Sumoylation of the nucleocapsid protein of severe acute respiratory syndrome coronavirus

Severe acute respiratory syndrome coronavirus (SARS-CoV) encodes a highly basic nucleocapsid (N) protein of 422 amino acids. Similar to other coronavirus N proteins, SARS-CoV N protein is predicted to be phosphorylated and may contain nuclear localization signals, serine/arginine-rich motif, RNA binding domain and regions responsible for self-association and homo-oligomerization. In this study, we demonstrate that the protein is posttranslationally modified by covalent attachment to the small ubiquitin-like modifier. The major sumoylation site was mapped to the (62)lysine residue of the N protein. Further expression and characterization of wild type N protein and K62A mutant reveal that sumoylation of the N protein drastically promotes its homo-oligomerization, and plays certain roles in the N protein-mediated interference of host cell division. This is the first report showing that a coronavirus N protein undergoes posttranslational modification by sumoylation, and the functional implication of this modification in the formation of coronavirus ribouncleoprotein complex, virion assembly and virus-host interactions.     

SR-rich motif plays a pivotal role in recombinant SARS coronavirus nucleocapsid protein multimerization

The nucleocapsid (N) protein of SARS coronavirus (SARS-CoV) is reported to function in encapsidating the viral genomic RNA into helical nucleocapsid, and its self-association is believed to be vital in coating the viral genomic RNA. Characterization of SARS-CoV N multimerization may thereby help us better understand the coronavirus assembly. In the current work, using the yeast two-hybrid technique, an unexpected interaction between residues 1-210 and 211-290 (central region) of the SARS-CoV N protein was detected, and SPR results further revealed that the SR-rich motif (amino acids 183-197) of SARS-CoV N protein is responsible for such an interaction. Chemical cross-linking and gel-filtration analyses indicated that the residues 283-422 of the SARS-CoV N protein have multimeric ability, although the full-length N protein is prone to exist predominantly as dimers. In addition, the multimeric ability of the C-terminal domain of SARS-CoV N protein could be weakened by the SR-rich motif interaction with the central region (amino acids 211-290). All of these data suggested that the SR-rich motif of the SARS-CoV N protein might play an import role in the transformation of the SARS-CoV N protein between the dimer and multimer during its binding to its central region for self-association or dissociation. This current paper will hopefully provide some new ideas in studying SARS-CoV N multimerization.     

Salicylate suppresses macrophage nitric-oxide synthase-2 and cyclo-oxygenase-2 expression by inhibiting CCAAT/enhancer-binding protein-beta binding via a common signaling pathway

We determined whether salicylate at pharmacological concentrations inhibits nitric-oxide synthase-2 (NOS-2) and cyclo-oxygenase-2 (COX-2) expressions in RAW 264.7 stimulated with lipopolysaccharide (LPS) and interferon-gamma (IFN-gamma). Cells were treated with sodium salicylate (10(-7)-10(-4) m) or vehicle for 30 min followed by LPS+IFN-gamma for up to 24 h. Salicylate suppressed NOS-2 and COX-2 protein levels and promoter activities stimulated by LPS+IFN-gamma for 4 h in a concentration-dependent manner but had no effect on NOS-2 expression stimulated by the combined agonists for 24 h. Results from promoter analysis indicate that the binding of CCAAT/enhancer-binding protein beta (C/EBPbeta) to its cognate site at -150/-142 on the NOS-2 promoter region was essential for NOS-2 expression at 4 h but not at 24 h. Salicylate reduced C/EBPbeta binding at 4 h and did not alter its binding at 24 h. NOS-2 and COX-2 protein levels and C/EBPbeta binding stimulated by LPS+IFN-gamma for 4 h were inhibited by a similar battery of signaling inhibitors, suggesting a common pathway for NOS-2 and COX-2 expression. Kinetic analysis indicates that NOS-2, similar to COX-2 expression, at 4 h was largely due to the action of LPS, which induced C/EBPbeta binding, whereas its expression at a longer time point was contributed by IFN-gamma. Our findings implicate two distinct pathways for NOS-2 expression induced by LPS+IFN-gamma. Salicylate at pharmacological concentrations is capable of suppressing the early phase of NOS-2 and COX-2 expression by blocking C/EBPbeta binding.     

