AIM To determine the variability/conservation of the domain of hepatitis B virus(HBV) pre S1 region that interacts with sodium-taurocholate cotransporting polypeptide(hereafter, NTCP-interacting domain) and the preval...AIM To determine the variability/conservation of the domain of hepatitis B virus(HBV) pre S1 region that interacts with sodium-taurocholate cotransporting polypeptide(hereafter, NTCP-interacting domain) and the prevalence of the rs2296651 polymorphism(S267 F, NTCP variant) in a Spanish population. METHODS Serum samples from 246 individuals were included and divided into 3 groups: patients with chronic HBV infection(CHB)(n = 41, 73% Caucasians), patients with resolved HBV infection(n = 100, 100% Caucasians) and an HBV-uninfected control group(n = 105, 100% Caucasians). Variability/conservation of the amino acid(aa) sequences of the NTCPinteracting domain,(aa 2-48 in viral genotype D) and a highly conserved pre S1 domain associated with virion morphogenesis(aa 92-103 in viral genotype D) were analyzed by next-generation sequencing and compared in 18 CHB patients with viremia > 4 log IU/mL. The rs2296651 polymorphism was determined in all individuals in all 3 groups using an in-house real-time PCR melting curve analysis.RESULTS The HBV pre S1 NTCP-interacting domain showed a high degree of conservation among the examined viral genomes especially between aa 9 and 21(in the genotype D consensus sequence). As compared with the virion morphogenesis domain, the NTCPinteracting domain had a smaller proportion of HBV genotype-unrelated changes comprising > 1% of the quasispecies(25.5% vs 31.8%), but a larger proportion of genotype-associated viral polymorphisms(34% vs 27.3%), according to consensus sequences from Gen Bank patterns of HBV genotypes A to H. Variation/conservation in both domains depended on viral genotype, with genotype C being the most highly conserved and genotype E the most variable(limited finding, only 2 genotype E included). Of note, proline residues were highly conserved in both domains, and serine residues showed changes only to threonine or tyrosine in the virion morphogenesis domain. The rs2296651 polymorphism was not detected in any participant.CONCLUSION In our CHB population, the NTCP-interactin展开更多
乙型肝炎病毒(Hepatitis B virus,HBV)是引起肝炎疾病的主要因素。HBV自身基因组极其简单,病毒复制生命过程都是在宿主因子协同作用下完成的。这些协同作用包括病毒包膜蛋白的加工对伴侣的依赖性、细胞因子对核衣壳的动力学修饰和转运...乙型肝炎病毒(Hepatitis B virus,HBV)是引起肝炎疾病的主要因素。HBV自身基因组极其简单,病毒复制生命过程都是在宿主因子协同作用下完成的。这些协同作用包括病毒包膜蛋白的加工对伴侣的依赖性、细胞因子对核衣壳的动力学修饰和转运、伴侣分子引发的反转录过程、宿主多泡体通路组分促进病毒粒子成熟与分泌以及X蛋白调控机制。本文综述了宿主因子对HBV以上几方面的影响最新研究进展,旨在为新型乙肝药物设计提供基础。展开更多
Hepatitis E virus (HEV), a fecal-orally transmitted foodborne viral pathogen,causes acute hepatitis in humans and is responsible for hepatitis E outbreaksworldwide. Since the identification of HEV as a zoonotic agent,...Hepatitis E virus (HEV), a fecal-orally transmitted foodborne viral pathogen,causes acute hepatitis in humans and is responsible for hepatitis E outbreaksworldwide. Since the identification of HEV as a zoonotic agent, this virus has beenisolated from a variety of hosts with an ever-expanding host range. HEV-openreading frame (ORF) 3, the smallest ORF in HEV genomes, initially had beenperceived as an unremarkable HEV accessory protein. However, as novel HEVORF3function has been discovered that is related to the existence of a putativethird virion structural form, referred to as “quasi-enveloped” HEV particles, HEVis challenging the conventional virion structure-based classification scheme,which assigns all viruses to two groups, “enveloped” or “non-enveloped”. In thisreview, we systematically describe recent progress that has identified multiplepathogenic roles of HEV-ORF3, including roles in HEV virion release, biogenesisof quasi-enveloped virus, regulation of the host innate immune response, andinterference with host signaling pathways. In addition, implications of HEVORF3-associated quasi-enveloped virions are discussed to guide futuredevelopment of improved vaccines against zoonotic HEV infection.展开更多
