Dengue virus(DENV) nonstructural protein 1(NS1) is a highly conserved 46-kDa protein that contains 2 glycosylation sites(Asn-130 and Asn-207) and 12 conserved cysteine(Cys) residues. Here, we performed site-directed m...Dengue virus(DENV) nonstructural protein 1(NS1) is a highly conserved 46-kDa protein that contains 2 glycosylation sites(Asn-130 and Asn-207) and 12 conserved cysteine(Cys) residues. Here, we performed site-directed mutagenesis to generate systematic mutants of viral strain TSV01. The results of the subsequent analysis showed that an alanine substitution at the second N-linked glycan Asn-207 in NS1 delayed viral RNA synthesis, reduced virus plaque size, and weakened the cytopathic effect. Three mutants at Cys sites(Cys-4, Cys-55, Cys-291) and a C-terminal deletion(ΔC) mutant significantly impaired RNA synthesis, and consequently abolished viral growth, whereas alanine mutations at Asn-130 and Glu-173 resulted in phenotypes that were similar to the wild-type(WT) virus. Further analysis showed that the Asn-207 mutation slightly delayed viral replication. These results suggest that the three conserved disulfide bonds and the second N-linked glycan in NS1 are required for DENV-2 replication.展开更多
基金supported by Important National Science & Technology Specific Projects (2012ZX10004219, 2012ZX10004403)the National Natural Scientific Fund of China (81072675)the Wuhan Key Laboratory on Emerging Infectious Diseases and Biosafety
文摘Dengue virus(DENV) nonstructural protein 1(NS1) is a highly conserved 46-kDa protein that contains 2 glycosylation sites(Asn-130 and Asn-207) and 12 conserved cysteine(Cys) residues. Here, we performed site-directed mutagenesis to generate systematic mutants of viral strain TSV01. The results of the subsequent analysis showed that an alanine substitution at the second N-linked glycan Asn-207 in NS1 delayed viral RNA synthesis, reduced virus plaque size, and weakened the cytopathic effect. Three mutants at Cys sites(Cys-4, Cys-55, Cys-291) and a C-terminal deletion(ΔC) mutant significantly impaired RNA synthesis, and consequently abolished viral growth, whereas alanine mutations at Asn-130 and Glu-173 resulted in phenotypes that were similar to the wild-type(WT) virus. Further analysis showed that the Asn-207 mutation slightly delayed viral replication. These results suggest that the three conserved disulfide bonds and the second N-linked glycan in NS1 are required for DENV-2 replication.
