Severe Acute Respiratory Syndrome Coronavirus Protein 6 Is Required for Optimal Replication

Severe acute respiratory syndrome coronavirus (SARS-CoV) encodes several accessory proteins of unknown function. One of these proteins, protein 6 (p6), which is encoded by ORF6, enhances virus replication when introduced into a heterologous murine coronavirus (mouse hepatitis virus [MHV]) but is not essential for optimal SARS-CoV replication after infection at a relatively high multiplicity of infection (MOI). Here, we reconcile these apparently conflicting results by showing that p6 enhances SARS-CoV replication to nearly the same extent as when expressed in the context of MHV if cells are infected at a low MOI and accelerates disease in mice transgenic for the human SARS-CoV receptor.The genome of severe acute respiratory syndrome coronavirus (SARS-CoV) encodes several structural proteins, including the spike, nucleocapsid, membrane, and envelope proteins (13). Integrated between and within these structural proteins are eight accessory proteins (6, 8, 10, 15, 16, 18, 21-27). Our laboratory showed previously that one of these SARS-CoV-specific accessory proteins, encoded by ORF6, showed a clearly recognizable phenotype when introduced into a heterologous attenuated murine coronavirus, mouse hepatitis virus (MHV) strain J2.2-V-1 (rJ2.2.6). rJ2.2.6 grew more rapidly and to higher titers in tissue culture cells and in the murine central nervous system than control viruses, and the presence of p6 increased mortality in mice from 10 to 20% to 80% (7, 19, 20). However, the absence of p6 did not diminish SARS-CoV growth in tissue culture cells when cells were infected with 1 PFU/cell (31). In addition to a role in enhancing virus replication, when expressed in the context of a SARS-CoV infection or by transfection, p6 blocked interferon (IFN)-induced STAT1 nuclear translocation by retention of the nuclear import adaptor molecule karyopherin alpha 2 in the cytoplasm, indicating a role in thwarting innate immune effectors (5, 11). In contrast, p6 did not significantly diminish IFN sensitivity when expressed in the context of rJ2.2 (20).The results described above were puzzling, because p6 seemed to be required for the optimal replication of a heterologous coronavirus but not for that of SARS-CoV. Thus, the objective of this study was to determine whether p6 could enhance SARS-CoV replication in tissue culture cells under any conditions. For this purpose, we examined its function by comparing the growth of a recombinant SARS-CoV (rSARS-CoV) in which p6 was deleted (rSARS-CoVΔ6) with that of wild-type rSARS-CoV at a range of multiplicities of infection (MOIs). Normal mice infected with SARS-CoV readily cleared the infection, making it difficult to detect a role for p6 in vivo. However, mice that are transgenic for expression of the human receptor angiotensin-converting enzyme 2 (hACE2) are exquisitely sensitive to infection with SARS-CoV and are useful for identifying an in vivo role for p6 (14).     

Monitoring of S Protein Maturation in the Endoplasmic Reticulum by Calnexin Is Important for the Infectivity of Severe Acute Respiratory Syndrome Coronavirus

Severe acute respiratory syndrome coronavirus (SARS-CoV) is the etiological agent of SARS, a fatal pulmonary disorder with no effective treatment. We found that SARS-CoV spike glycoprotein (S protein), a key molecule for viral entry, binds to calnexin, a molecular chaperone in the endoplasmic reticulum (ER), but not to calreticulin, a homolog of calnexin. Calnexin bound to most truncated mutants of S protein, and S protein bound to all mutants of calnexin. Pseudotyped virus carrying S protein (S-pseudovirus) produced by human cells that were treated with small interfering RNA (siRNA) for calnexin expression (calnexin siRNA-treated cells) showed significantly lower infectivity than S-pseudoviruses produced by untreated and control siRNA-treated cells. S-pseudovirus produced by calnexin siRNA-treated cells contained S protein modified with N-glycan side chains differently from other two S proteins and consisted of two kinds of viral particles: those of normal density with little S protein and those of high density with abundant S protein. Treatment with peptide-N-glycosidase F (PNGase F), which removes all types of N-glycan side chains from glycoproteins, eliminated the infectivity of S-pseudovirus. S-pseudovirus and SARS-CoV produced in the presence of α-glucosidase inhibitors, which disrupt the interaction between calnexin and its substrates, showed significantly lower infectivity than each virus produced in the absence of those compounds. In S-pseudovirus, the incorporation of S protein into viral particles was obviously inhibited. In SARS-CoV, viral production was obviously inhibited. These findings demonstrated that calnexin strictly monitors the maturation of S protein by its direct binding, resulting in conferring infectivity on SARS-CoV.     