Molecular analysis of BcrR, a membrane-bound bacitracin sensor and DNA-binding protein from Enterococcus faecalis

BcrR has been identified as a novel regulatory protein of high level bacitracin resistance encoded by the bcrABD operon in Enterococcus faecalis. The N-terminal domain of BcrR has similarity to the helix-turn-helix motif of DNA-binding proteins, and topological modeling predicts that the C-terminal domain contains four transmembrane alpha-helices. These data have led to the hypothesis that BcrR functions as both a membrane-bound sensor and transducer of bacitracin availability to regulate bcrABD expression. To characterize the bcrABD promoter and identify the promoter elements to which BcrR binds, a series of bcrA-lacZ fusions were constructed. A 69-bp region was identified that was essential for bacitracin-dependent bcrA-lacZ expression. Mutations that targeted this region were used to identify two inverted repeat sequences, each with the sequence 5'-GACA(N)(7)TGTC-3', on the bcrABD promoter that were required for bcrA-lacZ expression. To study BcrR binding to this region, we over-produced BcrR with a C-terminal hexa-histidine tag in Escherichia coli membranes, extracted the protein with n-dodecyl-beta-d-maltoside, and subsequently purified it via Ni(2+)-nitrilotriacetic acid and gel filtration chromatography to apparent homogeneity. Purified BcrR was reconstituted into liposomes, and BcrR binding to bcrABD promoter DNA was analyzed using electrophoretic mobility shift assays. Both inverted repeat sequences were required for BcrR binding, both in the presence and absence of bacitracin. These data demonstrate that membrane-bound BcrR binds specifically to the bcrABD promoter, irrespective of bacitracin concentration. We therefore propose that bacitracin-dependent induction of bcrABD expression by BcrR occurs after DNA binding.     

Induction of cyclooxygenase-2 by heat shock protein 60 in macrophages and endothelial cells

The 60-kDa heat shock protein (HSP60), an endogenous ligand for the toll-like 4 receptor, is generated in response to inflammation, tissue injury, and/or stress and stimulates macrophages to produce cytotoxic and proinflammatory mediators including nitric oxide, tumor necrosis factor (TNF)-alpha, interleukin (IL)-6, and IL-12. In the present studies we report that HSP60 is an effective inducer of cyclooxygenase-2 (COX-2) in macrophages, as well as endothelial cells. In both cell types, the synthesis of COX-2 was coordinate with induction of nitric oxide synthase (NOS)-2 and with nitric oxide production. With the use of promoter constructs in transient transfection assays, optimal expression of COX-2 in macrophages was found to require nuclear factor (NF)-kappaB, the cAMP-response element (CRE), and NF-IL-6, but not the E-box. Mobility shift assays revealed that HSP60 induced NF-kappaB and CRE binding activity, while CCAAT/enhancer binding protein (C/EBP), which binds to NF-IL-6, was constitutively active in the cells. Both c-Jun and CRE binding protein (CREB) bound to the CRE, while C/EBP-beta bound to NF-IL-6. These data indicate that NF-kappaB, C/EBP-beta, c-Jun, and CREB are important in HSP60-induced expression of COX-2. The c-Jun-NH(2)-terminal kinase (JNK), p44/42 mitogen-activated protein (MAP) kinase [extracellular signal-regulated kinase 1/2 (ERK1/2)], and p38 MAP kinase were rapidly activated by HSP60 in the macrophages. PD-98059, an inhibitor of phosphorylation of ERK1/2, caused a marked inhibition of HSP60-induced COX-2 and NOS-2 expression. Unexpectedly, SB-203580, a p38 kinase antagonist, was found to block HSP60-induced expression of COX-2, but not NOS-2. These data indicate that both ERK1/2 kinase and p38 kinase play a role in regulating HSP60-induced expression of COX-2.     

