Three crucial hurdles hinder studies on human cytomegalovirus(HCMV): strict species specificity, differences between in vivo and in vitro infection, and the complexity of gene regulation. Ever since the sequencing of ...Three crucial hurdles hinder studies on human cytomegalovirus(HCMV): strict species specificity, differences between in vivo and in vitro infection, and the complexity of gene regulation. Ever since the sequencing of the whole genome was first accomplished, functional studies on individual genes have been the mainstream in the CMV field. Gene regulation has therefore been elucidated in a more detailed fashion. However, viral gene regulation is largely controlled by both cellular and viral components. In other words, viral gene expression is determined by the virus–host interaction. Generally, cells respond to viral infection in a defensive pattern; at the same time, viruses try to counteract the cellular defense or else hide in the host(latency). Viruses evolve effective strategies against cellular defense in order to achieve replicative success. Whether or not they are successful, cellular defenses remain in the whole viral replication cycle: entry, immediate–early(IE) gene expression, early gene expression, DNA replication, late gene expression, and viral egress. Many viral strategies against cellular defense, and which occur in the immediate–early time of viral infection, have been documented. In this review, we will summarize the documented biological functions of IE1 and pp71 proteins, especially with regard to how they counteract cellular intrinsic defenses.展开更多
Objective： To investigate the effect of human cytomegalovirus （HCMV） IE1 protein on the secretory activity and apoptosis of macrophages. Methods： The eukaryotic expression vector pEGFP-C1/IE1 was used to transfect...Objective： To investigate the effect of human cytomegalovirus （HCMV） IE1 protein on the secretory activity and apoptosis of macrophages. Methods： The eukaryotic expression vector pEGFP-C1/IE1 was used to transfect THP-1-macrophages. 48 h after transfection, the expression and localization of GFP or GFP-IE1 was observed under fluorescent microscope. The levels of IL-1β and TNF-α in the culture media were examined by ELISA, and the mRNA expression of them was analyzed by RT-PCR. Cell undergoing apoptosis were determined by flow cytometry using the propidium iodide （PI） staining method. The data were analyzed by SPSSI3.0. Results： As observed under fluorescent microscope, the expressions of GFP-IEI and GFP by plasmid pEGFP-C1/IE1 or pEGFP-C1 in THP-1-macrophages could be found in nuclei or whole cells. Conclusion： As demonstrated by RT-PCR and ELISA, mRNA and protein expressions of IL-1β and TNF-α and promotes apoptosis in THP-1-macrophages.展开更多
In this paper, the function of the iel gene from baculovirus Spodoptera exigua multiple nucleopolyhedrovirus （SeMNPV）, belonging to group II nucleopolyhedrovirus, was studied in mammalian cells We amplified the SeMN...In this paper, the function of the iel gene from baculovirus Spodoptera exigua multiple nucleopolyhedrovirus （SeMNPV）, belonging to group II nucleopolyhedrovirus, was studied in mammalian cells We amplified the SeMNPV iel gene and expressed it by fusing to the C terminal of enhanced GFP protein in HEK 293 cells. Confocal microscopy revealed that the IE1-GFP fusion protein was localized in the nucleus of the mammalian cells. The promoter sequences of AcMNPV gp64, SeMNPV F protein and Drosophila hsp70 were also analyzed, to further study the function of SeMNPV IE1. The results showed that, in the absence of the hr sequence, IE1 improved the expression of the F promoter but didn＇t influence the gp64 promoter significantly, but IE1 moderately stimulated the hsp70 promoter.展开更多
目的探讨对虾白斑综合症病毒极早期基因IE1原核表达载体的构建与表达。方法将IE1编码序列克隆入pGEX-4T-2原核表达载体,并转染大肠埃希菌BL21,异丙基-β-D硫代半乳糖苷(IPTG)诱导谷胱甘肽硫转移酶(GST-IE1)融合蛋白表达,亲和层析法纯化...目的探讨对虾白斑综合症病毒极早期基因IE1原核表达载体的构建与表达。方法将IE1编码序列克隆入pGEX-4T-2原核表达载体,并转染大肠埃希菌BL21,异丙基-β-D硫代半乳糖苷(IPTG)诱导谷胱甘肽硫转移酶(GST-IE1)融合蛋白表达,亲和层析法纯化,并用聚丙烯酰胺变性凝胶电泳(SDS-PAGE)和蛋白印迹(western b lot)对融合蛋白进行鉴定。结果克隆到IE1基因序列长675 bp,编码224个氨基酸,理论分子量为25 kDa,等电点为4.87,所构建的重组体经PCR、双酶切及测序鉴定与目标基因相符,SDS-PAGE结果表明,目的基因在大肠埃希氏菌BL21中表达成功。结论克隆构建了对虾白斑综合症病毒极早期基因IE1,并在原核细胞中获得表达。展开更多
