The double-shelled grass carp reovirus （GCRV） is capable of endogenous RNA transcription and processing.Genome sequence analysis has revealed that the protein VP2,encoded by gene segment 2 （S2）,is the putative RNA...The double-shelled grass carp reovirus （GCRV） is capable of endogenous RNA transcription and processing.Genome sequence analysis has revealed that the protein VP2,encoded by gene segment 2 （S2）,is the putative RNA-dependent RNA polymerase （RdRp）.In previous work,we have ex-pressed the functional region of VP2 that is associated with RNA polymerase activity （denoted as rVP2390-900） in E.coil and have prepared a polyclonal antibody against VP2.To characterize the GCRV RNA polymerase,a recombinant full-length VP2 （rVP2） was first constructed and expressed in a baculovirus system,as a fusion protein with an attached His-tag.Immunofluorescence （IF） assays,together with immunoblot （IB） analyses from both expressed cell extracts and purified Histagged rVP2,showed that rVP2 was successfully expressed in Sf9 cells.Further characterization of the replicase activity showed that purified rVP2 and GCRV particles exhibited poly（C）-dependent poly（G） polymerase activity.The RNA enzymatic activity required the divalent cation Mg2＋,and was optimal at 28 ℃.The results provide a foundation for further studies on the RNA polymerases of aquareoviruses during viral transcription and replication.展开更多
As one of the immediate-early(IE)proteins of herpes simplex virus type 1(HSV-1),ICP22 is a multifunctional viral regulator that localizes in the nucleus of infected cells.It is required in experimental animal systems ...As one of the immediate-early(IE)proteins of herpes simplex virus type 1(HSV-1),ICP22 is a multifunctional viral regulator that localizes in the nucleus of infected cells.It is required in experimental animal systems and some nonhuman cell lines,but not in Vero or HEp-2 cells.ICP22 is extensively phosphorylated by viral and cellular kinases and nucleotidylylated by casein kinase Ⅱ.It has been shown to be required for efficient expression of early(E)genes and a subset of late(L)genes.ICP22,in conjunction with the UL13 kinase,mediates the phosphorylation of RNA polymerase Ⅱ.Both ICP22 and UL13 are required for the activation of cdc2,the degradation of cyclins A and B and the acquisition of a new cdc2 partner,the UL42 DNA polymerase processivity factor.The cdc2-UL42 complex mediates postranscriptional modification of topoisomerase Ⅱα in an ICP22-dependent manner to promote L gene expression.In addition,ICP22 interacts with cdk9 in a Us3 kinase dependent fashion to phosphorylate RNA polymerase Ⅱ.展开更多
The protein encoded by HSRG1(HSV-1 stimulation-related gene 1) is a virally induced protein expressed in HSV-1-infected cells.We have already reported that HSRG1 is capable of interacting with transcriptional regulato...The protein encoded by HSRG1(HSV-1 stimulation-related gene 1) is a virally induced protein expressed in HSV-1-infected cells.We have already reported that HSRG1 is capable of interacting with transcriptional regulator proteins.To further analyze the effects of HSRG1 on the regulation of viral gene transcription,we expressed the HSRG1 protein in transfected cells and found that it postpones the proliferation of HSV-1.CAT(chloramphenicol acetyltransferase) assays also revealed that HSRG1 reduces transcription from HSV-1 promoters.Yeast two-hybrid and immunoprecipitation assays indicated that HSRG1 interacts with Cyclin T2,the regulatory subunit of P-TEFb,which is required for transcription elongation by RNA Pol II(RNAP II) ,and that amino acid residues 1-420 in Cyclin T2 are important for binding with HSRG1.Fluorescence assays suggested that the cellular localizations of those two proteins are influenced by their interaction.Further analyses with CAT assays revealed that HSRG1 inhibits the transcriptional activation by Cyclin T2 of viral promoters.Our results suggested that the inhibitory effects of HSRG1 on viral replication and proliferation are probably induced by its binding to Cyclin T2.Therefore,it is likely that HSRG1 inhibits viral gene transcriptional elongation by interacting with Cyclin T2.展开更多