目的通过改变原噬菌体ms2包膜蛋白RNA包装位点（19碱基的茎环结构）的数量及亲和力，构建新的原核表达系统，探讨表达内含长片段（达到理论上的1900bp）RNA的耐RNase病毒样颗粒的可能性。方法首先设计含HindⅢ和NotⅠ酶切位点的引物，扩...目的通过改变原噬菌体ms2包膜蛋白RNA包装位点（19碱基的茎环结构）的数量及亲和力，构建新的原核表达系统，探讨表达内含长片段（达到理论上的1900bp）RNA的耐RNase病毒样颗粒的可能性。方法首先设计含HindⅢ和NotⅠ酶切位点的引物，扩增ms2包膜蛋白的编码成熟酶蛋白和衣壳蛋白的1700bp序列，并将原来的19mer的包装位点序列改变为C-5变异体（19 bp stem-loop结构中-5位的尿嘧啶改变为胞嘧啶），HindⅢ和NotⅠ酶切后，与用同样酶切的表达载体pET-28（b）相连接，得到重组载体pET-ms2-pac。应用重叠PCR扩增3种病毒的5段嵌合体序列（包括3段SARS-CoV基因、一段HCV基因和一段H5N1基因），并在SARS-CoV3和HCV序列之间插入一个19mer的变异体包装位点序列，在设计引物时，使嵌合体两端含有NotⅠ酶切位点，与NotⅠ酶切的重组载体pET-ms2-pac相连接，构建得到具有2个变异包装位点的表达载体pET-ms2-3V。同时构建3种对照重组表达载体，分别测定N-P3V-pET-P、N-P3V-pET-C、P-3V-pET-P和pET-ms2-3 V 4种重组表达质粒表达产物的260nm吸光度（A260）值，根据公式A260=0．125mg／ml计算4种表达产物的表达效率。结果成功构建了4种原核表达载体：pET-ms2-3V、P-3V-pET-P、N-P3V-pET-P和N-P3V-pET-C。pET-ms2-3V和P-3V-pET-P经原核表达后得到含全长为1891的5段嵌合体RNA的病毒样颗粒；N-P3V-pET-P、N-P3V-pET-C其原核表达产物病毒样颗粒中仅包装了1200bp的目的嵌合体RNA。N-P3V-pET-P、N-P3V-pET-C、P-3V-pET-P和pET-ms2-3V的表达效率分别为0．23、0．35、0．35和0．51mg／ml。N-P3V-pET-C比N-P3V-pET-P表达效率高52％，而pET-ms2-3V比P-3V-pET-P表达效率高38％。所包装的RNA具有耐RNase和DNase消化的特性以及良好的不同温度条件下的稳定性。结论通过改变噬菌体ms2 RNA包装位点（19碱基的茎环结构）的数量，可构建能表达内含达到理论上的约1900bp外源RNA�展开更多
Monkey B virus(Macacine herpesvirus 1; BV) is noted for its extreme neurovirulence in humans. Since the vhs protein encoded by the UL41 gene has been shown to be a neurovirulence factor in the related human herpes sim...Monkey B virus(Macacine herpesvirus 1; BV) is noted for its extreme neurovirulence in humans. Since the vhs protein encoded by the UL41 gene has been shown to be a neurovirulence factor in the related human herpes simplex viruses, the role of the UL41 gene in BV neurovirulence was investigated. BV mutants were constructed that lacked the entire UL41 ORF(Δ41) or had the RNase active site mutated(Δ41A). Neither mutant shut off host protein synthesis, degraded β-actin mRNA, or prevented an IFN-β response, indicating that the vhs protein and its RNase activity are both necessary for these activities. Replication of both mutants in primary mouse cells was impaired and they exhibited a prolonged disease course in mice. Whereas Δ41 infected mice were euthanized for symptoms related to central nervous system(CNS) infection, Δ41A infected mice were euthanized primarily for symptoms of autonomic nervous system dysfunction. While neuroinvasiveness was not affected, lesions in the CNS were more limited in size, anatomical distribution, and severity than for wild-type virus. These results indicate that the vhs protein affects the general replicative efficiency of BV in vivo rather than being a specific neurovirulence factor critical for invasion of or preferential replication in the CNS.展开更多
Viruses replicate and proliferate in host cells while continuously adjusting to and modulating the host environment.They encode a wide spectrum of multifunctional proteins,which interplay with and modify proteins in h...Viruses replicate and proliferate in host cells while continuously adjusting to and modulating the host environment.They encode a wide spectrum of multifunctional proteins,which interplay with and modify proteins in host cells.Viral genomes were chronologically the first to be sequenced.However,the corresponding viral proteomes,the alterations of host proteomes upon viral infection,and the dynamic nature of proteins,such as post-translational modifications,enzymatic cleavage,and activation or destruction by proteolysis,remain largely unknown.Emerging high-throughput techniques,in particular quantitative or semi-quantitative mass spectrometry-based proteomics analysis of viral and cellular proteomes,have been applied to define viruses and their interactions with their hosts.Here,we review the major areas of viral proteomics,including virion proteomics,structural proteomics,viral protein interactomics,and changes to the host cell proteome upon viral infection.展开更多