Small interfering RNA inhibits SARS-CoV nucleocapsid gene expression in cultured cells and mouse muscles

SARS-CoV is a newly identified coronavirus that causes severe acute respiratory syndrome (SARS). Currently, there is no effective method available for prophylaxis and treatment of SARS-CoV infections. In the present study, the influence of small interfering RNA (siRNA) on SARS-CoV nucleocapsid (N) protein expression was detected in cultured cells and mouse muscles. Four siRNA expression cassettes driven by mouse U6 promoter targeting SARS-CoV N gene were prepared, and their inhibitory effects on expression of N and enhanced green fluorescence protein (EGFP) fusion protein were observed. A candidate siRNA was proved to down-regulate N and EGFP expression actively in a sequence-specific manner. The expression vector of this siRNA was constructed and confirmed to reduce N and EGFP expression efficiently in both cultured cells and adult mouse muscles. Our findings suggest that the siRNA should provide the basis for prophylaxis and therapy of SARS-CoV infection in human.     

Selective binding of sterol regulatory element-binding protein isoforms and co-regulatory proteins to promoters for lipid metabolic genes in liver

Mice were subjected to different dietary manipulations to selectively alter expression of hepatic sterol regulatory element-binding protein 1 (SREBP-1) or SREBP-2. mRNA levels for key target genes were measured and compared with the direct binding of SREBP-1 and -2 to the associated promoters using isoform specific antibodies in chromatin immunoprecipitation studies. A diet supplemented with Zetia (ezetimibe) and lovastatin increased and decreased nuclear SREBP-2 and SREBP-1, respectively, whereas a fasting/refeeding protocol dramatically altered SREBP-1 but had modest effects on SREBP-2 levels. Binding of both SREBP-1 and -2 increased on promoters for 3-hydroxy-3-methylglutaryl-CoA reductase, fatty-acid synthase, and squalene synthase in livers of Zetia/lovastatin-treated mice despite the decline in total SREBP-1 protein. In contrast, only SREBP-2 binding was increased for the low density lipoprotein receptor promoter. Decreased SREBP-1 binding during fasting and a dramatic increase upon refeeding indicates that the lipogenic "overshoot" for fatty-acid synthase gene expression known to occur during high carbohydrate refeeding can be attributed to a similar overshoot in SREBP-1 binding. SREBP co-regulatory protein recruitment was also increased/decreased in parallel with associated changes in SREBP binding, and there were clear distinctions for different promoters in response to the dietary manipulations. Taken together, these studies reveal that there are alternative molecular mechanisms for activating SREBP target genes in response to the different dietary challenges of Zetia/lovastatin versus fasting/refeeding. This underscores the mechanistic flexibility that has evolved at the individual gene/promoter level to maintain metabolic homeostasis in response to shifting nutritional states and environmental fluctuations.     

The PDZ-Binding Motif of Severe Acute Respiratory Syndrome Coronavirus Envelope Protein Is a Determinant of Viral Pathogenesis

A recombinant severe acute respiratory syndrome coronavirus (SARS-CoV) lacking the envelope (E) protein is attenuated in vivo. Here we report that E protein PDZ-binding motif (PBM), a domain involved in protein-protein interactions, is a major determinant of virulence. Elimination of SARS-CoV E protein PBM by using reverse genetics caused a reduction in the deleterious exacerbation of the immune response triggered during infection with the parental virus and virus attenuation. Cellular protein syntenin was identified to bind the E protein PBM during SARS-CoV infection by using three complementary strategies, yeast two-hybrid, reciprocal coimmunoprecipitation and confocal microscopy assays. Syntenin redistributed from the nucleus to the cell cytoplasm during infection with viruses containing the E protein PBM, activating p38 MAPK and leading to the overexpression of inflammatory cytokines. Silencing of syntenin using siRNAs led to a decrease in p38 MAPK activation in SARS-CoV infected cells, further reinforcing their functional relationship. Active p38 MAPK was reduced in lungs of mice infected with SARS-CoVs lacking E protein PBM as compared with the parental virus, leading to a decreased expression of inflammatory cytokines and to virus attenuation. Interestingly, administration of a p38 MAPK inhibitor led to an increase in mice survival after infection with SARS-CoV, confirming the relevance of this pathway in SARS-CoV virulence. Therefore, the E protein PBM is a virulence domain that activates immunopathology most likely by using syntenin as a mediator of p38 MAPK induced inflammation.