Cells in G_2/M phase increased in human nasopharyngeal carcinoma cell line by EBV-LMP1 through activation of NF-кB and AP-1

Although previous studies showed that the principal oncoprotein encoded by Epstein-Barr virus, latentmembrane protein 1 (LMP1) 5 could induce the nasopharyngeal carcinoma cells in G_2/M phase increased, littleis known about the target molecules and mechanisms. The present study demonstrated that LMP1 couldinduce the accumulation of p53 protein and upregulate its transactivity in a dose dependent manner, whichresulted in the decrease of the kinase activity of cdc2/cyclin B complex and inducing arrest at G2/M phasethrough the activation of NF-κB and AP-1 signaling pathways, and the effect of NF-κB was more obviousthan that of AP-1. This study provided some significant evidence for further elucidating the molecularmechanisms that LMP1 had effects on the surveillance mechanism of cell cycle and promoting the survivalof transformed cells and tumorigenesis.     

The structure analysis and antigenicity study of the N protein of SARS-CoV

The Coronaviridae family is characterized by a nucleocapsid that is composed of the genome RNA molecule in combination with the nucleoprotein (N protein) within a virion. The most striking physiochemical feature of the N protein of SARS-CoV is that it is a typical basic protein with a high predicted pI and high hydrophilicity, which is consistent with its function of binding to the ribophosphate backbone of the RNA molecule. The predicted high extent of phosphorylation of the N protein on multiple candidate phosphorylation sites demonstrates that it would be related to important functions, such as RNA-binding and localization to the nucleolus of host cells. Subsequent study shows that there is an SR-rich region in the N protein and this region might be involved in the protein-protein interaction. The abundant antigenic sites predicted in the N protein, as well as experimental evidence with synthesized polypeptides, indicate that the N protein is one of the major antigens of the SARS-CoV. Compared with o     

Identification of an epitope of SARS-coronavirus nucleocapsid protein

The nucleocapsid (N) protein of severe acute respiratory syndrome-coronavirus (SARS-CoV) is a majorvirion structural protein. In this study, two epitopes (N1 and N2) of the N protein of SARS-CoV were predicted by bioinformatics analysis. After immunization with two peptides, the peptides-specific antibodies were isolated from the immunized rabbits. The further experiments demonstrated that N1 peptide-induced polyclonal antibodies had a high affinity to bind to E. coli expressed N protein of SARS-CoV. Furthermore, itwas confirmed that N1 peptide-specific IgG antibodies were detectable in the sera of severe acute respiratory syndrome (SARS) patients. The results indicated that an epitope of the N protein has been identified andN protein specific Abs were produced by peptide immunization, which will be useful for the study of SARS-CoV.     

TMEM43-S358L mutation enhances NF-κBTGFp signal cascade in arrhythmogenic right ventricular dysplasia/cardiomyopathy

Arrhythmogenic right ventricular dysplasia/cardiomyopathy(ARVD/C)is a genetic cardiac muscle disease that accounts for approximately 30%sudden cardiac death in young adults.The Ser358Leu mutation of transmembrane protein 43(TMEM43)was commonly identified in the patients of highly lethal and fully penetrant ARVD subtype,ARVD5.Here,we generated TMEM43 S358L mouse to explore the underlying mechanism.This mouse strain showed the classic patholo.gies of ARVD patients,including structural abnormalities and cardiac fibrofatty.TMEM43 S358L mutation led to hyper-activated nuclear factor kB(NFkB)activation in heart tissues and primary cardiomy.ocyte cells.Importantly,this hyper activation of NF-κB directly drove the expression of pro-fibrotic gene,transforming growth factor beta(TGFβ),and enhanced downstream signal,indicating that TMEM43 S358L mutation up-regulates NF-κB-TGFβ signal cascade during ARVD cardiac fibrosis.Our study partially reveals the regulatory mechanism of ARVD development.     