Dengue virus type 2 ThNH7/93 retained infectious activity after purification by ceramic hydroxyapatite chromatogra-phy. Dengue virus type 2 culture fluid was loaded onto the ceramic hydroxyapatite column and eluted wi...Dengue virus type 2 ThNH7/93 retained infectious activity after purification by ceramic hydroxyapatite chromatogra-phy. Dengue virus type 2 culture fluid was loaded onto the ceramic hydroxyapatite column and eluted with a linear gradient of sodium phosphate buffer. Culture fluid and protein contaminants derived from host cells were eluted initially, followed by elutions of dsDNA, and then dengue virus type 2. The recoveries of dengue virus type 2 were 64 ± 14% (n = 11) in the hemagglutination (HA) test and 60% (n = 2) determined by focus assay for viral infectivity. This protocol was highly reproducible, simple, rapid, and appears applicable to other virus species such as influenza virus, Japanese encephalitis virus and adenovirus.展开更多
AIM: To probe the organizational structure of the adsorption apparatus of bacteriophage epsilon 15(E15) using genetic and biochemical methodology METHODS: Hydroxylamine was used to create nonsense mutants of bacteriop...AIM: To probe the organizational structure of the adsorption apparatus of bacteriophage epsilon 15(E15) using genetic and biochemical methodology METHODS: Hydroxylamine was used to create nonsense mutants of bacteriophage E15. The mutants were then screened for defects in their adsorption apparatus proteins, initially by measuring the concentrations of free tail spike proteins in lysates of cells that had been infected by the phage mutants under nonpermissive growth conditions. Phage strains whose infected cell lysates contained above-average levels of free tail spike protein under non-permissive growth conditions were assumed to contain nonsense mutations in genes coding for adsorption apparatus proteins.These mutants were characterized by classical genetic mapping methods as well as automated sequencing of several of their genes. Finally, sodium dodecyl sulfatepolyacrylamide gel electrophoresis and autoradiography were used to examine the protein compositions of the radioactive particles produced when the various mutants were grown on a non-permissive host cell in the presence of 35S-methionine and co-purified along with E15 wt phage on Cs Cl block gradients.RESULTS: Our results are consistent with gp4 forming the portal ring structure of E15. In addition, they show that proteins gp15 and gp17 likely comprise the central tube portion of the E15 adsorption apparatus, with gp17 being more distally positioned than gp15 and dependent upon both gp15 and gp16 for its attachment. Finally, our data indicates that tail spike proteins comprised of gp20 can assemble onto nascent virions that contain gp7, gp10, gp4 and packaged DNA, but which lack both gp15 and gp17, thereby forming particles that are of sufficient stability to survive Cs Cl buoyant density centrifugation.CONCLUSION: The portal ring(gp4) of E15 is bound to tail spikes(gp20) and the tail tube(gp15 and gp17); gp17's attachment requires both gp15 and gp16.展开更多
