The envelope protein of severe acute respiratory syndrome coronavirus interacts with the non-structural protein 3 and is ubiquitinated

To analyze the proteins interacting with the severe acute respiratory syndrome coronavirus (SARS-CoV) envelope (E) protein, a SARS-CoV was engineered including two tags associated to the E protein. Using this virus, complexes of SARS-CoV E and other proteins were purified using a tandem affinity purification system. Several viral and cell proteins including spike, membrane, non-structural protein 3 (nsp3), dynein heavy chain, fatty acid synthase and transmembrane protein 43 bound E protein. In the present work, we focused on the binding of E protein to nsp3 in infected cells and cell-free systems. This interaction was mediated by the N-terminal acidic domain of nsp3. Moreover, nsp3 and E protein colocalized during the infection. It was shown that E protein was ubiquitinated in vitro and in cell culture, suggesting that the interaction between nsp3 and E protein may play a role in the E protein ubiquitination status and therefore on its turnover.     

Potential enhancement of osteoclastogenesis by severe acute respiratory syndrome coronavirus 3a/X1 protein

Severe acute respiratory syndrome coronavirus (SARS-CoV) causes a lung disease with high mortality. In addition, osteonecrosis and bone abnormalities with reduced bone density have been observed in patients following recovery from SARS, which were partly but not entirely explained by the short-term use of steroids. Here, we demonstrate that human monocytes, potential precursors of osteoclasts, partly express angiotensin converting enzyme 2 (ACE2), a cellular receptor of SARS-CoV, and that expression of an accessory protein of SARS-CoV, 3a/X1, in murine macrophage cell line RAW264.7 cells, enhanced NF-κB activity and differentiation into osteoclast-like cells in the presence of receptor activator of NF-κB ligand (RANKL). Furthermore, human epithelial A549 cells expressed ACE2, and expression of 3a/X1 in these cells up-regulated TNF-α, which is known to accelerate osteoclastogenesis. 3a/X1 also enhanced RANKL expression in mouse stromal ST2 cells. These findings indicate that SARS-CoV 3a/X1 might promote osteoclastogenesis by direct and indirect mechanisms.     

Severe acute respiratory syndrome coronavirus accessory protein 9b is a virion-associated protein

Eight accessory proteins have been identified in severe acute respiratory syndrome-associated coronavirus (SARS-CoV). They are believed to play roles in the viral life cycle and may contribute to the pathogenesis and virulence. ORF9b as one of these accessory proteins is located in subgenomic mRNA9 and encodes a 98 amino acid protein. However, whether 9b protein is a structural component of SARS-CoV particles remains unknown. In this study, we demonstrate that 9b protein is translated from bicistronic mRNA9 via leaky ribosome scanning and it is incorporated into both virus-like particles (VLPs) and purified SARS-CoV virions. Further analysis shows that sufficient incorporation of 9b protein into VLPs is dependent upon the co-expression of E and M proteins, but not upon the presence of either S or N protein. Our data indicate that 9b protein of SARS-CoV is another virion-associated accessory protein. This finding will lead to a better understanding of the properties of the SARS-CoV 9b protein.     

Immunological characterization of the spike protein of the severe acute respiratory syndrome coronavirus

Severe acute respiratory syndrome (SARS) is a novel infectious disease caused by the SARS-associated coronavirus (SARS-CoV). There are four major structural proteins in the SARS-CoV, including the nucleocapsid, spike, membrane, and small envelope proteins. In this study, two sets of truncated fragments of spike protein were generated, the first were approximately 210-bp nonoverlapping fragments and the second were overlapping segments of 750 to 900 bp. From these 23 fragments, we identified a fragment of 259 amino acids (amino acids 441 to 700) that is a major immunodominant epitope. This fragment was highly expressed, and the purified fragment C could detect all 33 SARS patient serum samples tested, collected from 7 to 60 days after the onset of fever, but had no reactivity with all 66 healthy human serum samples tested. Thus, fragment C of spike protein was identified as an immunodominant antigen and could be used for serological detection of SARS-CoV infection.     

