Inhibitor design for SARS coronavirus main protease based on "distorted key theory"

In order to find effective peptide inhibitors against SARS CoV M(pro), an analysis was performed for 11 oligo-peptides that can be cleaved by the SARS coronavirus main protease (CoV M(pro), or 3CL(pro)). Flexible molecular alignments of the 11 cleavable peptides have provided useful insights into the chemical properties of their amino acid residues close to the cleavage site. Moreover, it was found through the ligand-receptor docking studies that of the 11 cleavable peptides, NH2-ATLQ / AIAS-COOH and NH2-ATLQ / AENV-COOH had the highest affinity with SARS CoV M(pro). The two octapeptides were selected as initial templates for further chemical modification to make them become effective inhibitors against the SARS enzyme according to the "distorted key" theory [K. C. Chou, Analytical Biochemistry 233 (1996) 1-14]. The possible chemical modification methods are proposed and examined. The approach developed in this study and the findings thus obtained might stimulate new strategies and provide useful information for drug design against SARS.     

Molecular modeling studies of peptide drug candidates against SARS

Flexible alignment and docking studies were conducted for the three octapeptides, ATLQANEV, AVLQSGFR, and ATLQAIAS, that were cleavable by SARS-CoV Mpro. It has been observed that all pharmacophores of the three peptides overlap very well, and that ATLQANEV binds best with the receptor, followed by AVLQSGFR, and then ATLQAIAS. During the process of docking the octapeptides to the SARS enzyme, the residues of the catalytic dyad, i.e., His-41 and Cys-145 are actively involved in forming the hydrogen bonds, so is the center residue (Gln) of all the three octapeptides. The findings are fully consistent with experimental observations. The present studies suggest that the octapeptides ATLQANEV and ATLQAIAS, like AVLQSGFR, might also be the good starting points for designing potential drugs against SARS.     

Selection of SARS-coronavirus-specific B cell epitopes by phage peptide library screening and evaluation of the immunological effect of epitope-based peptides on mice

Antibodies to SARS-Coronavirus (SARS-CoV)-specific B cell epitopes might recognize the pathogen and interrupt its adherence to and penetration of host cells. Hence, these epitopes could be useful for diagnosis and as vaccine constituents. Using the phage-displayed peptide library screening method and purified Fab fragments of immunoglobulin G (IgG Fab) from normal human sera and convalescent sera from SARS-CoV-infected patients as targets, 11 B cell epitopes of SARS-CoV spike glycoprotein (S protein) and membrane protein (M protein) were screened. After a bioinformatics tool was used to analyze these epitopes, four epitope-based S protein dodecapeptides corresponding to the predominant epitopes were chosen for synthesis. Their antigenic specificities and immunogenicities were studied in vitro and in vivo. Flow cytometry and ELISPOT analysis of lymphocytes as well as a serologic analysis of antibody showed that these peptides could trigger a rapid, highly effective, and relatively safe immune response in BALB/c mice. These findings might aid development of SARS diagnostics and vaccines. Moreover, the role of S and M proteins as important surface antigens is confirmed.     

Tracking the evolution of the SARS coronavirus using high-throughput, high-density resequencing arrays

Mutations in the SARS-Coronavirus (SARS-CoV) can alter its clinical presentation, and the study of its mutation patterns in human populations can facilitate contact tracing. Here, we describe the development and validation of an oligonucleotide resequencing array for interrogating the entire 30-kb SARS-CoV genome in a rapid, cost-effective fashion. Using this platform, we sequenced SARS-CoV genomes from Vero cell culture isolates of 12 patients and directly from four patient tissues. The sequence obtained from the array is highly reproducible, accurate (>99.99% accuracy) and capable of identifying known and novel variants of SARS-CoV. Notably, we applied this technology to a field specimen of probable SARS and rapidly deduced its infectious source. We demonstrate that array-based resequencing-by-hybridization is a fast, reliable, and economical alternative to capillary sequencing for obtaining SARS-CoV genomic sequence on a population scale, making this an ideal platform for the global monitoring of SARS-CoV and other small-genome pathogens.     

