Viral nonstructural proteins in both enveloped and non-enveloped viruses play important roles in viral replication. Protein NS38 of Grass carp reovirus (GCRV), has been deduced to be a non-structural protein, and, con...Viral nonstructural proteins in both enveloped and non-enveloped viruses play important roles in viral replication. Protein NS38 of Grass carp reovirus (GCRV), has been deduced to be a non-structural protein, and, consistent with other reoviruses, is considered to cooperate with the NS80 protein in viral particle assembly. To investigate the molecular basis of the role of NS38, a complete protein was expressed in E.coli for the first time. It was found that there is a better expression of NS38 induced with IPTG at 28 ℃ rather than 37 ℃. In addition, the antiserum of NS38 prepared with purified fusion protein and injected into rabbit could be used for detecting NS38 protein expression in GCRV infected cell lysate, while there is not any reaction crossed with purified virus particle, confirming NS38 is not a component of the viral structural protein. The result reported in this study will provide evidence for further viral protein-protein and protein-RNA interaction in dsRNA viruses replication.展开更多
AIM： To investigate whether DNA-dependent activator of interferon-regulatory factors （DAI） inhibits hepatitis B virus （HBV） replication and what the mechanism is. METHODS： After the human hepatoma cell line Huh7...AIM： To investigate whether DNA-dependent activator of interferon-regulatory factors （DAI） inhibits hepatitis B virus （HBV） replication and what the mechanism is. METHODS： After the human hepatoma cell line Huh7 was cotransfected with DAI and HBV expressing plas- mid, viral protein （HBV surface antigen and HBV e an- tigen） secretion was detected by enzyme-linked immu- nosorbent assay, and HBV RNA was analyzed by real- time polymerase chain reaction and Northern blotting, and viral DNA replicative intermediates were examined by Southern blotting. Interferon regulatory factor 3 （IRF3） phosphorylation and nuclear translocation were analyzed via Western blotting and immunofluorescence staining respectively. Nuclear factor-KB （NF-KB） activity induced by DAI was detected by immunofluorescence staining of P65 and dual luciferase reporter assay. Tran- swell co-culture experiment was performed in order to investigate whether the antiviral effects of DAI were dependent on the secreted cytokines. RESULTS： Viral protein secretion was significantly re- duced by 57% （P 〈 0.05）, and the level of total HBV RNA was reduced by 67% （P 〈 0.05）. The viral core particle-associated DNA was also dramatically down- regulated in DAI-expressing Huh7 cells. Analysis of involved signaling pathways revealed that activation of NF-KB signaling was essential for DAI to elicit antivi- ral response in Huh7 cells. When the NF-KB signaling pathway was blocked by a NF-KB signaling suppressor （I~：B^-SR）, the anti-HBV activity of DAI was remarkably abrogated. The inhibitory effect of DAI was indepen- dent of IRF3 signaling and secreted cytokines. CONCLUSION： This study demonstrates that DAI can inhibit HBV replication and the inhibitory effect is asso- ciated with activation of NF-KB but independent of IRF3 and secreted cytokines.展开更多
基金National Basic Research Program (973) of China ( 2009CB118701)National Natural Scientific Foundation of China (30871940, 30671615)
文摘Viral nonstructural proteins in both enveloped and non-enveloped viruses play important roles in viral replication. Protein NS38 of Grass carp reovirus (GCRV), has been deduced to be a non-structural protein, and, consistent with other reoviruses, is considered to cooperate with the NS80 protein in viral particle assembly. To investigate the molecular basis of the role of NS38, a complete protein was expressed in E.coli for the first time. It was found that there is a better expression of NS38 induced with IPTG at 28 ℃ rather than 37 ℃. In addition, the antiserum of NS38 prepared with purified fusion protein and injected into rabbit could be used for detecting NS38 protein expression in GCRV infected cell lysate, while there is not any reaction crossed with purified virus particle, confirming NS38 is not a component of the viral structural protein. The result reported in this study will provide evidence for further viral protein-protein and protein-RNA interaction in dsRNA viruses replication.
基金Supported by Grants of The Chinese State Basic Research, No.2009CB522504National Mega Projects for Infectious Diseases, No. 2008ZX10203
文摘AIM： To investigate whether DNA-dependent activator of interferon-regulatory factors （DAI） inhibits hepatitis B virus （HBV） replication and what the mechanism is. METHODS： After the human hepatoma cell line Huh7 was cotransfected with DAI and HBV expressing plas- mid, viral protein （HBV surface antigen and HBV e an- tigen） secretion was detected by enzyme-linked immu- nosorbent assay, and HBV RNA was analyzed by real- time polymerase chain reaction and Northern blotting, and viral DNA replicative intermediates were examined by Southern blotting. Interferon regulatory factor 3 （IRF3） phosphorylation and nuclear translocation were analyzed via Western blotting and immunofluorescence staining respectively. Nuclear factor-KB （NF-KB） activity induced by DAI was detected by immunofluorescence staining of P65 and dual luciferase reporter assay. Tran- swell co-culture experiment was performed in order to investigate whether the antiviral effects of DAI were dependent on the secreted cytokines. RESULTS： Viral protein secretion was significantly re- duced by 57% （P 〈 0.05）, and the level of total HBV RNA was reduced by 67% （P 〈 0.05）. The viral core particle-associated DNA was also dramatically down- regulated in DAI-expressing Huh7 cells. Analysis of involved signaling pathways revealed that activation of NF-KB signaling was essential for DAI to elicit antivi- ral response in Huh7 cells. When the NF-KB signaling pathway was blocked by a NF-KB signaling suppressor （I~：B^-SR）, the anti-HBV activity of DAI was remarkably abrogated. The inhibitory effect of DAI was indepen- dent of IRF3 signaling and secreted cytokines. CONCLUSION： This study demonstrates that DAI can inhibit HBV replication and the inhibitory effect is asso- ciated with activation of NF-KB but independent of IRF3 and secreted cytokines.
