Various lipid metabolism-related factors are essential for Zika virus(ZIKV)replication.In this study,we revealed a crucial role of diacylglycerol O-acyltransferase 2(DGAT2)in ZIKV replication using a short hairpin RNA...Various lipid metabolism-related factors are essential for Zika virus(ZIKV)replication.In this study,we revealed a crucial role of diacylglycerol O-acyltransferase 2(DGAT2)in ZIKV replication using a short hairpin RNA-based gene knockdown technique.The replication of ZIKV was significantly inhibited by DGAT2 depletion in multiple cell lines and restored by trans-complementation with DGAT2.Mechanistically,DGAT2 is recruited in the viral replication complex by interacting with non-structural(NS)proteins.Among them,both human and murine DGAT2s can be cleaved by NS2B3 at the 122R-R-S124 site.Interestingly,the cleavage product of DGAT2 becomes more stable and is sufficient to promote the lipid droplet(LD)formation independent of its enzymatic activity.This work identifies DGAT2 as a novel target of the viral protease NS2B3 and elucidates that DGAT2 is recruited by viral proteins into the replication complex,thereby playing a proviral role by promoting LD formation,which advances our understanding of host–flavivirus interaction.展开更多
Objective To study the mechanism of the cellular proteins involved in the process of replication of hepatitis C virus (HCV) negative-strand RNA.Methods Ultraviolet (UV) cross-linking was used to identify the cellular ...Objective To study the mechanism of the cellular proteins involved in the process of replication of hepatitis C virus (HCV) negative-strand RNA.Methods Ultraviolet (UV) cross-linking was used to identify the cellular proteins that would bind to the 3' -end of HCV negative-strand RNA. Competition experiment was used to confirm the specificity of this binding, in which excess nonhomologous protein and RNA transcripts were used as competitors. The required binding sequence was determined by mapping, then the binding site was predicted through secondary structure analysis.Results A cellular protein of 45 kD (p45) was found to bind specifically to the 3' -end of HCV negative-strand RNA by UV cross-linking, nhomologous proteins and RNA transcripts could not compete out this binding, whereas the unlabeled 3' -end of HCV negative-strand RNA could. Mapping of the protein-binding site suggested that the 3' -end 131-278nt of HCV negative-strand RNA was the possible protein-binding region. Analysis of RNA secondary structure presumed that the potential binding site was located at 194-GAAAGAAC-201.Conclusion The cellular protein p45 could specifically bind to the secondary structure of the 3' -end of HCV intermediate negative-strand RNA, and may play an important role in HCV RNA replication.展开更多
基金supported by the National Natural Science Foundation of China(82471389,92169110,and 82271385)Natural Science Foundation of Guangdong Province(2022A1515010451,2024A1515010471,and 2023A1515010476)Guangzhou Municipal Science and Technology Program(202206010114 and 2023A04J2235).
文摘Various lipid metabolism-related factors are essential for Zika virus(ZIKV)replication.In this study,we revealed a crucial role of diacylglycerol O-acyltransferase 2(DGAT2)in ZIKV replication using a short hairpin RNA-based gene knockdown technique.The replication of ZIKV was significantly inhibited by DGAT2 depletion in multiple cell lines and restored by trans-complementation with DGAT2.Mechanistically,DGAT2 is recruited in the viral replication complex by interacting with non-structural(NS)proteins.Among them,both human and murine DGAT2s can be cleaved by NS2B3 at the 122R-R-S124 site.Interestingly,the cleavage product of DGAT2 becomes more stable and is sufficient to promote the lipid droplet(LD)formation independent of its enzymatic activity.This work identifies DGAT2 as a novel target of the viral protease NS2B3 and elucidates that DGAT2 is recruited by viral proteins into the replication complex,thereby playing a proviral role by promoting LD formation,which advances our understanding of host–flavivirus interaction.
基金This work was supported by Scientific Research Grant of Guangdong Province(No.990098,990101).
文摘Objective To study the mechanism of the cellular proteins involved in the process of replication of hepatitis C virus (HCV) negative-strand RNA.Methods Ultraviolet (UV) cross-linking was used to identify the cellular proteins that would bind to the 3' -end of HCV negative-strand RNA. Competition experiment was used to confirm the specificity of this binding, in which excess nonhomologous protein and RNA transcripts were used as competitors. The required binding sequence was determined by mapping, then the binding site was predicted through secondary structure analysis.Results A cellular protein of 45 kD (p45) was found to bind specifically to the 3' -end of HCV negative-strand RNA by UV cross-linking, nhomologous proteins and RNA transcripts could not compete out this binding, whereas the unlabeled 3' -end of HCV negative-strand RNA could. Mapping of the protein-binding site suggested that the 3' -end 131-278nt of HCV negative-strand RNA was the possible protein-binding region. Analysis of RNA secondary structure presumed that the potential binding site was located at 194-GAAAGAAC-201.Conclusion The cellular protein p45 could specifically bind to the secondary structure of the 3' -end of HCV intermediate negative-strand RNA, and may play an important role in HCV RNA replication.