Adenovirus-mediated expression of the C-terminal domain of SARS-CoV spike protein is sufficient to induce apoptosis in Vero E6 cells

The pro-apoptotic properties of severe acute respiratory syndrome coronavirus (SARS-CoV) structural proteins were studied in vitro. By monitoring apoptosis indicators including chromatin condensation, cellular DNA fragmentation and cell membrane asymmetry, we demonstrated that the adenovirus-mediated over-expression of SARS-CoV spike (S) protein and its C-terminal domain (S2) induce apoptosis in Vero E6 cells in a time- and dosage-dependent manner, whereas the expression of its N-terminal domain (S1) and other structural proteins, including envelope (E), membrane (M) and nucleocapsid (N) protein do not. These findings suggest a possible role of S and S2 protein in SARS-CoV induced apoptosis and the molecular pathogenesis of SARS.     

The orientations of core antenna chlorophylls in photosystem II are optimized to maximize the quantum yield of photosynthesis

The open reading frame 3 of the severe acute respiratory syndrome coronavirus (SARS-CoV) genome encodes a predicted protein 3a, consisting of 274 amino acids, that lacks any significant similarities to any known protein. We generated specific antibodies against SARS protein 3a by using a synthetic peptide (P2) corresponding to amino acids 261-274 of the putative protein. Anti-P2 antibodies and the sera from SARS patients could specifically detect the recombinant SARS protein 3a expressed in Escherichia coli and in Vero E6 cells. Expression of SARS protein 3a was detected at 8-12 h after infection and reached a higher level after approximately 24 h in SARS-CoV-infected Vero E6 cells. Protein 3a was also detected in the alveolar lining pneumocytes and some intra-alveolar cells of a SARS-CoV-infected patient's lung specimen. Recombinant protein 3a expressed in Vero E6 cells and protein 3a in the SARS-CoV-infected cells was distributed over the cytoplasm in a fine punctate pattern with partly concentrated staining in the Golgi apparatus. Our study demonstrates that SARS-CoV indeed expresses a novel protein 3a, which is present only in SARS-CoV and not in other known CoVs.     

Cell type-specific cleavage of nucleocapsid protein by effector caspases during SARS coronavirus infection

The epidemic outbreak of severe acute respiratory syndrome (SARS) in 2003 was caused by a novel coronavirus (CoV), designated SARS-CoV. The RNA genome of SARS-CoV is complexed by the nucleocapsid protein (N) to form a helical nucleocapsid. Besides this primary function, N seems to be involved in apoptotic scenarios. We show that upon infection of Vero E6 cells with SARS-CoV, which elicits a pronounced cytopathic effect and a high viral titer, N is cleaved by caspases. In contrast, in SARS-CoV-infected Caco-2 cells, which show a moderate cytopathic effect and a low viral titer, this processing of N was not observed. To further verify these observations, we transiently expressed N in different cell lines. Caco-2 and N2a cells served as models for persistent SARS-CoV infection, whereas Vero E6 and A549 cells did as prototype cell lines lytically infected by SARS-CoV. The experiments revealed that N induces the intrinsic apoptotic pathway, resulting in processing of N at residues 400 and 403 by caspase-6 and/or caspase-3. Of note, caspase activation is highly cell type specific in SARS-CoV-infected as well as transiently transfected cells. In Caco-2 and N2a cells, almost no N-processing was detectable. In Vero E6 and A549 cells, a high proportion of N was cleaved by caspases. Moreover, we examined the subcellular localization of SARS-CoV N in these cell lines. In transfected Vero E6 and A549 cells, SARS-CoV N was localized both in the cytoplasm and nucleus, whereas in Caco-2 and N2a cells, nearly no nuclear localization was observed. In addition, our studies indicate that the nuclear localization of N is essential for its caspase-6-mediated cleavage. These data suggest a correlation among the replication cycle of SARS-CoV, subcellular localization of N, induction of apoptosis, and the subsequent activation of caspases leading to cleavage of N.     