Sec22 Regulates Endoplasmic Reticulum Morphology but Not Autophagy and Is Required for Eye Development in Drosophila

The endoplasmic reticulum (ER) is a highly dynamic organelle that plays a critical role in many cellular processes. Abnormal ER morphology is associated with some human diseases, although little is known regarding how ER morphology is regulated. Using a forward genetic screen to identify genes that regulated ER morphology in Drosophila, we identified a mutant of Sec22, the orthologs of which in yeast, plants, and humans are required for ER to Golgi trafficking. However, the physiological function of Sec22 has not been previously investigated in animal development. A loss of Sec22 resulted in ER proliferation and expansion, enlargement of late endosomes, and abnormal Golgi morphology in mutant larvae fat body cells. However, starvation-induced autophagy was not affected by a loss of Sec22. Mosaic analysis of the eye revealed that Sec22 was required for photoreceptor morphogenesis. In Sec22 mutant photoreceptor cells, the ER was highly expanded and gradually lost normal morphology with aging. The rhabdomeres in mutants were small and sometimes fused with each other. The morphology of Sec22 mutant eyes resembled the eye morphology of flies with overexpressed eyc (eyes closed). eyc encodes for a Drosophila p47 protein that is required for membrane fusion. A loss of Syntaxin5 (Syx5), encoding for a t-SNARE on Golgi, also phenocopied the Sec22 mutant. Sec22 formed complexes with Syx5 and Eyc. Thus, we propose that appropriate trafficking between the ER and Golgi is required for maintaining ER morphology and for Drosophila eye morphogenesis.     

冠状病毒是引起广州地区严重急性呼吸综合征(SARS)的主要病因

为查找引起广州地区流行的严重急性呼吸综合征(SARS)的病原体,采集患者漱口液及尸解标本,用组织培养法接种人胚肺细胞、MDCK细胞、Hep-2细胞和鸡胚分离病毒,用间接免疫荧光法检测患者恢复期血清lgG抗体,确定分离的病原是SARS的主要病因,再用套式RT—PCR、免疫电镜法鉴定病原。结果用人胚肺、Hep-2细胞在75份漱口液和3例尸解组织中分离出13株病原体,经套式RT—PCR扩增出110bp的特异产物,经测序证实为冠状病毒。制备冠状病毒的抗原,检测30份SARS病人恢复期血,其中26份血清lgG抗体阳性。同时检测30份普通发热病人血清作对照,IgG抗体全部阴性。由此证明,经组织培养分离到的病原体是引起SARS的致病因子,用分子生物学方法测序后证实为冠状病毒。     

The PDZ-Binding Motif of Severe Acute Respiratory Syndrome Coronavirus Envelope Protein Is a Determinant of Viral Pathogenesis

A recombinant severe acute respiratory syndrome coronavirus (SARS-CoV) lacking the envelope (E) protein is attenuated in vivo. Here we report that E protein PDZ-binding motif (PBM), a domain involved in protein-protein interactions, is a major determinant of virulence. Elimination of SARS-CoV E protein PBM by using reverse genetics caused a reduction in the deleterious exacerbation of the immune response triggered during infection with the parental virus and virus attenuation. Cellular protein syntenin was identified to bind the E protein PBM during SARS-CoV infection by using three complementary strategies, yeast two-hybrid, reciprocal coimmunoprecipitation and confocal microscopy assays. Syntenin redistributed from the nucleus to the cell cytoplasm during infection with viruses containing the E protein PBM, activating p38 MAPK and leading to the overexpression of inflammatory cytokines. Silencing of syntenin using siRNAs led to a decrease in p38 MAPK activation in SARS-CoV infected cells, further reinforcing their functional relationship. Active p38 MAPK was reduced in lungs of mice infected with SARS-CoVs lacking E protein PBM as compared with the parental virus, leading to a decreased expression of inflammatory cytokines and to virus attenuation. Interestingly, administration of a p38 MAPK inhibitor led to an increase in mice survival after infection with SARS-CoV, confirming the relevance of this pathway in SARS-CoV virulence. Therefore, the E protein PBM is a virulence domain that activates immunopathology most likely by using syntenin as a mediator of p38 MAPK induced inflammation.     