It is suggested that the isolates of Oryctes Nudivirus (OrNV), cultured for decades in cells of Heteronychus arator (F.) (HA), be checked to verify genomic changes have not occurred which adapt them to culture but red...It is suggested that the isolates of Oryctes Nudivirus (OrNV), cultured for decades in cells of Heteronychus arator (F.) (HA), be checked to verify genomic changes have not occurred which adapt them to culture but reduce or cancel their ability to infect the target pest, the coconut rhinoceros beetle (CRB), Oryctes rhinoceros (L.). Full genomes of field-caught OrNV isolates, and their infectivity against larvae and adults, could be compared with those of HA-cultured isolates. Further data to correlate OrNV dosage indices with doses in number of virions/ml could be advantageous so as to explore if CRB larvae or adults may resist infection by a sub-threshold dose. Also the possibility of changes in the HA culture cells which alter the outer coat of the resulting virion, hence perhaps its infectivity towards CRB cells, could be checked. Might it be possible to move beyond HA-culture and develop tissue culture of Oryctes rhinoceros cells for mass production of OrNV as this beetle species is the target? Nuclear genomes of OrNV-resistant and OrNV-susceptible strains of the CRB could be examined for changes perhaps correlated with resistance. The possibility of endosymbiotic bacteria affecting CRB susceptibility to OrNV might be checked.展开更多
AIM To explore hepatitis C virus(HCV) adaptive mutations or combinations thereof responsible for enhanced viral production and investigate the underlying mechanisms.METHODS A series of plasmids with adaptive mutations...AIM To explore hepatitis C virus(HCV) adaptive mutations or combinations thereof responsible for enhanced viral production and investigate the underlying mechanisms.METHODS A series of plasmids with adaptive mutations were constructed. After the plasmids were transfected into Huh7.5 cells, we determined the infectious HCV particle titers by NS5 A immunofluorescence assays, and detected HCV RNA replication by real-time PCR and protein expression by Western blot. Then we carried out immunoblotting of supernatants and celllysates with anti-NS3 to analyze the virus release level. In addition, co-localization of lipid droplets(LDs) with NS5 A was measured using confocal laser scanning microscopy. The ratio between the p56 and p58 phosphoforms of NS5 A was analyzed further.RESULTS The plasmids named JFH1-m E2, JFH1-mp7, JFH1-m NS4 B, JFH1-m NS5 A, JFH1-m E2/NS5 A, JFH1-mp7/NS5 A, JFH1-m NS4 B/NS5 A, JFH1-m E2/p7/NS5 A, and m JFH1 were constructed successfully. This study generated infectious HCV particles with a robust titer of 1.61 × 106 focus-forming units(FFUs)/m L. All of the six adaptive mutations increased the HCV particle production at varying levels. The NS5 A(C2274 R, I2340 T, and V2440 L) and p7(H781 Y) were critical adaptive mutations. The effect of NS5 A(C2274 R, I2340 T, and V2440 L), p7(H781 Y), and NS4 B(N1931 S) on infectious HCV titers was investigated by measuring the HCV RNA replication, protein expression, and virion release. However, the six adaptive mutations were not required for the LD localization of NS5 A proteins or the phosphorylation of NS5 A.CONCLUSION In this study, we generated infectious HCV particles with a robust titer of 1.61 × 106 FFUs/m L, and found that the viral replication and release levels could be enhanced by some of the adaptive mutations.展开更多
基金Supported by Instituto de Salud Carlos Ⅲ,No.PI14/01416 and No.PI15/00856cofinanced by the European Regional Development Fund(ERDF)the Gilead Fellowship Program,No.GLD14-00296