A universal microarray for detection of SARS coronavirus

Severe acute respiratory syndrome (SARS) is caused by the SARS coronavirus (SARS-CoV). There are many point mutations among SARS-CoV genome sequences. Previous studies suggested that the mutations are correlated closely with the SARS epidemic. It was found that the bases of six nucleotide positions (nt9404, nt9479, nt19838, nt21721, nt22222 and nt27827) with high-mutation rate have an important relationship with the SARS epidemic. For viral detection as well as genotyping, a universal microarray system was developed that combines RT-PCR and ligase detection reaction (LDR). The Zip Codes attached covalently to a slide remain constant and their complementary Zip Codes (cZip Codes) can be used for tagging target sequence, making the microarrays universal. The discriminating oligonucleotides contain on the 5' end "cZip Codes" that are used to direct LDR product to specific Zip Codes attached covalently to a slide. Since Zip Codes have no homology to either the target sequence or to other sequences in the genomes of both human host and SARS-CoV, there was no false signal due to mismatch hybridizations. 20 samples assayed with the universal microarray were confirmed by DNA sequencing, demonstrating that this microarray system is a promising diagnostic tool for detection and genotyping of the SARS-CoV.     

Recombinant protein-based ELISA and immuno-cytochemical assay for the diagnosis of SARS

A new Coronavirus (SARS-CoV) is the aetiological agent of the severe acute respiratory syndrome (SARS). Because of the critical role played by serological assays for SARS diagnosis, an in-house ELISA based on SARS-CoV recombinant antigens was developed. The SARS-CoV nucleocapsid protein (N), three N fragments (N1, N2, and N3) and the intraviral domain of the membrane protein (M2) were cloned and expressed in Escherichia coli as histidine-tagged proteins. Six reference sera from SARS patients were used to detect virus-specific IgG in an ELISA using each recombinant protein as coating antigen. High-titre positive reactions were detected in all SARS positive sera. The specificity of the assay appears to be high as no positive reaction was detected in the sera of 20 healthy subjects and 73 patients with non-SARS, low-tract respiratory infections. Specific hyper-immune sera to SARS-CoV and the recombinant proteins, N, N1, N2, N3, and M2 were also generated in mice and rabbits. The specificity of these sera was confirmed by an immunocytochemical assay on biochips of SARS-CoV infected and uninfected cells.     

严重急性呼吸综合征患者相关病毒的随访监测及意义

目的 随访监测确诊SARS患者,研究SARS-CoV、新型呼肠病毒和脊髓灰质炎病毒在患者体内的存在状况及与SARS的可能相关性.方法 采集8例SARS患者痊愈2年后的咽拭子、唾液（或痰液）、尿液、血清及连续3天的粪便标本,用RT-PCR的方法分别扩增SARS-CoV、呼肠病毒和脊髓灰质炎病毒;采用ELISA的方法检测患者血清中的SARS-CoV抗体和脊髓灰质炎病毒IgG抗体.结果 8例患者中有4例的粪便中脊髓灰质炎病毒RNA阳性,均为Sabin 1毒株,在5′端非编码区的第480nt及VP1蛋白编码区的第2795nt两个重要的减毒位点上均发生了回复突变;SARS-CoV和新型呼肠病毒均阴性.对照组标本中均未检测到上述三种病毒.3例随访患者血清中SARS-CoV抗体阳性;4例患者血清中脊髓灰质炎IgG抗体阳性.25例健康体检者的血清中SARS-CoV抗体均阴性,其中23例的血清中脊髓灰质炎病毒抗体阳性.结论SARS患者痊愈2年后的血清中特异性脊髓灰质炎病毒抗体与健康人有显著差异,其粪便标本中仍有较高的脊髓灰质炎病毒阳性率,而且病毒核酸在两个重要的减毒位点上均发生了回复突变.     

