The means of survival of genomic dsRNA of reoviruses from dsRNA-triggered and Dicer-initiated RNAi pathway remains to be defined. The present study aimed to investigate the effect of Grass carp reovirus (GCRV) replica...The means of survival of genomic dsRNA of reoviruses from dsRNA-triggered and Dicer-initiated RNAi pathway remains to be defined. The present study aimed to investigate the effect of Grass carp reovirus (GCRV) replication on the RNAi pathway of grass carp kidney cells (CIK). The dsRNA-triggered RNAi pathway was demonstrated unimpaired in CIK cells through RNAi assay. GCRV-specific siRNA was generated in CIK cells transfected with purified GCRV genomic dsRNA in Northern blot analysis; while in GCRV-infected CIK cells, no GCRV-specific siRNA could be detected. Infection and transfection experiments further indicated that replication of GCRV correlated with the increased transcription level of the Dicer gene and functional inhibition of in vitro synthesized egfp-siRNA in silencing the EGFP reporter gene. These data demonstrated that although only the genomic dsRNA of GCRV was sensitive to the cellular RNAi pathway, unidentified RNAi suppressor protein(s) might contribute to the survival of the viral genome and efficient viral replication.展开更多
Hepatitis C virus (HCV), a positive single-stranded RNA virus, is a major cause of liver disease in humans. Herein we report a novel strategy to inhibit the reproduction and translation of HCV using a short RNA, named...Hepatitis C virus (HCV), a positive single-stranded RNA virus, is a major cause of liver disease in humans. Herein we report a novel strategy to inhibit the reproduction and translation of HCV using a short RNA, named an Additional RNA, to activate the endonuclease activity of Argonaute 2 (Ago2). In the presence of the Additional RNA, the HCV genome RNA has the requisite 12 nucleotides of base-pairing with microRNA-122. This activates the endonuclease activity of Ago2, resulting in cleavage and release of the HCV genome RNA from Ago2 and microRNA-122. The free HCV genome RNA would be susceptible to intracellular degradation, effectively inhibiting its reproduction and translation. This study presents a new method to inhibit HCV that may hold great potential for HCV treatment in the future.展开更多
The nucleocapsid of vesicular stomatitis virus serves as the genomic template for transcription and replication. The viral genomic RNA is sequestered in the nucleocapsid in every step of the virus replication cycle. T...The nucleocapsid of vesicular stomatitis virus serves as the genomic template for transcription and replication. The viral genomic RNA is sequestered in the nucleocapsid in every step of the virus replication cycle. The structure of the nucleocapsid and the entire virion revealed how the viral genomic RNA is encapsidated and packaged in the virus. A unique mechanism for viral RNA synthesis is derived from the structure of the nuleocapsid and its interactions with the viral RNA-dependent RNA polymerase.展开更多
Within Baculoviridae, little is known about the molecular mechanisms of replication in betabaculoviruses, despite extensive studies in alphabaculoviruses. In this study, the promoters of nine late genes of the betabac...Within Baculoviridae, little is known about the molecular mechanisms of replication in betabaculoviruses, despite extensive studies in alphabaculoviruses. In this study, the promoters of nine late genes of the betabaculovirus Plutella xylostella granulovirus(Plxy GV) were cloned into a transient expression vector and the alphabaculovirus Autographa californica multiple nucleopolyhedrovirus(Ac MNPV) genome, and compared with homologous late gene promoters of Ac MNPV in Sf9 cells. In transient expression assays, all Plxy GV late promoters were activated in cells transfected with the individual reporter plasmids together with an Ac MNPV bacmid. In infected cells, reporter gene expression levels with the promoters of Plxy GV e18 and Ac MNPV vp39 and gp41 were significantly higher than those of the corresponding Ac MNPV or Plxy GV promoters,which had fewer late promoter motifs. Observed expression levels were lower for the Plxy GV p6.9,pk1, gran, p10 a, and p10 b promoters than for the corresponding Ac MNPV promoters, despite equal numbers of late promoter motifs, indicating that species-specific elements contained in some late promoters were favored by the native viral RNA polymerases for optimal transcription. The 8-nt sequence TAAATAAG encompassing the ATAAG motif was conserved in the Ac MNPV polh, p10,and pk1 promoters. The 5-nt sequence CAATT located 4 or 5 nt upstream of the T/ATAAG motif was conserved in the promoters of Plxy GV gran, p10 c, and pk1. The results of this study demonstrated that Plxy GV late gene promoters could be effectively activated by the RNA polymerase from Ac MNPV, implying that late gene expression systems are regulated by similar mechanisms in alphabaculoviruses and betabaculoviruses.展开更多
基金The Shanghai committee of Science and Technology(Grant No.10PJ1404800)the National Natural Science Foundation of China(Grant No.31072244)
文摘The means of survival of genomic dsRNA of reoviruses from dsRNA-triggered and Dicer-initiated RNAi pathway remains to be defined. The present study aimed to investigate the effect of Grass carp reovirus (GCRV) replication on the RNAi pathway of grass carp kidney cells (CIK). The dsRNA-triggered RNAi pathway was demonstrated unimpaired in CIK cells through RNAi assay. GCRV-specific siRNA was generated in CIK cells transfected with purified GCRV genomic dsRNA in Northern blot analysis; while in GCRV-infected CIK cells, no GCRV-specific siRNA could be detected. Infection and transfection experiments further indicated that replication of GCRV correlated with the increased transcription level of the Dicer gene and functional inhibition of in vitro synthesized egfp-siRNA in silencing the EGFP reporter gene. These data demonstrated that although only the genomic dsRNA of GCRV was sensitive to the cellular RNAi pathway, unidentified RNAi suppressor protein(s) might contribute to the survival of the viral genome and efficient viral replication.