血中检测SARS冠状病毒N蛋白在SARS实验室早期诊断中的作用

为明确严重急性呼吸综合症(SARS)冠状病毒N蛋白在SARS实验室早期诊断中的作用,通过微量中和试验及酶联免疫方法、间接免疫荧光法检测疑似病人恢复期血清(大于28天)中SARS-IgG抗体,确诊SARS患者。同时收集发病不同时期SARS及普通发热病人血清,利用酶联免疫方法检测SARS-CoVN蛋白,并与荧光定量PCR早期诊断方法相比较。共检测：广州地区2003年12月～2004．年1月新发4例确诊SARS患者不同时期的血液和咽漱液标本;恢复期血清SARS-CoV中和抗体阳性病人不同时期的血清46份;广州地区2003年1月～4月临床确诊SARS患者159例的血清和56例疑似患者血清;非SARS普通发热病人血清97份;正常人体检血清100份。结果：4例新发SARS患者的不同时期标本中,3例患者急性期血均检出N蛋白,优于常用的荧光定量PCR检测方法。46份SARS-CoV中和抗体阳性的血清标本,N蛋白检出率为100％。159例临床确诊病例中,发病早期5天以内SARS-CoVN蛋白的检出率为92．3％,随后呈现逐步下降的趋势,在发病第18天仍可检出。56例临床疑似患者发病早期也有23．2％检出率。而97例普通发热病人及100份正常人血清中均未能检测出SARS-CoVN蛋白。表明在血清中检测SARS冠状病毒N蛋白的方法敏感性和特异性都好,对SARS实验室早期诊断具有重要作用。     

Severe Acute Respiratory Syndrome Coronavirus Replication Is Severely Impaired by MG132 due to Proteasome-Independent Inhibition of M-Calpain

The ubiquitin-proteasome system (UPS) is involved in the replication of a broad range of viruses. Since replication of the murine hepatitis virus (MHV) is impaired upon proteasomal inhibition, the relevance of the UPS for the replication of the severe acute respiratory syndrome coronavirus (SARS-CoV) was investigated in this study. We demonstrate that the proteasomal inhibitor MG132 strongly inhibits SARS-CoV replication by interfering with early steps of the viral life cycle. Surprisingly, other proteasomal inhibitors (e.g., lactacystin and bortezomib) only marginally affected viral replication, indicating that the effect of MG132 is independent of proteasomal impairment. Induction of autophagy by MG132 treatment was excluded from playing a role, and no changes in SARS-CoV titers were observed during infection of wild-type or autophagy-deficient ATG5(-/-) mouse embryonic fibroblasts overexpressing the human SARS-CoV receptor, angiotensin-converting enzyme 2 (ACE2). Since MG132 also inhibits the cysteine protease m-calpain, we addressed the role of calpains in the early SARS-CoV life cycle using calpain inhibitors III (MDL28170) and VI (SJA6017). In fact, m-calpain inhibition with MDL28170 resulted in an even more pronounced inhibition of SARS-CoV replication (>7 orders of magnitude) than did MG132. Additional m-calpain knockdown experiments confirmed the dependence of SARS-CoV replication on the activity of the cysteine protease m-calpain. Taken together, we provide strong experimental evidence that SARS-CoV has unique replication requirements which are independent of functional UPS or autophagy pathways compared to other coronaviruses. Additionally, this work highlights an important role for m-calpain during early steps of the SARS-CoV life cycle.     