基金supported by funding from the National Natural Science Foundation of China (grants: 31172434, 31372565)
文摘The double-shelled grass carp reovirus （GCRV） is capable of endogenous RNA transcription and processing.Genome sequence analysis has revealed that the protein VP2,encoded by gene segment 2 （S2）,is the putative RNA-dependent RNA polymerase （RdRp）.In previous work,we have ex-pressed the functional region of VP2 that is associated with RNA polymerase activity （denoted as rVP2390-900） in E.coil and have prepared a polyclonal antibody against VP2.To characterize the GCRV RNA polymerase,a recombinant full-length VP2 （rVP2） was first constructed and expressed in a baculovirus system,as a fusion protein with an attached His-tag.Immunofluorescence （IF） assays,together with immunoblot （IB） analyses from both expressed cell extracts and purified Histagged rVP2,showed that rVP2 was successfully expressed in Sf9 cells.Further characterization of the replicase activity showed that purified rVP2 and GCRV particles exhibited poly（C）-dependent poly（G） polymerase activity.The RNA enzymatic activity required the divalent cation Mg2＋,and was optimal at 28 ℃.The results provide a foundation for further studies on the RNA polymerases of aquareoviruses during viral transcription and replication.
基金The Startup Fund of the Hundred Talents Program of the Chinese Academy of Science(20071010141)National Natural Science Foundation of China (30870120)+1 种基金Open Research Fund Program of the State Key Laboratory of Virology of China(2007003,2009 007)Hubei Province Natural Science Foundation of Innovation Groups Project(2008CDA013)
文摘As one of the immediate-early(IE)proteins of herpes simplex virus type 1(HSV-1),ICP22 is a multifunctional viral regulator that localizes in the nucleus of infected cells.It is required in experimental animal systems and some nonhuman cell lines,but not in Vero or HEp-2 cells.ICP22 is extensively phosphorylated by viral and cellular kinases and nucleotidylylated by casein kinase Ⅱ.It has been shown to be required for efficient expression of early(E)genes and a subset of late(L)genes.ICP22,in conjunction with the UL13 kinase,mediates the phosphorylation of RNA polymerase Ⅱ.Both ICP22 and UL13 are required for the activation of cdc2,the degradation of cyclins A and B and the acquisition of a new cdc2 partner,the UL42 DNA polymerase processivity factor.The cdc2-UL42 complex mediates postranscriptional modification of topoisomerase Ⅱα in an ICP22-dependent manner to promote L gene expression.In addition,ICP22 interacts with cdk9 in a Us3 kinase dependent fashion to phosphorylate RNA polymerase Ⅱ.
基金supported by the National Natural Science Foundation of China(Grant Nos.30700028,30670094)the Doctoral Fund of Ministry of Education of China(Grant No.0060023008)
文摘The protein encoded by HSRG1(HSV-1 stimulation-related gene 1) is a virally induced protein expressed in HSV-1-infected cells.We have already reported that HSRG1 is capable of interacting with transcriptional regulator proteins.To further analyze the effects of HSRG1 on the regulation of viral gene transcription,we expressed the HSRG1 protein in transfected cells and found that it postpones the proliferation of HSV-1.CAT(chloramphenicol acetyltransferase) assays also revealed that HSRG1 reduces transcription from HSV-1 promoters.Yeast two-hybrid and immunoprecipitation assays indicated that HSRG1 interacts with Cyclin T2,the regulatory subunit of P-TEFb,which is required for transcription elongation by RNA Pol II(RNAP II) ,and that amino acid residues 1-420 in Cyclin T2 are important for binding with HSRG1.Fluorescence assays suggested that the cellular localizations of those two proteins are influenced by their interaction.Further analyses with CAT assays revealed that HSRG1 inhibits the transcriptional activation by Cyclin T2 of viral promoters.Our results suggested that the inhibitory effects of HSRG1 on viral replication and proliferation are probably induced by its binding to Cyclin T2.Therefore,it is likely that HSRG1 inhibits viral gene transcriptional elongation by interacting with Cyclin T2.