Assembly of severe acute respiratory syndrome coronavirus RNA packaging signal into virus-like particles is nucleocapsid dependent

The severe acute respiratory syndrome coronavirus (SARS-CoV) was recently identified as the etiology of SARS. The virus particle consists of four structural proteins: spike (S), small envelope (E), membrane (M), and nucleocapsid (N). Recognition of a specific sequence, termed the packaging signal (PS), by a virus N protein is often the first step in the assembly of viral RNA, but the molecular mechanisms involved in the assembly of SARS-CoV RNA are not clear. In this study, Vero E6 cells were cotransfected with plasmids encoding the four structural proteins of SARS-CoV. This generated virus-like particles (VLPs) of SARS-CoV that can be partially purified on a discontinuous sucrose gradient from the culture medium. The VLPs bearing all four of the structural proteins have a density of about 1.132 g/cm(3). Western blot analysis of the culture medium from transfection experiments revealed that both E and M expressed alone could be released in sedimentable particles and that E and M proteins are likely to form VLPs when they are coexpressed. To examine the assembly of the viral genomic RNA, a plasmid representing the GFP-PS580 cDNA fragment encompassing the viral genomic RNA from nucleotides 19715 to 20294 inserted into the 3' noncoding region of the green fluorescent protein (GFP) gene was constructed and applied to the cotransfection experiments with the four structural proteins. The SARS-CoV VLPs thus produced were designated VLP(GFP-PS580). Expression of GFP was detected in Vero E6 cells infected with the VLP(GFP-PS580), indicating that GFP-PS580 RNA can be assembled into the VLPs. Nevertheless, when Vero E6 cells were infected with VLPs produced in the absence of the viral N protein, no green fluorescence was visualized. These results indicate that N protein has an essential role in the packaging of SARS-CoV RNA. A filter binding assay and competition analysis further demonstrated that the N-terminal and C-terminal regions of the SARS-CoV N protein each contain a binding activity specific to the viral RNA. Deletions that presumably disrupt the structure of the N-terminal domain diminished its RNA-binding activity. The GFP-PS-containing SARS-CoV VLPs are powerful tools for investigating the tissue tropism and pathogenesis of SARS-CoV.     

The putative protein 6 of the severe acute respiratory syndrome-associated coronavirus: expression and functional characterization

The SARS-CoV open reading frame 6 (ORF6) is transcribed into mRNA6 and encodes a putative 7.5 kDa accessory protein, SARS 6, with unknown function. In this study, we have confirmed the SARS 6 protein expression in lung and intestine tissues of the SARS patients and in SARS-CoV infected Vero E6 cells by immunohistochemistry. Further studies by immunoblot and confocal microscopy analyses revealed the expression and the endoplasmic reticulum (ER) localization of the recombinant SARS 6 protein in mammalian cells. Expression of SARS 6 protein in mammalian cells elicits biological activity of stimulating cellular DNA synthesis.     

Antisense downregulation of SARS-CoV gene expression in Vero E6 cells

BACKGROUND: Severe acute respiratory syndrome (SARS) is caused by a novel coronavirus (SARS-CoV). It is an enveloped, single-stranded, plus-sense RNA virus with a genome of approximately 30 kb. The structural proteins E, M and N of SARS-CoV play important roles during host cell entry and viral morphogenesis and release. Therefore, we have studied whether expression of these structural proteins can be down-regulated using an antisense technique. METHODS: Vero E6 cells were transfected with plasmid constructs containing exons of the SARS-CoV structural protein E, M or N genes or their exons in frame with the reporter protein EGFP. The transfected cell cultures were treated with antisense phosphorothioated oligonucleotides (antisense PS-ODN, 20mer) or a control oligonucleotide by addition to the culture medium. RESULTS: Among a total of 26 antisense PS-ODNs targeting E, M and N genes, we obtained six antisense PS-ODNs which could sequence-specifically reduce target genes expression by over 90% at the concentration of 50 microM in the cell culture medium tested by RT-PCR. The antisense effect was further proved by down-regulating the expression of the fusion proteins containing the structural proteins E, M or N in frame with the reporter protein EGFP. In Vero E6 cells, the antisense effect was dependent on the concentrations of the antisense PS-ODNs in a range of 0-10 microM or 0-30 microM. CONCLUSIONS: The antisense PS-ODNs are effective in downregulation of SARS. The findings indicate that antisense knockdown of SARS could be a useful strategy for treatment of SARS, and could also be suitable for studies of the pathological function of SARS genes in a cellular model system.