Identification of a B-cell antigenic epitope at the N-terminus of SARS-CoV M protein and characterization of monoclonal antibody against the protein

To identify the potential B-cell antigenic epitopes within the N-terminus of SARS-CoV (SARS-associated coronavirus, SARS-CoV) M protein and characterize monoclonal antibody (MAb) against the protein as well as its recognizing region, we expressed and purified a portion of SARS-CoV M protein (amino acid 1–43) in Escherichia coli (E. coli). By using Western blot and enzyme-linked immunosorbent assay (ELISA), we showed that the purified recombinant M protein could be recognized by four SARS-CoV-positive human sera even when those sera were 12,800-fold diluted. Furthermore, we characterized one representative IgG2 MAb, 3H9, which exhibited a strong immunoreaction to both recombinant M protein and native viral protein of SARS-CoV. We found a B-cell antigenic epitope located between amino acid 1–15 and defined the MAb recognizing region within amino acid 16–28 of M. These findings not only suggest that both recombinant M protein and its specific MAbs may be used as the diagnostic reagents for SARS, but also provide a potential target site for the design of an epitope-based vaccine against SARS. Chao Qian and Di Qin contributed equally to this work.     

Structure-based preliminary analysis of immunity and virulence of SARS coronavirus

The research on SARS-associated coronavirus (SARS-CoV) has not stopped since its discovery, but the pathogenesis of SARS is still unclear. To explore the possible molecular mechanisms of the invasion and virulence of SARS-CoV, we investigated the structural basis of the viral proteins using computational biology. Forty-five motifs relating to superantigens, toxins and other bioactive molecules were detected in the proteins of SARS-CoV. The results showed that the distribution of the motifs varied in different proteins. Enzyme-like motifs were located in the R protein, while ICAM- 1-like and toxin-like molecules were located in the spike, envelop, nucleocapsid, PUP1, PUP 2 and PUP 4 proteins. Comparison of SARS-CoV with other viruses (OC43, PEDV, HRSV, HHerpV and HAdenoV) showed that each group of motifs was different for each type of virus. Data suggest that the proteins of SARS-CoV with toxic motifs might play crucial roles in targeting host cells and interfering with the immune system. This study provides new information for drug and vaccine design, as well as therapeutic strategies against SARS.     

Furin cleavage of the SARS coronavirus spike glycoprotein enhances cell-cell fusion but does not affect virion entry

The fusogenic potential of Class I viral envelope glycoproteins is activated by proteloytic cleavage of the precursor glycoprotein to generate the mature receptor-binding and transmembrane fusion subunits. Although the coronavirus (CoV) S glycoproteins share membership in this class of envelope glycoproteins, cleavage to generate the respective S1 and S2 subunits appears absent in a subset of CoV species, including that responsible for the severe acute respiratory syndrome (SARS). To determine whether proteolytic cleavage of the S glycoprotein might be important for the newly emerged SARS-CoV, we introduced a furin recognition site at single basic residues within the putative S1-S2 junctional region. We show that furin cleavage at the modified R667 position generates discrete S1 and S2 subunits and potentiates membrane fusion activity. This effect on the cell-cell fusion activity by the S glycoprotein is not, however, reflected in the infectivity of pseudotyped lentiviruses bearing the cleaved glycoprotein. The lack of effect of furin cleavage on virion infectivity mirrors that observed in the normally cleaved S glycoprotein of the murine coronavirus and highlights an additional level of complexity in coronavirus entry.     

Expression of elevated levels of pro-inflammatory cytokines in SARS-CoV-infected ACE2+ cells in SARS patients: relation to the acute lung injury and pathogenesis of SARS

The authors have previously shown that acute lung injury (ALI) produces a wide spectrum of pathological processes in patients who die of severe acute respiratory syndrome (SARS) and that the SARS coronavirus (SARS-CoV) nucleoprotein is detectable in the lungs, and other organs and tissues, in these patients. In the present study, immunohistochemistry (IHC) and in situ hybridization (ISH) assays were used to analyse the expression of angiotensin-converting enzyme 2 (ACE2), SARS-CoV spike (S) protein, and some pro-inflammatory cytokines (PICs) including MCP-1, TGF-beta1, TNF-alpha, IL-1beta, and IL-6 in autopsy tissues from four patients who died of SARS. SARS-CoV S protein and its RNA were only detected in ACE2+ cells in the lungs and other organs, indicating that ACE2-expressing cells are the primary targets for SARS-CoV infection in vivo in humans. High levels of PICs were expressed in the SARS-CoV-infected ACE2+ cells, but not in the uninfected cells. These results suggest that cells infected by SARS-CoV produce elevated levels of PICs which may cause immuno-mediated damage to the lungs and other organs, resulting in ALI and, subsequently, multi-organ dysfunction. Therefore application of PIC antagonists may reduce the severity and mortality of SARS.     