Over-expression of severe acute respiratory syndrome coronavirus 3b protein induces both apoptosis and necrosis in Vero E6 cells

The genome of the severe acute respiratory syndrome coronavirus encodes for eight accessory viral proteins with no known homologues in other coronaviruses. One of these is the 3b protein, which is encoded by the second open reading frame in subgenomic RNA 3 and contains 154 amino acids. Here, a detailed time-course study was performed to compare the apoptosis and necrosis profiles induced by full-length 3b, a 3b mutant that was deleted by 30 amino acids from the C terminus (3bDelta124-154) and the classical apoptosis inducer, Bax. Our results showed that Vero E6 cells transfected with a construct for expressing 3b underwent necrosis as early as 6h after transfection and underwent simultaneous necrosis and apoptosis at later time-points. At all the time-points analysed, the apoptosis induced by the expression of 3b was less than the level induced by Bax but the level of necrosis was comparable. The 3bDelta124-154 mutant behaves in a similar manner indicating that the localization of the 3b protein does not seems to be important for the cell-death pathways since full-length 3b is localized predominantly to the nucleolus, while the mutant is found to be concentrated in the peri-nuclear regions. To our knowledge, this is the first report of the induction of necrosis by a SARS-CoV protein.     

Subcellular location and topology of severe acute respiratory syndrome coronavirus envelope protein

Severe acute respiratory syndrome (SARS) coronavirus (CoV) envelope (E) protein is a transmembrane protein. Several subcellular locations and topological conformations of E protein have been proposed. To identify the correct ones, polyclonal and monoclonal antibodies specific for the amino or the carboxy terminus of E protein, respectively, were generated. E protein was mainly found in the endoplasmic reticulum-Golgi intermediate compartment (ERGIC) of cells transfected with a plasmid encoding E protein or infected with SARS-CoV. No evidence of E protein presence in the plasma membrane was found by using immunofluorescence, immunoelectron microscopy and cell surface protein labeling. In addition, measurement of plasma membrane voltage gated ion channel activity by whole-cell patch clamp suggested that E protein was not present in the plasma membrane. A topological conformation in which SARS-CoV E protein amino terminus is oriented towards the lumen of intracellular membranes and carboxy terminus faces cell cytoplasm is proposed.     

Vaccine design for severe acute respiratory syndrome coronavirus

Severe acute respiratory syndrome (SARS) is an emerging infectious disease caused by a new coronavirus (SARS-CoV). Recent studies suggest that SARS-CoV is zoonotic and may have a broad host range besides humans. Although the global outbreak of SARS has been contained, there are serious concerns over its re-emergence and bioterrorism potential. As a part of preparedness, development of a safe and effective vaccine is one of the highest priorities in fighting SARS. A number of candidate vaccines, using a variety of approaches, are under development. The first vaccine tested in clinical trial is made from the inactivated form of SARS-CoV. Several live attenuated, genetically engineered or vector vaccines encoding the SARS-CoV spike (S) protein have been in pre-clinical studies. These vaccine candidates are effective in terms of eliciting protective immunity in the vaccinated animals. However, caution should be taken with the safety of whole virus or full-length S protein-based immunogens in humans because they may induce harmful immune or inflammatory responses. We propose to use the receptor-binding domain (RBD) of SARS-CoV S protein (residues 318--510) for developing a safe and effective subunit SARS vaccine, as it is not only a functional domain that mediates virus-receptor binding but also a major neutralization determinant of SARSCoV. It has been demonstrated that the RBD of SARS-CoV S protein contains multiple conformational epitopes capable of inducing highly potent neutralizing antibody responses and protective immunity.     

Inactivation of the coronavirus that induces severe acute respiratory syndrome, SARS-CoV

Severe acute respiratory syndrome (SARS) is a life-threatening disease caused by a novel coronavirus termed SARS-CoV. Due to the severity of this disease, the World Health Organization (WHO) recommends that manipulation of active viral cultures of SARS-CoV be performed in containment laboratories at biosafety level 3 (BSL3). The virus was inactivated by ultraviolet light (UV) at 254 nm, heat treatment of 65 degrees C or greater, alkaline (pH > 12) or acidic (pH < 3) conditions, formalin and glutaraldehyde treatments. We describe the kinetics of these efficient viral inactivation methods, which will allow research with SARS-CoV containing materials, that are rendered non-infectious, to be conducted at reduced safety levels.     

