A convenient cell fusion assay for the study of SARS-CoV entry and inhibition

SARS-CoV spike (S) protein-mediated cell fusion is important for the viral entry mechanism and identification of SARS-CoV entry inhibitors. In order to avoid the high risks involved in handling SARS-CoV and to facilitate the study of viral fusion mechanism, we established the cell lines: SR-COS7 cells that stably express both SARS-CoV S protein and red fluorescence protein, R-COS7 cells that stably express red fluorescence protein, and AG-COS7 cells that stably express both ACE2 and green fluorescence protein, respectively. When SR-COS7 cells or R-COS7 cells were cocultured with AG-COS7 cells, syncytia with yellow fluorescence were conveniently observed after 12 h in SR-COS7 cells plus AG-COS7 cells, but not in R-COS7 cells plus AG-COS7 cells. The cell-to-cell fusion efficiency was simply determined for quantitative analysis based on the number of syncytium detected by flow cytometry. Such new cell-to-cell fusion model was further assessed by the potent HR2 peptide inhibitor, which led to the obvious decrease of the cell-to-cell fusion efficiency. The successful fusion and inhibition of cell-based binding assay shows that it can be well used for the study of SARS-CoV entry and inhibition.     

针对leader序列的siRNA能够有效抑制SARS冠状病毒多个mRNA的表达

小干扰RNAs(siRNAs)能够有效降解具有互补序列的RNA.在SARS-CoV的基因组RNA和所有亚基因组RNA的5′端均有一段共同的leader序列,而且该leader序列在不同的病毒分离物中高度保守,因此leader序列可作为一个用于抑制SARS-CoV复制的有效靶点.研究表明,针对leader序列化学合成的siRNA和DNA载体表达的shRNA都可以有效抑制SARS-CoV mRNA的表达.Leader序列特异的siRNA或shRNA不仅可以有效抑制leader与报告基因EGFP融合基因的表达,而且还可以有效抑制leader与刺突蛋白(spikeprotein)、膜蛋白(membrane protein)和核衣壳蛋白(nucleocapsid protein)基因的融合转录产物的表达.结果表明,针对leader序列的RNA干扰可以发展成为一种抗SARS-CoV治疗的有效策略.     

siRNA干扰HSV1 gD糖蛋白基因的研究

以HSV1 gD糖蛋白基因为靶点,设计合成5对siRNA,并构建pEGFP-N1-gD融合表达质粒,脂质体法共转染Vero细胞,利用绿色荧光蛋白(EGFP)报告基因的特征,流式细胞仪筛选特异沉默gD表达的siRNA。然后有效siRNA转染Vero细胞并感染HSV1,通过空斑减数实验,Real-time PCR和子代病毒滴度评价其对HSV-1感染的作用。结果显示siRNA能有效抑制gD-EGFP融合蛋白和感染细胞内gD糖蛋白的表达,但对HSV-1的感染性无明显抑制作用,故选择gD作为siRNA抗病毒靶点还需进一步的论证。     

siRNA对SARS冠状病毒复制的抑制作用

为探讨siRNA在哺乳动物细胞中对SARS冠状病毒复制的抑制作用,针对BJ0 1株SARS冠状病毒复制酶基因(Pol)和刺突蛋白基因(S) ,设计4个siRNA ,并构建相应的siRNA表达载体及克隆细胞系.利用间接免疫荧光法及实时定量反转录PCR法,检测所设计的siRNA对SARS冠状病毒复制的抑制作用.结果表明,针对Pol基因的siRNA(psOe)在Vero细胞中可阻断BJ0 1株SARS病毒RNA的复制及其蛋白的表达.该结果为深入阐明SARS冠状病毒的致病机理及探讨SARS病毒防治新途径奠定了基础.     

Identification of two antigenic epitopes on SARS-CoV spike protein

The spike (S) protein of severe acute respiratory syndrome-coronavirus (SARS-CoV) is a major virion structural protein. It plays an important role in interaction with receptor and inducing neutralizing antibodies. In the study, six tentative antigenic epitopes (S1 S2 S3 S4 S5 S6) of the spike protein of SARS-CoV were predicted by bio-informatics analysis, and a multi-epitope chimeric gene of S1-S2-S3-S4-S5-S6 was synthesized and fused to downstream GST gene in pGEX-6p-1. The Western blotting demonstrated that SARS patient convalescent serum could recognize the recombinant fusion protein. A number of monoclonal antibodies were developed against the fusion protein. In further, the six predicted epitope genes were individually fused to GST of pGEX-6p-1 and expressed in Escherichia coli BL21, respectively. Among six fusion peptides, S5 reacted with monoclonal antibody D3C5 and S2 reacted with monoclonal antibody D3D1 against spike protein of SARS-CoV. The epitopes recognized by monoclonal antibodies D3C5 and D3D1 are linear, and correspond to 447-458 and 789-799 amino acids of spike protein of SARS-CoV, respectively. Identification of antigenic epitope of spike protein of SARS-CoV could provide the basis for the development of immunity-based prophylactic, therapeutic, and diagnostic techniques for the control of severe acute respiratory syndrome.     