瞬时转染SARS-CoV M基因对大鼠肺成纤维细胞生长特性及生物学功能的影响

目的 通过观察转染SARS-CoVM基因的大鼠肺间质成纤维细胞（fibroblast,FB）生长及功能的改变,初步探讨SARS病毒通过肺组织损伤而引发肺纤维化的可能机制和途径。方法 碱裂解法扩增纯化质粒,酶消化法分离大鼠肺间质成纤维细胞;采用脂质体介导的方法瞬时转染M基因入FB;采用RT-PCR法和Western blot鉴定转染结果;四唑盐（MTT）比色法和流式细胞技术（FCM）检测转染后的大鼠肺成纤维细胞的生长情况;采用半定量RT-PCR和Realtime PCR检测转染后基质金属蛋白酶-2（MMP-2）基因转录水平的变化。结果 与转染空载体的成纤维细胞相比,转染M基因的成纤维细胞在转染48h时生长明显增强,G0／G1期比例下降,S期比例增高;转染M基因后MMP-2的转录明显增强。结论 成功地将携带有M基因的真核表达质粒瞬时转染入大鼠肺间质成纤维细胞,目的基因在肺间质成纤维细胞中表达并具有与SARS-CoVM相似的抗原性;SARS-CoVM蛋白可能促进肺间质成纤维细胞生长;并促进MMP-2的表达。     

5株SARS-CoV部分基因序列比较分析

目的　分析SARS CoV部分结构区的基因序列 ,了解其变异程度。方法　采用套式PCR法扩增各结构区基因 ,对阳性PCR产物进行克隆和测序 ,并对序列进行分析。结果　完成了LC1株病毒的M、N、E和S基因的扩增和克隆 ,对LC2、LC3、LC4和LC5株病毒的M区基因进行了扩增和克隆。序列分析显示各结构基因的核苷酸序列与已报道的 18株序列的同源性在 99%以上。结论SARS CoV的基因序列较保守 ,有利于PCR诊断试剂和预防用疫苗的研制。     

Identification of a critical neutralization determinant of severe acute respiratory syndrome (SARS)-associated coronavirus: importance for designing SARS vaccines

The spike (S) protein of severe acute respiratory syndrome-associated coronavirus (SARS-CoV) is not only responsible for receptor binding, but also a major antigenic determinant capable of inducing protective immunity. In this study, we demonstrated that the receptor-binding domain (RBD) of S protein is an important immunogenic site in patients with SARS and rabbits immunized with inactivated SARS-CoV. Serum samples from convalescent SARS patients and immunized rabbits had potent neutralizing activities against infection by pseudovirus expressing SARS-CoV S protein. Depletion of RBD-specific antibodies from patient or rabbit immune sera by immunoadsorption significantly reduced serum-mediated neutralizing activity, while affinity-purified anti-RBD antibodies had relatively higher potency neutralizing infectivity of SARS pseudovirus, indicating that the RBD of S protein is a critical neutralization determinant of SARS-CoV during viral infection and immunization. Two monoclonal antibodies (1A5 and 2C5) targeting at the RBD of S protein were isolated from mice immunized with inactivated SARS-CoV. Both 1A5 and 2C5 possessed potent neutralizing activities, although they directed against distinct conformation-dependant epitopes as shown by ELISA and binding competition assay. We further demonstrated that 2C5, but not 1A5, was able to block binding of the RBD to angiotensin-converting enzyme 2 (ACE2), the functional receptor on targeted cells. These data provide important information for understanding the antigenicity and immunogenicity of SARS-CoV and for designing SARS vaccines.     

