Nitric oxide is an important molecule playing a key role in a broad range of biological process such as neurotransmission, vasodilatation and immune responses. While the anti-microbiological properties of nitric oxide-derived reactive nitrogen intermediates (RNI) such as peroxynitrite, are known, the mechanism of these effects are as yet poorly studied. Severe Acute Respiratory Syndrome coronavirus (SARS-CoV) belongs to the family Coronaviridae, was first identified during 2002-2003. Mortality in SARS patients ranges from between 6 to 55%. We have previously shown that nitric oxide inhibits the replication cycle of SARS-CoV in vitro by an unknown mechanism. In this study, we have further investigated the mechanism of the inhibition process of nitric oxide against SARS-CoV. We found that peroxynitrite, an intermediate product of nitric oxide in solution formed by the reaction of NO with superoxide, has no effect on the replication cycle of SARS-CoV, suggesting that the inhibition is either directly effected by NO or a derivative other than peroxynitrite. Most interestingly, we found that NO inhibits the replication of SARS-CoV by two distinct mechanisms. Firstly, NO or its derivatives cause a reduction in the palmitoylation of nascently expressed spike (S) protein which affects the fusion between the S protein and its cognate receptor, angiotensin converting enzyme 2. Secondly, NO or its derivatives cause a reduction in viral RNA production in the early steps of viral replication, and this could possibly be due to an effect on one or both of the cysteine proteases encoded in Orf1a of SARS-CoV.

We have previously shown that an Escherichia coli-expressed, denatured spike (S) protein fragment of the severe acute respiratory coronavirus, containing residues 1029 to 1192 which include the heptad repeat 2 (HR2) domain, was able to induce neutralizing polyclonal antibodies (C. The virus-cell membrane fusion event is an essential step in the entry process of all enveloped animal viruses, including important human pathogens such as influenza virus, human immunodeficiency virus (HIV) (8, 23), and the newly emerged severe acute respiratory syndrome coronavirus (SARS-CoV) (9). Following the binding to their receptors on the cell surface, virus-encoded membrane fusion proteins mediate the fusion process. In many but not all cases, the viral fusion proteins are proteolytically processed by host proteases into 2 subunits that remain closely associated with each other: a surface subunit with a receptor-binding site and a transmembrane subunit with a fusion peptide consisting of two or more heptad repeat domains. Upon interaction of the fusion protein with a cellular receptor, the buried fusion peptide is exposed and inserted into the membrane of the target cell. A series of conformational changes trigger virus-cell fusion activity (9) and lead to the unloading of the viral genome into cells. Additionally, many viral fusion proteins also induce cell-cell fusion, i.e., the formation of multinucleated syncytia, facilitating the rapid spread of virus infection.TThe spike (S) protein of coronaviruses is responsible for receptor binding and membrane fusion. It shares similarity with class I virus fusion proteins (2, 3). Typically, it is a type I integral membrane protein, which is N-glycosylated and trimerized in the endoplasmic reticulum. The N-terminal S1 protein contains the receptor-binding site (10,18,22,34). The C-terminal S2 protein is a fusion subunit and anchors on the viral envelope through a transmembrane domain. The S2 protein ectodomain contains two 4,3 hydrophobic heptad repeats (HR1 and HR2) and a putative, internal fusion peptide (3, 23). For the SARS-CoV S protein, the HR2 is located adjacent to the transmembrane domain, whereas the HR1 is 140 to 170 residues upstream of the HR2.Crystallographic, biophysical, and biochemical analysis of the fusion core of SARS-CoV S protein (2,12,19,27,30,35) and other class I fusion proteins (8, 25) supports a model of membrane fusion probably adopted by these enveloped viruses. After the attachment of the receptor-binding subunit to the receptor, the HR1 and HR2 domains in the membrane fusion subunit interact with each other and form a six-helix bundle, a complex consisting of a homotrimeric HR1 coiled coil surrounded by three HR2 helices. The spacer domain (or link, or interhelical domain) between HR1 and HR2 forms a loop and reverses the direction of the polypeptide chain so that the HR2 helices pack against the HR1 coiled coil in an antiparallel manner. This conformational change results in a close apposition of the fusion peptide, already exposed and inserted into th...

The spike (S) protein of the severe acute respiratory syndrome coronavirus (SARS-CoV) interacts with cellular receptors to mediate membrane fusion, allowing viral entry into host cells; hence it is recognized as the primary target of neutralizing antibodies, and therefore knowledge of antigenic determinants that can elicit neutralizing antibodies could be beneficial for the development of a protective vaccine. Here, we expressed five different fragments of S, covering the entire ectodomain (amino acids 48 to 1192), as glutathione S-transferase fusion proteins in Escherichia coli and used the purified proteins to raise antibodies in rabbits. By Western blot analysis and immunoprecipitation experiments, we showed that all the antibodies are specific and highly sensitive to both the native and denatured forms of the full-length S protein expressed in virus-infected cells and transfected cells, respectively. Indirect immunofluorescence performed on fixed but unpermeabilized cells showed that these antibodies can recognize the mature form of S on the cell surface. All the antibodies were also able to detect the maturation of the 200-kDa form of S to the 210-kDa form by pulse-chase experiments. When the antibodies were tested for their ability to inhibit SARS-CoV propagation in Vero E6 culture, it was found that the anti-S⌬10 antibody, which was targeted to amino acid residues 1029 to 1192 of S, which include heptad repeat 2, has strong neutralizing activities, suggesting that this region of S carries neutralizing epitopes and is very important for virus entry into cells.A novel coronavirus (CoV) was identified as the etiological agent of severe acute respiratory syndrome (SARS) (8,9,15,20). CoVs are positive-strand RNA viruses, and the virion consists of a nucleocapsid (N) core surrounded by an envelope containing three membrane proteins, spike (S), membrane (M), and envelope (E), that are common to all members of the genus (for reviews, see references 16 and 24). The S protein, which forms morphologically characteristic projections on the virion surface, binds to host receptors and mediates membrane fusion. The M and E proteins are important for viral particle assembly, while N is important for viral RNA packaging.The S protein of CoV is a type 1 integral membrane glycoprotein. It is cotranslationally glycosylated and oligomerized at the endoplasmic reticulum. Its N-linked high-mannose side chains are trimmed and modified and become endoglycosidase H (EndoH) resistant during the transportation to the Golgi apparatus. For some but not all CoVs, the S protein is cleaved into the N-terminal S1 and C-terminal S2 subunits, which contain receptor binding and membrane fusion domains (10, 32), respectively. The mature forms of S are assembled into virions, which release from infected cells. A portion of S is transported to the plasma membrane, resulting in cell-cell fusion or formation of syncytia. The S protein belongs to the class 1 viral fusion proteins and contains two heptad repeat domains (HR1 and HR2) in S2 or the C-...