p53/58 Binds COPI and Is Required for Selective Transport through the Early Secretory Pathway

p53/58 is a transmembrane protein that continuously recycles between the ER and pre-Golgi intermediates composed of vesicular-tubular clusters (VTCs) found in the cell periphery and at the cis face of the Golgi complex. We have generated an antibody that uniquely recognizes the p53/58 cytoplasmic tail. Here we present evidence that this antibody arrests the anterograde transport of vesicular stomatitis virus glycoprotein and leads to the accumulation of p58 in preGolgi intermediates. Consistent with a role for the KKXX retrieval motif found at the cytoplasmic carboxyl terminus of p53/58 in retrograde traffic, inhibition of transport through VTCs correlates with the ability of the antibody to block recruitment of COPI coats to the p53/58 cytoplasmic tail and to p53/58-containing membranes. We suggest that p53/58 function may be required for the coupled exchange of COPII for COPI coats during segregation of anterograde and retrograde transported proteins.     

Upregulation of the Chemokine (C-C Motif) Ligand 2 via a Severe Acute Respiratory Syndrome Coronavirus Spike-ACE2 Signaling Pathway

Severe acute respiratory syndrome coronavirus (SARS-CoV) was identified to be the causative agent of SARS with atypical pneumonia. Angiotensin-converting enzyme 2 (ACE2) is the major receptor for SARS-CoV. It is not clear whether ACE2 conveys signals from the cell surface to the nucleus and regulates expression of cellular genes upon SARS-CoV infection. To understand the pathogenesis of SARS-CoV, human type II pneumocyte (A549) cells were incubated with the viral spike protein or with SARS-CoV virus-like particles containing the viral spike protein to examine cytokine modulation in lung cells. Results from oligonucleotide-based microarray, real-time PCR, and enzyme-linked immunosorbent assays indicated an upregulation of the fibrosis-associated chemokine (C-C motif) ligand 2 (CCL2) by the viral spike protein and the virus-like particles. The upregulation of CCL2 by SARS-CoV spike protein was mainly mediated by extracellular signal-regulated kinase 1 and 2 (ERK1/2) and AP-1 but not the IκBα-NF-κB signaling pathway. In addition, Ras and Raf upstream of the ERK1/2 signaling pathway were involved in the upregulation of CCL2. Furthermore, ACE2 receptor was activated by casein kinase II-mediated phosphorylation in cells pretreated with the virus-like particles containing spike protein. These results indicate that SARS-CoV spike protein triggers ACE2 signaling and activates fibrosis-associated CCL2 expression through the Ras-ERK-AP-1 pathway.Severe acute respiratory syndrome (SARS) is an atypical pneumonia that occurred in several countries during late 2002 and the first half of 2003. A novel coronavirus, SARS-coronavirus (SARS-CoV), isolated from SARS patients was identified to be the causative agent of SARS. SARS-CoV infected more than 8,000 people, with a worldwide mortality rate of 9.6% (8, 20). The virus contains a positive-sense single-stranded RNA genome of approximately 30,000 nucleotides. Four major structural proteins including spike (S), membrane (M), envelope (E), and nucleocapsid (N) make up the SARS-CoV particles (31, 36). Angiotensin (Ang)-converting enzyme 2 (ACE2) and CD209L (L-SIGN) have been identified to be the receptors for SARS-CoV (15, 27). SARS-CoV spike protein induced ACE2-mediated interleukin-8 (IL-8) release from lung cells via activation of activation protein 1 (AP-1) (4). Nevertheless, involvement of ACE2 in virus pathogenesis is not fully understood.Dysregulation of inflammatory cytokines and adhesion molecules may be involved in lung injury that causes acute respiratory distress syndrome. High levels of proinflammatory cytokines such as interleukin-6, transforming growth factor β (TGF-β), and tumor necrosis factor alpha (TNF-α) were detected in the sera and ACE2⁺ cells of SARS patients (12, 45). Elevated levels of cytokines, including alpha interferon (IFN-α), IFN-β, IFN-γ, CCL3, CCL5, and CXCL10, were also detected in SARS-CoV-infected macrophages, dendritic cells, and a colon carcinoma cell line (1, 5, 25). It is possible that the high fatality rate of SARS results from a severe immune response caused by cytokines and chemokines.CCL2 [chemokine (C-C motif) ligand 2; monocyte chemoattractant protein-1, (MCP-1)] is a CC chemokine that attracts monocytes, memory T lymphocytes, and basophils. CCL2 and its receptor CCR2 are involved in inflammatory reactions, including monocyte/macrophage migration, Th2 cell polarization, and the production of TGF-β and procollagen in fibroblast cells (9, 10). CCL2 is thus associated with several lung inflammatory disorders including acute respiratory distress syndrome, asthma, and pulmonary fibrosis (35). These inflammatory disorders and pulmonary infiltration are known to account for the progressive respiratory failure and death of SARS patients. In addition, upregulation of CCL2 was detected in the sera of SARS patients and the supernatant of a SARS-CoV-infected culture system (5, 16). However, mechanisms by which SARS-CoV is involved in the upregulation of CCL2 are not known.In this study, we have taken a step forward in understanding the pathogenesis of SARS-CoV by examining SARS-CoV-mediated cytokine modulation in human type II pneumocyte (A549) cells and monkey kidney Vero E6 cells. Both pretreatment of A549 cells with SARS-CoV virus-like particles (VLPs) and preincubation of the cells with the viral spike protein upregulate the expression of fibrosis-associated CCL2. SARS-CoV may interact with ACE2 receptor and activate casein kinase II-mediated ACE2 phosphorylation, which is critical for SARS-CoV-induced CCL2 upregulation. In addition, Ras, Raf, MEK, extracellular signal-regulated kinase 1 and 2 (ERK1/2), and AP-1 are directly involved in SARS-CoV-induced CCL2 upregulation. These data suggest that the intracellular ACE2 signaling pathway in the pneumocytes of SARS-CoV-infected patients confers risks of lung fibrosis leading to respiratory failure.     