Comparison of immunoglobulin G responses to the spike and nucleocapsid proteins of severe acute respiratory syndrome (SARS) coronavirus in patients with SARS

Both the nucleocapsid (N) and the spike (S) proteins of severe acute respiratory syndrome (SARS)-associated coronavirus (SARS-CoV) are able to induce strong humoral responses in humans following an infection. To compare the immunoglobulin G (IgG) responses to the S and N proteins of SARS-CoV in SARS patients during the manifestation/convalescent period with those during the postinfection period, serum samples were collected from hospitalized SARS patients within 6 weeks after the onset of illness (set 1; 57 sequential samples from 19 patients) or 2 to 3 months after their recovery (set 2; 33 postinfection samples from 33 subjects). Serum samples from 100 healthy blood donors (set 3), collected in 2002, were also included. The specific IgG response to whole virus, the fragment from positions 450 to 650 of the S protein (S450-650), and the full-length N protein of SARS-CoV were measured by enzyme-linked immunosorbent assays (ELISAs). Western blot assays were carried out to confirm the ELISA results. Fifty-one of the serum samples in set 1 (89%) bound to the N protein, a proportion similar to that which recognized whole virus (79%) and the S-protein fragment (77%). All 33 serum samples from set 2 were strongly positive for N-protein-specific IgG, while 27 (82%) were positive for anti-S450-650 IgG. Two of the serum samples from set 3 were strongly positive for anti-N-protein IgG but not anti-S450-650 IgG. Similar levels of IgG responses to the S and N proteins were observed in SARS patients during the manifestation and convalescent stages. In the postinfection period, however, a number of patients had much lower serum IgG levels against S450-650 than against the N protein.     

A novel cell-based binding assay system reconstituting interaction between SARS-CoV S protein and its cellular receptor

Severe acute respiratory syndrome (SARS), a life-threatening disease, is caused by the newly identified virus SARS coronavirus (SARS-CoV). In order to study the spike (S) protein of this highly contagious virus, we established a clonal cell-line, CHO-SG, from the Chinese hamster ovary cells that stably expresses C-terminally EGFP-tagged SARS-CoV S protein (S-EGFP). The ectodomain of the S glycoprotein is localized on the surface of CHO-SG cells with N-acetyl-glucosamine-terminated carbohydrate structure. CHO-SG cells associated tightly with Vero E6 cells, a SARS-CoV receptor (ACE2) expressing cell-line, and the interaction remained stable under highly stringent condition (1M NaCl). This interaction could be blocked by either the serum from a SARS convalescent patient or a goat anti-ACE2 antibody, indicating that the interaction is specific. A binding epitope with lesser degree of glycosylation and native conformation was localized by using rabbit anti-sera raised against five denatured recombinant S protein fragments expressed in Escherichia coli. One of the sera obtained from the fragment encompassing amino acids 48-358 significantly blocked the interaction between CHO-SG and Vero E6 cells. The region is useful for studying neutralizing antibodies in future vaccine development. This paper describes an easy and safe cell-based assay suitable for studying the binding between SARS-CoV S protein and its receptor.     

SARS-coronavirus spike S2 domain flanked by cysteine residues C822 and C833 is important for activation of membrane fusion

The S2 domain of the coronavirus spike (S) protein is known to be responsible for mediating membrane fusion. In addition to a well-recognized cleavage site at the S1-S2 boundary, a second proteolytic cleavage site has been identified in the severe acute respiratory syndrome coronavirus (SARS-CoV) S2 domain (R797). C-terminal to this S2 cleavage site is a conserved region flanked by cysteine residues C822 and C833. Here, we investigated the importance of this well conserved region for SARS-CoV S-mediated fusion activation. We show that the residues between C822-C833 are well conserved across all coronaviruses. Mutagenic analysis of SARS-CoV S, combined with cell-cell fusion and pseudotyped virion infectivity assays, showed a critical role for the core-conserved residues C822, D830, L831, and C833. Based on available predictive models, we propose that the conserved domain flanked by cysteines 822 and 833 forms a loop structure that interacts with components of the SARS-CoV S trimer to control the activation of membrane fusion.