Evaluate severe acute respiratory syndrome coronavirus 2 infectivity by pseudoviral particles

Since the outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in humans in late 2019, it has rapidly spread worldwide. To identify the biological characteristics of SARS-CoV-2 in a normal laboratory environment (biosafety level 2 [BSL-2]), a lentiviral-based nucleocapsid was used to carry the spike protein of SARS-CoV-2 onto the surface of pseudoviral particles as a surrogate model to evaluate the infective characterization of SARS-CoV-2. This study indicated that SARS-CoV-2 has extensive tissue tropism for humans and may infect monkeys and tree shrews but not rodents. More importantly, the use of pseudoviral particles in this study allows rapid assessment of neutralizing antibodies in serum in a BSL-2 laboratory. This study will provide a quick and easy tool for evaluating neutralizing antibodies in the serum of recovering patients and assessing the potency of candidate vaccines.     

Mapping of antigenic sites on the nucleocapsid protein of the severe acute respiratory syndrome coronavirus

Antigenic sites on the nucleocapsid (N) protein of severe acute respiratory syndrome (SARS) coronavirus (SARS-CoV) were mapped by Pepscan analysis with overlapping peptides that span the N protein sequence. Two major immunodominant epitopes located in the C-terminal region (amino acids [aa] 362 to 412) and middle region (aa 153 to 178) reacted with more than 75% of sera from SARS patients. Several minor immunodominant epitopes were reactive with about 50% of the SARS sera. Antisera from mice immunized with inactivated SARS-CoV recognized the two major immunodominant epitopes and one antigenic site located adjacent to the N-terminal region (aa 76 to 101), which did not react with the sera from SARS patients. Several monoclonal antibodies against SARS-CoV bound to the N- or C-terminal antigenic sites. These results suggest that the above antigenic sites on the N protein are important in eliciting humoral immune response against SARS-CoV in humans and animals and can be used as antigens for developing diagnostic tests.     

Identification of the severe acute respiratory syndrome coronavirus by simultaneous multigene DNA sequencing

The recent severe acute respiratory syndrome (SARS) outbreak resulted in calls for an accurate diagnostic test that can be used not only for routine testing but also for generating nucleotide sequences to monitor the epidemic. Although the identity of the SARS coronavirus (SARS-CoV) genome was confirmed by DNA sequencing, it is impractical to sequence the entire 29-kb SARS-CoV genome on a routine basis. Therefore, alternative assay methods such as the enzyme-linked immunosorbent assay and PCR have been pursued for routine testing, primarily to resolve probable cases. We report here a modification of standard DNA sequencing technology for accurate identification of SARS-CoV in routine testing. Instead of requiring the sequencing of the whole SARS-CoV genome, our modification enables the simultaneous sequencing of three regions of the SARS-CoV genome, the spike protein-encoding gene (35 nucleotides), gene M (43 nucleotides), and gene N (45 nucleotides), in a single electropherogram. Comparing these nucleotide sequences to DNA databank entries (National Institutes of Health) conclusively identified them as SARS-CoV sequences.     

Development, characterization, and application of monoclonal antibodies against severe acute respiratory syndrome coronavirus nucleocapsid protein

Five monoclonal antibodies (MAbs) against recombinant nucleocapsid protein (NP) of severe acute respiratory syndrome (SARS)-causing coronavirus (CoV) were developed by hybridoma technology. Epitope mapping by Western blotting showed that these anti-SARS-CoV NP MAbs bind to distinct domains of NP. These anti-SARS-CoV NP MAbs, with their high specificity, are potentially ideal candidates for developing early and sensitive diagnostic assays for SARS-CoV.     