基金supported by the National Science Fund for Distinguished Young Scholars (20925520)the National Natural Science Foundation of China (21235003)the Leading Academic Discipline Project of Shanghai Municipal Education Commission (J50108)
文摘Hepatitis C virus (HCV), a positive single-stranded RNA virus, is a major cause of liver disease in humans. Herein we report a novel strategy to inhibit the reproduction and translation of HCV using a short RNA, named an Additional RNA, to activate the endonuclease activity of Argonaute 2 (Ago2). In the presence of the Additional RNA, the HCV genome RNA has the requisite 12 nucleotides of base-pairing with microRNA-122. This activates the endonuclease activity of Ago2, resulting in cleavage and release of the HCV genome RNA from Ago2 and microRNA-122. The free HCV genome RNA would be susceptible to intracellular degradation, effectively inhibiting its reproduction and translation. This study presents a new method to inhibit HCV that may hold great potential for HCV treatment in the future.
基金supported by the National Institutes of Health (Grant No. AI050066)
文摘The nucleocapsid of vesicular stomatitis virus serves as the genomic template for transcription and replication. The viral genomic RNA is sequestered in the nucleocapsid in every step of the virus replication cycle. The structure of the nucleocapsid and the entire virion revealed how the viral genomic RNA is encapsidated and packaged in the virus. A unique mechanism for viral RNA synthesis is derived from the structure of the nuleocapsid and its interactions with the viral RNA-dependent RNA polymerase.
基金supported by the fund of Hubei Key Laboratory of Genetic Regulation and Integrative Biology
文摘Within Baculoviridae, little is known about the molecular mechanisms of replication in betabaculoviruses, despite extensive studies in alphabaculoviruses. In this study, the promoters of nine late genes of the betabaculovirus Plutella xylostella granulovirus(Plxy GV) were cloned into a transient expression vector and the alphabaculovirus Autographa californica multiple nucleopolyhedrovirus(Ac MNPV) genome, and compared with homologous late gene promoters of Ac MNPV in Sf9 cells. In transient expression assays, all Plxy GV late promoters were activated in cells transfected with the individual reporter plasmids together with an Ac MNPV bacmid. In infected cells, reporter gene expression levels with the promoters of Plxy GV e18 and Ac MNPV vp39 and gp41 were significantly higher than those of the corresponding Ac MNPV or Plxy GV promoters,which had fewer late promoter motifs. Observed expression levels were lower for the Plxy GV p6.9,pk1, gran, p10 a, and p10 b promoters than for the corresponding Ac MNPV promoters, despite equal numbers of late promoter motifs, indicating that species-specific elements contained in some late promoters were favored by the native viral RNA polymerases for optimal transcription. The 8-nt sequence TAAATAAG encompassing the ATAAG motif was conserved in the Ac MNPV polh, p10,and pk1 promoters. The 5-nt sequence CAATT located 4 or 5 nt upstream of the T/ATAAG motif was conserved in the promoters of Plxy GV gran, p10 c, and pk1. The results of this study demonstrated that Plxy GV late gene promoters could be effectively activated by the RNA polymerase from Ac MNPV, implying that late gene expression systems are regulated by similar mechanisms in alphabaculoviruses and betabaculoviruses.