文摘AIM To determine the variability/conservation of the domain of hepatitis B virus(HBV) pre S1 region that interacts with sodium-taurocholate cotransporting polypeptide(hereafter, NTCP-interacting domain) and the prevalence of the rs2296651 polymorphism(S267 F, NTCP variant) in a Spanish population. METHODS Serum samples from 246 individuals were included and divided into 3 groups: patients with chronic HBV infection(CHB)(n = 41, 73% Caucasians), patients with resolved HBV infection(n = 100, 100% Caucasians) and an HBV-uninfected control group(n = 105, 100% Caucasians). Variability/conservation of the amino acid(aa) sequences of the NTCPinteracting domain,(aa 2-48 in viral genotype D) and a highly conserved pre S1 domain associated with virion morphogenesis(aa 92-103 in viral genotype D) were analyzed by next-generation sequencing and compared in 18 CHB patients with viremia > 4 log IU/mL. The rs2296651 polymorphism was determined in all individuals in all 3 groups using an in-house real-time PCR melting curve analysis.RESULTS The HBV pre S1 NTCP-interacting domain showed a high degree of conservation among the examined viral genomes especially between aa 9 and 21(in the genotype D consensus sequence). As compared with the virion morphogenesis domain, the NTCPinteracting domain had a smaller proportion of HBV genotype-unrelated changes comprising > 1% of the quasispecies(25.5% vs 31.8%), but a larger proportion of genotype-associated viral polymorphisms(34% vs 27.3%), according to consensus sequences from Gen Bank patterns of HBV genotypes A to H. Variation/conservation in both domains depended on viral genotype, with genotype C being the most highly conserved and genotype E the most variable(limited finding, only 2 genotype E included). Of note, proline residues were highly conserved in both domains, and serine residues showed changes only to threonine or tyrosine in the virion morphogenesis domain. The rs2296651 polymorphism was not detected in any participant.CONCLUSION In our CHB population, the NTCP-interactin
文摘乙型肝炎病毒(Hepatitis B virus,HBV)是引起肝炎疾病的主要因素。HBV自身基因组极其简单,病毒复制生命过程都是在宿主因子协同作用下完成的。这些协同作用包括病毒包膜蛋白的加工对伴侣的依赖性、细胞因子对核衣壳的动力学修饰和转运、伴侣分子引发的反转录过程、宿主多泡体通路组分促进病毒粒子成熟与分泌以及X蛋白调控机制。本文综述了宿主因子对HBV以上几方面的影响最新研究进展,旨在为新型乙肝药物设计提供基础。
基金National Natural Science Foundation of China,No.31672534Key Project supported by Medical Science and Technology Development Foundation of Nanjing Department of Health,No.ZKX19026.
文摘Hepatitis E virus (HEV), a fecal-orally transmitted foodborne viral pathogen,causes acute hepatitis in humans and is responsible for hepatitis E outbreaksworldwide. Since the identification of HEV as a zoonotic agent, this virus has beenisolated from a variety of hosts with an ever-expanding host range. HEV-openreading frame (ORF) 3, the smallest ORF in HEV genomes, initially had beenperceived as an unremarkable HEV accessory protein. However, as novel HEVORF3function has been discovered that is related to the existence of a putativethird virion structural form, referred to as “quasi-enveloped” HEV particles, HEVis challenging the conventional virion structure-based classification scheme,which assigns all viruses to two groups, “enveloped” or “non-enveloped”. In thisreview, we systematically describe recent progress that has identified multiplepathogenic roles of HEV-ORF3, including roles in HEV virion release, biogenesisof quasi-enveloped virus, regulation of the host innate immune response, andinterference with host signaling pathways. In addition, implications of HEVORF3-associated quasi-enveloped virions are discussed to guide futuredevelopment of improved vaccines against zoonotic HEV infection.