Receptor-binding domain of SARS-CoV spike protein induces highly potent neutralizing antibodies: implication for developing subunit vaccine

The spike (S) protein of severe acute respiratory syndrome (SARS) coronavirus (CoV), a type I transmembrane envelope glycoprotein, consists of S1 and S2 domains responsible for virus binding and fusion, respectively. The S1 contains a receptor-binding domain (RBD) that can specifically bind to angiotensin-converting enzyme 2 (ACE2), the receptor on target cells. Here we show that a recombinant fusion protein (designated RBD-Fc) containing 193-amino acid RBD (residues 318-510) and a human IgG1 Fc fragment can induce highly potent antibody responses in the immunized rabbits. The antibodies recognized RBD on S1 domain and completely inhibited SARS-CoV infection at a serum dilution of 1:10,240. Rabbit antisera effectively blocked binding of S1, which contains RBD, to ACE2. This suggests that RBD can induce highly potent neutralizing antibody responses and has potential to be developed as an effective and safe subunit vaccine for prevention of SARS.     

Amino acids 270 to 510 of the severe acute respiratory syndrome coronavirus spike protein are required for interaction with receptor

A novel coronavirus, severe acute respiratory syndrome coronavirus (SARS-CoV), has recently been identified as the causative agent of severe acute respiratory syndrome (SARS). SARS-CoV appears similar to other coronaviruses in both virion structure and genome organization. It is known for other coronaviruses that the spike (S) glycoprotein is required for both viral attachment to permissive cells and for fusion of the viral envelope with the host cell membrane. Here we describe the construction and expression of a soluble codon-optimized SARS-CoV S glycoprotein comprising the first 1,190 amino acids of the native S glycoprotein (S(1190)). The codon-optimized and native S glycoproteins exhibit similar molecular weight as determined by Western blot analysis, indicating that synthetic S glycoprotein is modified correctly in a mammalian expression system. S(1190) binds to the surface of Vero E6 cells, a cell permissive to infection, as demonstrated by fluorescence-activated cell sorter analysis, suggesting that S(1190) maintains the biologic activity present in native S glycoprotein. This interaction is blocked with serum obtained from recovering SARS patients, indicating that the binding is specific. In an effort to map the ligand-binding domain of the SARS-CoV S glycoprotein, carboxy- and amino-terminal truncations of the S(1190) glycoprotein were constructed. Amino acids 270 to 510 were the minimal receptor-binding region of the SARS-CoV S glycoprotein as determined by flow cytometry. We speculate that amino acids 1 to 510 of the SARS-CoV S glycoprotein represent a unique domain containing the receptor-binding site (amino acids 270 to 510), analogous to the S1 subunit of other coronavirus S glycoproteins.     

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.     

SARS—CoVSars7a和EGFP融合蛋白真核表达载体构建及其表达

根据SARS-CoV sars7a基因设计并化学合成部分重叠引物,经二轮PCR获得sars7a基因片段,以此片段为模板并利用一对带有Kozak序列及删除终止密码的引物进行PCR,获得产物与pEGFP-N1载体连接,使sars7a基因位于．EGFP的基因上游,得到含编码Sars7a-EGFP融合蛋白基因的哺乳动物细胞表达载体。采用细胞核转染技术将重组表达载体转染K562细胞,以流式细胞仪和共聚焦显微镜分析,可检测到EGFP的绿色荧光,表明Sars7a—EGFP得到表达,该蛋白分布于整个细胞,提示Sars7a并非膜蛋白,更可能是胞浆蛋白。此外,该蛋白的表达对K562细胞凋亡无明显影响。     

Silencing SARS-CoV Spike protein expression in cultured cells by RNA interference

The severe acute respiratory syndrome (SARS) has been one of the most epidemic diseases threatening human health all over the world. Based on clinical studies, SARS-CoV (the SARS-associated coronavirus), a novel coronavirus, is reported as the pathogen responsible for the disease. To date, no effective and specific therapeutic method can be used to treat patients suffering from SARS-CoV infection. RNA interference (RNAi) is a process by which the introduced small interfering RNA (siRNA) could cause the degradation of mRNA with identical sequence specificity. The RNAi methodology has been used as a tool to silence genes in cultured cells and in animals. Recently, this technique was employed in anti-virus infections in human immunodeficiency virus and hepatitis C/B virus. In this study, RNAi technology has been applied to explore the possibility for prevention of SARS-CoV infection. We constructed specific siRNAs targeting the S gene in SARS-CoV. We demonstrated that the siRNAs could effectively and specifically inhibit gene expression of Spike protein in SARS-CoV-infected cells. Our study provided evidence that RNAi could be a tool for inhibition of SARS-CoV.     