The severe acute respiratory syndrome coronavirus (SARS-CoV), isolated from humans infected during the peak of epidemic, encodes two accessory proteins termed as 8a and 8b. Interestingly, the SARS-CoV isolated from animals contains an extra 29-nucleotide in this region such that these proteins are fused to become a single protein, 8ab. Here, we compared the cellular properties of the 8a, 8b and 8ab proteins by examining their cellular localizations and their abilities to interact with other SARS-CoV proteins. These results may suggest that the conformations of 8a and 8b are different from 8ab although nearly all the amino acids in 8a and 8b are found in 8ab. In addition, the expression of the structural protein, envelope (E), was down-regulated by 8b but not 8a or 8ab. Consequently, E was not detectable in SARS-CoV-infected cells that were expressing high levels of 8b. These findings suggest that 8b may modulate viral replication and/or pathogenesis.

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The most striking difference between the subgenomic mRNA8 of severe acute respiratory syndrome coronavirus isolated from human and some animal species is the deletion of 29 nucleotides, resulting in splitting of a single ORF (ORF8) into two ORFs (ORF8a and ORF8b). ORF8a and ORF8b are predicted to encode two small proteins, 8a and 8b, and ORF8 a single protein, 8ab (a fusion form of 8a and 8b). To understand the functions of these proteins, we cloned cDNA fragments covering these ORFs into expression plasmids, and expressed the constructs in both in vitro and in vivo systems. Expression of a construct containing ORF8a and ORF8b generated only a single protein, 8a; no 8b protein expression was obtained. Expression of a construct containing ORF8 generated the 8ab fusion protein. Site‐directed mutagenesis and enzymatic treatment revealed that protein 8ab is modified by N‐linked glycosylation on the N81 residue and by ubiquitination. In the absence of the 8a region, protein 8b undergoes rapid degradation by proteasomes, and addition of proteasome inhibitors inhibits the degradation of protein 8b as well as the protein 8b‐induced rapid degradation of the severe acute respiratory syndrome coronavirus E protein. Glycosylation could also stabilize protein 8ab. More interestingly, the two proteins could bind to monoubiquitin and polyubiquitin, suggesting the potential involvement of these proteins in the pathogenesis of severe acute respiratory syndrome coronavirus.

Mutations in the LaminA gene are a common cause of monogenic dilated cardiomyopathy. Here we show that mice with a cardiomyocyte-specific Lmna deletion develop cardiac failure and die within 3–4 weeks after inducing the mutation. When the same Lmna mutations are induced in mice genetically deficient in the LINC complex protein SUN1, life is extended to more than one year. Disruption of SUN1’s function is also accomplished by transducing and expressing a dominant-negative SUN1 miniprotein in Lmna deficient cardiomyocytes, using the cardiotrophic Adeno Associated Viral Vector 9. The SUN1 miniprotein disrupts binding between the endogenous LINC complex SUN and KASH domains, displacing the cardiomyocyte KASH complexes from the nuclear periphery, resulting in at least a fivefold extension in lifespan. Cardiomyocyte-specific expression of the SUN1 miniprotein prevents cardiomyopathy progression, potentially avoiding the necessity of developing a specific therapeutic tailored to treating each different LMNA cardiomyopathy-inducing mutation of which there are more than 450.

ObjectiveHCV infection affects millions of people worldwide, and many patients develop chronic infection leading to liver cancers. For decades, the lack of a small animal model that can recapitulate HCV infection, its immunopathogenesis and disease progression has impeded the development of an effective vaccine and therapeutics. We aim to provide a humanised mouse model for the understanding of HCV-specific human immune responses and HCV-associated disease pathologies.DesignRecently, we have established human liver cells with a matched human immune system in NOD-scid Il2rg−/− (NSG) mice (HIL mice). These mice are infected with HCV by intravenous injection, and the pathologies are investigated.ResultsIn this study, we demonstrate that HIL mouse is capable of supporting HCV infection and can present some of the clinical symptoms found in HCV-infected patients including hepatitis, robust virus-specific human immune cell and cytokine responses as well as liver fibrosis and cirrhosis. Similar to results obtained from the analysis of patient samples, the human immune cells, particularly T cells and macrophages, play critical roles during the HCV-associated liver disease development in the HIL mice. Furthermore, our model is demonstrated to be able to reproduce the therapeutic effects of human interferon alpha 2a antiviral treatment.ConclusionsThe HIL mouse provides a model for the understanding of HCV-specific human immune responses and HCV-associated disease pathologies. It could also serve as a platform for antifibrosis and immune-modulatory drug testing.