A Single Tyrosine in the Severe Acute Respiratory Syndrome Coronavirus Membrane Protein Cytoplasmic Tail Is Important for Efficient Interaction with Spike Protein

Severe acute respiratory syndrome coronavirus (SARS-CoV) encodes 3 major envelope proteins: spike (S), membrane (M), and envelope (E). Previous work identified a dibasic endoplasmic reticulum retrieval signal in the cytoplasmic tail of SARS-CoV S that promotes efficient interaction with SARS-CoV M. The dibasic signal was shown to be important for concentrating S near the virus assembly site rather than for direct interaction with M. Here, we investigated the sequence requirements of the SARS-CoV M protein that are necessary for interaction with SARS-CoV S. The SARS-CoV M tail was shown to be necessary for S localization in the Golgi region when the proteins were exogenously coexpressed in cells. This was specific, since SARS-CoV M did not retain an unrelated glycoprotein in the Golgi. Importantly, we found that an essential tyrosine residue in the SARS-CoV M cytoplasmic tail, Y₁₉₅, was important for S-M interaction. When Y₁₉₅ was mutated to alanine, M_Y195A no longer retained S intracellularly at the Golgi. Unlike wild-type M, M_Y195A did not reduce the amount of SARS-CoV S carbohydrate processing or surface levels when the two proteins were coexpressed. Mutating Y₁₉₅ also disrupted SARS-CoV S-M interaction in vitro. These results suggest that Y₁₉₅ is necessary for efficient SARS-CoV S-M interaction and, thus, has a significant involvement in assembly of infectious virus.Coronaviruses are enveloped positive-strand RNA viruses that infect a wide variety of mammalian and avian species. These viruses generally cause mild disease in humans and are one major cause of the common cold (34). However, severe acute respiratory syndrome coronavirus (SARS-CoV), a novel human coronavirus which emerged in the Guangdong province in China in 2002 (30, 48), caused a widespread pandemic. SARS-CoV caused severe disease with a mortality rate of approximately 10%, the highest for any human coronavirus to date (62). The phylogeny and group classification of SARS-CoV remain controversial (17), but it is widely accepted to be a distant member of group 2. While SARS-CoV is no longer a major health threat, understanding the basic biology of this human pathogen remains important.Coronaviruses encode three major envelope proteins in addition to various nonstructural and accessory proteins. The envelope protein (E) is the least abundant structural protein in the virion envelope, although it is expressed at robust levels during infection (21). E plays an essential role in assembly for some but not all coronaviruses (31-33, 45) and may also be a viroporin (reviewed in reference 21). The spike glycoprotein (S) is the second most abundant protein in the envelope. S determines host cell tropism, binds the host receptor, and is responsible for virus-cell, as well as cell-cell, fusion (15). The S protein is a type I membrane protein with a large, heavily glycosylated luminal domain and a short cytoplasmic tail that has been shown to be palmitoylated in some coronaviruses (47, 58). The membrane protein (M) is the most abundant protein in the virion envelope and acts as a scaffold for virus assembly. M has three transmembrane domains, a long cytoplasmic tail, and a short glycosylated luminal domain (reviewed in reference 21). Unlike many enveloped viruses, coronaviruses assemble at and bud into the lumen of the endoplasmic reticulum (ER)-Golgi intermediate compartment (ERGIC) and exit the infected cell by exocytosis (29). In order to accomplish this, the envelope proteins