Immunological, structural, and preliminary X-ray diffraction characterizations of the fusion core of the SARS-coronavirus spike protein

The SARS-CoV spike protein, a glycoprotein essential for viral entry, is a primary target for vaccine and drug development. Two peptides denoted HR-N(SN50) and HR-C(SC40), corresponding to the Leu/Ile/Val-rich heptad-repeat regions from the N-terminal and C-terminal segments of the SARS-CoV spike S2 sequence, respectively, were synthesized and predicted to form trimeric assembly of hairpin-like structures. The polyclonal antibodies produced by recombinant S2 protein were tested for antigenicity of the two heptad repeats. We report here the first crystallographic study of the SARS spike HR-N/HR-C complex. The crystal belongs to the triclinic space group P1 and the data-set collected to 2.98 A resolution showed noncrystallographic pseudo-222 and 3-fold symmetries. Based on these data, comparative modeling of the SARS-CoV fusion core was performed. The immunological and structural information presented herein may provide a more detailed understanding of the viral fusion mechanism as well as the development of effective therapy against SARS-CoV infection.     

Solution structure of the severe acute respiratory syndrome-coronavirus heptad repeat 2 domain in the prefusion state

The envelope glycoprotein, termed the spike protein, of severe acute respiratory syndrome coronavirus (SARS-CoV) is known to mediate viral entry. Similar to other class 1 viral fusion proteins, the heptad repeat regions of SARS-CoV spike are thought to undergo conformational changes from a prefusion form to a subsequent post-fusion form that enables fusion of the viral and host membranes. Recently, the structure of a post-fusion form of SARS-CoV spike, which consists of isolated domains of heptad repeats 1 and 2 (HR1 and HR2), has been determined by x-ray crystallography. To date there is no structural information for the prefusion conformations of SARS-CoV HR1 and HR2. In this work we present the NMR structure of the HR2 domain (residues 1141-1193) from SARS-CoV (termed S2-HR2) in the presence of the co-solvent trifluoroethanol. We find that in the absence of HR1, S2-HR2 forms a coiled coil symmetric trimer with a complex molecular mass of 18 kDa. The S2-HR2 structure, which is the first example of the prefusion form of coronavirus envelope, supports the current model of viral membrane fusion and gives insight into the design of structure-based antagonists of SARS.     

SARS冠状病毒N蛋白激活hfgl2凝血酶原酶基因的研究

研究鉴定激活hfgl2凝血酶原酶基因的SARS冠状病毒结构蛋白。从SARS尸检肺组织中抽提RNA后制备cDNA,分别扩增SARS-CoV的N、S2和M全长基因序列,再分别克隆到真核表达载体pcDNA3.1( )上。应用免疫组织化学分析鉴定pcDNA3.1-N、pcDNA3.1-M和pcDNA3.1-S2的表达。构建人纤维介素(hfgl2)启动子荧光素酶报告基因质粒,并将SARS冠状病毒结构蛋白表达质粒分别与其共转染以明确激活hfgl2基因转录的SARS冠状病毒结构蛋白。将目的片段克隆至pcDNA3.1( ),经酶切鉴定和测序鉴定无误;免疫组织化学染色可见明显的CHO细胞胞浆棕染。与hfgl2启动子共转染实验阐明SARS冠状病毒膜(M)蛋白和刺突糖(S2)蛋白对hfgl2基因的激活与对照组无显著差异,而SARS冠状病毒核心(N)蛋白可激活hfgl2启动子,使其转染活性提高4.6倍。SARS冠状病毒N蛋白可增强hfgl2基因的转录活性。     

RNA Interference Induces Effective Inhibition of mRNA Accumulation and Protein Expression of SHEV ORF3 Gene In vitro

RNA interference (RNAi) provides a powerful promising approach to inhibit viral infection specifically. To explore the possibility of using RNAi as a strategy against HEV infection, which is a serious public health problem in developing countries of Asia, Middle East, Africa, and in Mexico, after the fusion protein expression plasmids pEGFP-ORF3 which contain the EGFP reporter gene and SHEV ORF3 as silencing target, were constructed; EGFP-ORF3 fusion protein expressing HEK 293 cell lines were established; and four siRNAs targeting ORF3 gene were designed, synthesized, and used to transfect the stable cell lines. At 24, 48, and 72 h after transfection, flow cytometry, real-time quantitative PCR, and Western blot were used to assess the expression level of ORF3. The results demonstrated that specific siRNAs which are sequence dependant effectively inhibited mRNA accumulation and protein expression of SHEV ORF3 in HEK 293 cells. These findings provide useful information for the development of RNAi-based prophylaxis and therapy for SHEV infection.