Translational control of the subgenomic RNAs of severe acute respiratory syndrome coronavirus

Nitric oxide (NO) has been shown to suppress Japanese encephalitis virus (JEV) RNA synthesis, viral protein accumulation, and virus release from infected cells. In this article, the potential viral structural proteins as the activators of NO product were studied at the molecular level. First, the genomic region encoding the JEV structural proteins was cloned into a prokaryotic expression vector pET for high-level expression. After purification, these JEV recombinant proteins were added to macrophages to examine the productions of NO and pro-inflammatory mediators. In this study, the recombinant core protein, but not envelope (E), could trigger NO and pro-inflammatory mediators (TNF-α, IL-6, and IL-12) productions on macrophages. And their effects were about 85–95% relative to LPS-stimulated macrophages in a dose-dependent manner. Meanwhile, the rCore-2D could up regulate promoters of IL-8 and TNF-α via EGFP expression in reporter plasmid (IL-8p–EGFP and TNF-αp–EGFP)-transfected cells by flow cytometric analysis. These results suggest that JEV core protein could regulate pro-inflammatory mediators and NO production, and may play a crucial role in the innate immunity for the host to restrict the initial stage of JEV infection.     

一种新型冠状病毒假病毒的制备及初步应用

目的 制备一种新型冠状病毒(SARS-CoV-2)假病毒,并将其应用于抗体中和能力检测和抗体广谱性评估.方法 整合2种近期出现的SARS-CoV-2变异病毒株(20A.EU1和B1.1.7)刺突(spike,S)蛋白的突变序列、以及对胞浆区肽段19个氨基酸进行局部缺失突变,构建突变型S蛋白表达质粒,转染293T细胞进行...     

Assessment of synthetic peptides of severe acute respiratory syndrome coronavirus recognized by long-lasting immunity

In order to determine highly immunogenic severe acute respiratory syndrome coronavirus (SARS-CoV) epitope peptides capable of inducing long-lasting immunity, we first screened immunoglobulin-G (IgG) antibodies reactive to 197 different overlapping 15-mers from the SARS-CoV proteins in the sera of three infected patients. Forty-two peptides among them were reactive to the sera from all three patients. Consequently, we tested for the reactivity of these 42 peptides to patients' sera (n = 45) at 6-month post-infection. The significantly higher levels of IgG antibodies specific to three (S791, M207 and N161) of 42 peptides were detectable in the post-infection sera from 23 (51%), 27 (60%) and 19 (42%) of 45 patients, respectively. These three peptides, recognized by their long-lasting immunity, may provide a better understanding of the immunogenicity of SARS-CoV.     

Immunization of mice with a DNA vaccine based on severe acute respiratory syndrome coronavirus spike protein fragment 1

According to data in GenBank, a gene encoding SARS spike protein fragment 1 (S1) was synthesized. After recombination with an immunostimulatory sequence (ISS), the gene was cloned into the plasmid pIRES to produce pIRES-ISS-S1. On confirmation of the expression of S1 protein by indirect immunofluorescence assay (IFA), after the transfection of pIRES-ISS-S1 into BHK-21 cells, the DNA vaccine was repeatedly administrated to BALB/c mice. CD4+ and CD8+ spleen T lymphocytes were analyzed by flow cytometry (FCM) to evaluate T cell-mediated immune responses, the antigen-specific responses of T cells were evaluated by cytotoxic T lymphocyte (CTL) assay, and the level of IgG in antisera from immunized mice was determined by enzyme-linked immunosorbent assay. Results showed that the counts of spleen CD4+ and CD8+ T lymphocytes were increased, that the T cell-mediated immune responses showed antigen specificity, and that IgG was significantly induced with DNA vaccines pIRES-ISS-S1 and pIRES-S1 at titers of 1:320 and 1:160, respectively. These results are promising for the protective immunization of humans.     

Rapid and sensitive detection of severe acute respiratory syndrome coronavirus by rolling circle amplification

The severe acute respiratory syndrome (SARS) epidemic of 2003 was responsible for 774 deaths and caused significant economic damage worldwide. Since July 2003, a number of SARS cases have occurred in China, raising the possibility of future epidemics. We describe here a rapid, sensitive, and highly efficient assay for the detection of SARS coronavirus (SARS-CoV) in cultured material and a small number (n = 7) of clinical samples. Using rolling circle amplification (RCA), we were able to achieve sensitive detection levels of SARS-CoV RNA in both solid and liquid phases. The main advantage of RCA is that it can be performed under isothermal conditions with minimal reagents and avoids the generation of false-positive results, a problem that is frequently encountered in PCR-based assays. Furthermore, the RCA technology provides a faster, more sensitive, and economical option to currently available PCR-based methods.