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
Co-infection of maize chlorotic mottle virus(MCMV)with a virus in the Potyviridae family,such as sugarcane mosaic virus,usually leads to maize lethal necrosis(MLN).Over the past decade,MCMV/MLN has emerged in many cou...Co-infection of maize chlorotic mottle virus(MCMV)with a virus in the Potyviridae family,such as sugarcane mosaic virus,usually leads to maize lethal necrosis(MLN).Over the past decade,MCMV/MLN has emerged in many countries/regions of the world and resulted in serious yield loss in maize production.Although partial functions of some MCMV-encoded proteins have been identified,the host factors related to MCMV replication are poorly understood.Here,we show that maize peroxisomes can form aggregated bodies in MCMV-infected leaf cells.The dsRNA binding-dependent fluorescence complementation assay indicated that the aggregated peroxisomes in maize served as the major replication site of MCMV.In addition,our results revealed that all the three maize catalases were present mostly in peroxisomes in the presence or absence of MCMV.Furthermore,we determined that inhibition of catalase activity or induction of reactive oxygen species(ROS)in maize protoplasts significantly reduced the accumulation of MCMV RNA.In summary,this research reveals the replication site of MCMV and an important role of maize catalases in supporting virus replication.Our results are conducive to understanding the pathogenesis of MCMV and identifying targets for resistance breeding or gene regulation strategies.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.
基金supported by grants from the Ministry of Science and Technology(2016YFD0300710)the National Natural Science Foundation of China(no.31571980)+1 种基金the Ministry of Education(the 111 Project B13006)State Key Laboratory of Agrobiotechnology(2019SKLAB6–19).
文摘Co-infection of maize chlorotic mottle virus(MCMV)with a virus in the Potyviridae family,such as sugarcane mosaic virus,usually leads to maize lethal necrosis(MLN).Over the past decade,MCMV/MLN has emerged in many countries/regions of the world and resulted in serious yield loss in maize production.Although partial functions of some MCMV-encoded proteins have been identified,the host factors related to MCMV replication are poorly understood.Here,we show that maize peroxisomes can form aggregated bodies in MCMV-infected leaf cells.The dsRNA binding-dependent fluorescence complementation assay indicated that the aggregated peroxisomes in maize served as the major replication site of MCMV.In addition,our results revealed that all the three maize catalases were present mostly in peroxisomes in the presence or absence of MCMV.Furthermore,we determined that inhibition of catalase activity or induction of reactive oxygen species(ROS)in maize protoplasts significantly reduced the accumulation of MCMV RNA.In summary,this research reveals the replication site of MCMV and an important role of maize catalases in supporting virus replication.Our results are conducive to understanding the pathogenesis of MCMV and identifying targets for resistance breeding or gene regulation strategies.
