoV and palm civet SARS-CoV, respectively. These data suggest that the recombinant baculovirus-expressed S protein vaccines possess excellent immunogenicity, thereby inducing highly potent neutralizing responses against human and animal SARS-CoV variants. The antigenic structure of the S protein was characterized by a panel of 38 monoclonal antibodies (MAbs) isolated from the immunized mice. The epitopes of most anti-S MAbs (32 of 38) were localized within the S1 domain, and those of the remaining 6 MAbs were mapped to the S2 domain. Among the anti-S1 MAbs, 17 MAbs targeted the N-terminal region (amino acids [aa] 12 to 327), 9 MAbs recognized the receptor-binding domain (RBD; aa 318 to 510), and 6 MAbs reacted with the C-terminal region of S1 domain that contains the major immunodominant site (aa 528 to 635). Strikingly, all of the RBD-specific MAbs had potent neutralizing activity, 6 of which efficiently blocked the receptor binding, confirming that the RBD contains the main neutralizing epitopes and that blockage of the receptor association is the major mechanism of SARS-CoV neutralization. Five MAbs specific for the S1 N-terminal region exhibited moderate neutralizing activity, but none of the MAbs reacting with the S2 domain and the major immunodominant site in S1 showed neutralizing activity. All of the neutralizing MAbs recognize conformational epitopes. These data provide important information for understanding the antigenicity and immunogenicity of S protein and for designing SARS vaccines. This panel of anti-S MAbs can be used as tools for studying the structure and function of the SARS-CoV S protein.     

Mechanisms of host receptor adaptation by severe acute respiratory syndrome coronavirus

The severe acute respiratory syndrome coronavirus (SARS-CoV) from palm civets has twice evolved the capacity to infect humans by gaining binding affinity for human receptor angiotensin-converting enzyme 2 (ACE2). Numerous mutations have been identified in the receptor-binding domain (RBD) of different SARS-CoV strains isolated from humans or civets. Why these mutations were naturally selected or how SARS-CoV evolved to adapt to different host receptors has been poorly understood, presenting evolutionary and epidemic conundrums. In this study, we investigated the impact of these mutations on receptor recognition, an important determinant of SARS-CoV infection and pathogenesis. Using a combination of biochemical, functional, and crystallographic approaches, we elucidated the molecular and structural mechanisms of each of these naturally selected RBD mutations. These mutations either strengthen favorable interactions or reduce unfavorable interactions with two virus-binding hot spots on ACE2, and by doing so, they enhance viral interactions with either human (hACE2) or civet (cACE2) ACE2. Therefore, these mutations were viral adaptations to either hACE2 or cACE2. To corroborate the above analysis, we designed and characterized two optimized RBDs. The human-optimized RBD contains all of the hACE2-adapted residues (Phe-442, Phe-472, Asn-479, Asp-480, and Thr-487) and possesses exceptionally high affinity for hACE2 but relative low affinity for cACE2. The civet-optimized RBD contains all of the cACE2-adapted residues (Tyr-442, Pro-472, Arg-479, Gly-480, and Thr-487) and possesses exceptionally high affinity for cACE2 and also substantial affinity for hACE2. These results not only illustrate the detailed mechanisms of host receptor adaptation by SARS-CoV but also provide a molecular and structural basis for tracking future SARS-CoV evolution in animals.     

Functional characterization of heptad repeat 1 and 2 mutants of the spike protein of severe acute respiratory syndrome coronavirus

To understand the roles of heptad repeat 1(HR1) and HR2 of the spike (S) protein of the severe acute respiratory syndrome coronavirus (SARS-CoV) in virus-cell interactions, the conserved Leu or Ile residues located at positions 913, 927, 941, and 955 in HR1 and 1151, 1165, and 1179 in HR2 were individually replaced with an alpha-helix-breaker Pro residue. The 913P mutant was expressed mainly as a faster-migrating, lower-molecular-weight S(L) form, while the wild type and all other mutants produced similar levels of both the S(L) form and the slower-migrating, higher-molecular-weight S(H) form. The wild-type S(L) form was processed to the S(H) form, whereas the S(L) form of the 913P mutant was inefficiently converted to the S(H) form after biosynthesis. None of these mutations affected cell surface expression or binding to its cognate ACE2 receptor. In a human immunodeficiency virus type 1/SARS S coexpression study, all mutants except the 913P mutant incorporated the S(H) form into the virions as effectively as did the wild-type S(H) form. The mutation at Ile-1151 did not affect membrane fusion or viral entry. The impaired viral entry of the 927P, 941P, 955P, and 1165P mutants was due to their inability to mediate membrane fusion, whereas the defect in viral entry of the 1179P mutant occurred not at the stage of membrane fusion but rather at a postfusion stage. Our study demonstrates the functional importance of HR1 and HR2 of the SARS-CoV spike protein in membrane fusion and viral entry.     

The cytoplasmic tail of the severe acute respiratory syndrome coronavirus spike protein contains a novel endoplasmic reticulum retrieval signal that binds COPI and promotes interaction with membrane protein

Like other coronaviruses, severe acute respiratory syndrome coronavirus (SARS CoV) assembles at and buds into the lumen of the endoplasmic reticulum (ER)-Golgi intermediate compartment (ERGIC). Accumulation of the viral envelope proteins at this compartment is a prerequisite for virus assembly. Previously, we reported the identification of a dibasic motif (KxHxx) in the cytoplasmic tail of the SARS CoV spike (S) protein that was similar to a canonical dilysine ER retrieval signal. Here we demonstrate that this motif is a novel and functional ER retrieval signal which reduced the rate of traffic of the full-length S protein through the Golgi complex. The KxHxx motif also partially retained two different reporter proteins in the ERGIC region and reduced their rates of trafficking, although the motif was less potent than the canonical dilysine signal. The dibasic motif bound the coatomer complex I (COPI) in an in vitro binding assay, suggesting that ER retrieval may contribute to the accumulation of SARS CoV S protein near the virus assembly site for interaction with other viral structural proteins. In support of this, we found that the dibasic motif on the SARS S protein was required for its localization to the ERGIC/Golgi region when coexpressed with SARS membrane (M) protein. Thus, the cycling of SARS S through the ER-Golgi system may be required for its incorporation into assembling virions in the ERGIC.