基金supported by a pilot grant from the Research Center for Minority Institutes (RCMI) program (2G12RR003050-24/8G12MD007579-27) (Q.T.)an American Cancer Society grant (RSG-090289-01MPC) (Q.T)+1 种基金NIH/NIAID SC1AI112785 (Q.T.)the Ponce Health Sciences University/RCMI Publications Office (G12 RR003050/8G12MD007579-27)
文摘Three crucial hurdles hinder studies on human cytomegalovirus(HCMV): strict species specificity, differences between in vivo and in vitro infection, and the complexity of gene regulation. Ever since the sequencing of the whole genome was first accomplished, functional studies on individual genes have been the mainstream in the CMV field. Gene regulation has therefore been elucidated in a more detailed fashion. However, viral gene regulation is largely controlled by both cellular and viral components. In other words, viral gene expression is determined by the virus–host interaction. Generally, cells respond to viral infection in a defensive pattern; at the same time, viruses try to counteract the cellular defense or else hide in the host(latency). Viruses evolve effective strategies against cellular defense in order to achieve replicative success. Whether or not they are successful, cellular defenses remain in the whole viral replication cycle: entry, immediate–early(IE) gene expression, early gene expression, DNA replication, late gene expression, and viral egress. Many viral strategies against cellular defense, and which occur in the immediate–early time of viral infection, have been documented. In this review, we will summarize the documented biological functions of IE1 and pp71 proteins, especially with regard to how they counteract cellular intrinsic defenses.
基金the Education Committee of Hunan Province(No.05C472)
文摘Objective： To investigate the effect of human cytomegalovirus （HCMV） IE1 protein on the secretory activity and apoptosis of macrophages. Methods： The eukaryotic expression vector pEGFP-C1/IE1 was used to transfect THP-1-macrophages. 48 h after transfection, the expression and localization of GFP or GFP-IE1 was observed under fluorescent microscope. The levels of IL-1β and TNF-α in the culture media were examined by ELISA, and the mRNA expression of them was analyzed by RT-PCR. Cell undergoing apoptosis were determined by flow cytometry using the propidium iodide （PI） staining method. The data were analyzed by SPSSI3.0. Results： As observed under fluorescent microscope, the expressions of GFP-IEI and GFP by plasmid pEGFP-C1/IE1 or pEGFP-C1 in THP-1-macrophages could be found in nuclei or whole cells. Conclusion： As demonstrated by RT-PCR and ELISA, mRNA and protein expressions of IL-1β and TNF-α and promotes apoptosis in THP-1-macrophages.
基金The knowledge innovation program of the Chinese Academy of Sciences (KSCX2-YW-Z-0938)
文摘In this paper, the function of the iel gene from baculovirus Spodoptera exigua multiple nucleopolyhedrovirus （SeMNPV）, belonging to group II nucleopolyhedrovirus, was studied in mammalian cells We amplified the SeMNPV iel gene and expressed it by fusing to the C terminal of enhanced GFP protein in HEK 293 cells. Confocal microscopy revealed that the IE1-GFP fusion protein was localized in the nucleus of the mammalian cells. The promoter sequences of AcMNPV gp64, SeMNPV F protein and Drosophila hsp70 were also analyzed, to further study the function of SeMNPV IE1. The results showed that, in the absence of the hr sequence, IE1 improved the expression of the F promoter but didn＇t influence the gp64 promoter significantly, but IE1 moderately stimulated the hsp70 promoter.
文摘目的探讨对虾白斑综合症病毒极早期基因IE1原核表达载体的构建与表达。方法将IE1编码序列克隆入pGEX-4T-2原核表达载体,并转染大肠埃希菌BL21,异丙基-β-D硫代半乳糖苷(IPTG)诱导谷胱甘肽硫转移酶(GST-IE1)融合蛋白表达,亲和层析法纯化,并用聚丙烯酰胺变性凝胶电泳(SDS-PAGE)和蛋白印迹(western b lot)对融合蛋白进行鉴定。结果克隆到IE1基因序列长675 bp,编码224个氨基酸,理论分子量为25 kDa,等电点为4.87,所构建的重组体经PCR、双酶切及测序鉴定与目标基因相符,SDS-PAGE结果表明,目的基因在大肠埃希氏菌BL21中表达成功。结论克隆构建了对虾白斑综合症病毒极早期基因IE1,并在原核细胞中获得表达。