must be targeted to the budding compartment for assembly.For most coronaviruses, the E and M proteins localize in the Golgi region near the budding site independently of other viral structural proteins. We have previously shown for infectious bronchitis virus (IBV) E protein that the cytoplasmic tail contains Golgi targeting information (9). IBV M contains Golgi targeting information in its first transmembrane domain (57), while the transmembrane domains and cytoplasmic tail of mouse hepatitis virus (MHV) M appear to play a role in Golgi targeting (1, 36). Some coronavirus S proteins contain targeting information in their cytoplasmic tails; however, some do not (38, 39, 52, 63). Since S proteins can escape to the cell surface when highly expressed, S may rely on lateral interactions with other viral envelope proteins to localize to the budding site and be incorporated into newly assembling virions.In line with its role in virus assembly, M is necessary for virus-like particle (VLP) formation (3, 10, 26, 40, 55, 59). M has been shown to interact with itself to form homo-oligomers (12). In addition, M interacts with E, S, and the viral nucleocapsid and is essential for virion assembly (reviewed in reference 21). Lateral interactions between the coronavirus envelope proteins are critical for efficient virus assembly. The interaction of S and M has been studied for MHV, and the cytoplasmic tail of each protein is important for interaction (16, 44). Specifically, deletion of an amphipathic region in the MHV M cytoplasmic tail abrogates efficient interaction with MHV S (11). The S and M proteins of IBV, bovine coronavirus, feline infectious peritonitis virus, and SARS-CoV have been shown to interact; however, information about the specific regions that are important for interaction remains elusive (16, 22, 26, 42, 64). Due to the presence of several accessory proteins in the virion envelope (23-25, 28, 51, 53), it is possible that the requirements for SARS-CoV S and M interaction could be different from those of previously studied coronaviruses.In earlier work, we reported that SARS-CoV M retains SARS-CoV S intracellularly at the Golgi region when both proteins are expressed exogenously (39). We also demonstrated that the SARS-CoV S cytoplasmic tail interacts with in vitro-transcribed and -translated SARS-CoV M (39). Here, we show that the SARS-CoV M cytoplasmic tail is necessary for specific retention of SARS-CoV S at the Golgi region. We found a critical tyrosine residue at position 195 to be important for retaining SARS-CoV S Golgi membranes when coexpressed with M. When Y₁₉₅ was mutated to alanine, the mutant protein, M_Y195A, did not reduce the amount of SARS-CoV S at the plasma membrane or reduce the extent of S carbohydrate processing as well as wild-type SARS-CoV M does. Additionally, mutation of Y₁₉₅ in SARS-CoV M disrupted the S-M interaction in vitro. Thus, Y₁₉₅ is likely to play a critical role in the assembly of infectious SARS-CoV.     

Isolation and Characterization of a Novel Bat Coronavirus Closely Related to the Direct Progenitor of Severe Acute Respiratory Syndrome Coronavirus

We report the isolation and characterization of a novel bat coronavirus which is much closer to the severe acute respiratory syndrome coronavirus (SARS-CoV) in genomic sequence than others previously reported, particularly in its S gene. Cell entry and susceptibility studies indicated that this virus can use ACE2 as a receptor and infect animal and human cell lines. Our results provide further evidence of the bat origin of the SARS-CoV and highlight the likelihood of future bat coronavirus emergence in humans.