文摘目的通过改变原噬菌体ms2包膜蛋白RNA包装位点（19碱基的茎环结构）的数量及亲和力，构建新的原核表达系统，探讨表达内含长片段（达到理论上的1900bp）RNA的耐RNase病毒样颗粒的可能性。方法首先设计含HindⅢ和NotⅠ酶切位点的引物，扩增ms2包膜蛋白的编码成熟酶蛋白和衣壳蛋白的1700bp序列，并将原来的19mer的包装位点序列改变为C-5变异体（19 bp stem-loop结构中-5位的尿嘧啶改变为胞嘧啶），HindⅢ和NotⅠ酶切后，与用同样酶切的表达载体pET-28（b）相连接，得到重组载体pET-ms2-pac。应用重叠PCR扩增3种病毒的5段嵌合体序列（包括3段SARS-CoV基因、一段HCV基因和一段H5N1基因），并在SARS-CoV3和HCV序列之间插入一个19mer的变异体包装位点序列，在设计引物时，使嵌合体两端含有NotⅠ酶切位点，与NotⅠ酶切的重组载体pET-ms2-pac相连接，构建得到具有2个变异包装位点的表达载体pET-ms2-3V。同时构建3种对照重组表达载体，分别测定N-P3V-pET-P、N-P3V-pET-C、P-3V-pET-P和pET-ms2-3 V 4种重组表达质粒表达产物的260nm吸光度（A260）值，根据公式A260=0．125mg／ml计算4种表达产物的表达效率。结果成功构建了4种原核表达载体：pET-ms2-3V、P-3V-pET-P、N-P3V-pET-P和N-P3V-pET-C。pET-ms2-3V和P-3V-pET-P经原核表达后得到含全长为1891的5段嵌合体RNA的病毒样颗粒；N-P3V-pET-P、N-P3V-pET-C其原核表达产物病毒样颗粒中仅包装了1200bp的目的嵌合体RNA。N-P3V-pET-P、N-P3V-pET-C、P-3V-pET-P和pET-ms2-3V的表达效率分别为0．23、0．35、0．35和0．51mg／ml。N-P3V-pET-C比N-P3V-pET-P表达效率高52％，而pET-ms2-3V比P-3V-pET-P表达效率高38％。所包装的RNA具有耐RNase和DNase消化的特性以及良好的不同温度条件下的稳定性。结论通过改变噬菌体ms2 RNA包装位点（19碱基的茎环结构）的数量，可构建能表达内含达到理论上的约1900bp外源RNA�
基金supported in part by PHS grants 2P40 OD010988 and 1P40 OD010431
文摘Monkey B virus(Macacine herpesvirus 1; BV) is noted for its extreme neurovirulence in humans. Since the vhs protein encoded by the UL41 gene has been shown to be a neurovirulence factor in the related human herpes simplex viruses, the role of the UL41 gene in BV neurovirulence was investigated. BV mutants were constructed that lacked the entire UL41 ORF(Δ41) or had the RNase active site mutated(Δ41A). Neither mutant shut off host protein synthesis, degraded β-actin mRNA, or prevented an IFN-β response, indicating that the vhs protein and its RNase activity are both necessary for these activities. Replication of both mutants in primary mouse cells was impaired and they exhibited a prolonged disease course in mice. Whereas Δ41 infected mice were euthanized for symptoms related to central nervous system(CNS) infection, Δ41A infected mice were euthanized primarily for symptoms of autonomic nervous system dysfunction. While neuroinvasiveness was not affected, lesions in the CNS were more limited in size, anatomical distribution, and severity than for wild-type virus. These results indicate that the vhs protein affects the general replicative efficiency of BV in vivo rather than being a specific neurovirulence factor critical for invasion of or preferential replication in the CNS.
基金supported by the National Project on Major Infectious Diseases Prevention (Grant No. 2008ZX10002-009)the National Basic Research Program of China (Grant No. 2011CB910703)
文摘Viruses replicate and proliferate in host cells while continuously adjusting to and modulating the host environment.They encode a wide spectrum of multifunctional proteins,which interplay with and modify proteins in host cells.Viral genomes were chronologically the first to be sequenced.However,the corresponding viral proteomes,the alterations of host proteomes upon viral infection,and the dynamic nature of proteins,such as post-translational modifications,enzymatic cleavage,and activation or destruction by proteolysis,remain largely unknown.Emerging high-throughput techniques,in particular quantitative or semi-quantitative mass spectrometry-based proteomics analysis of viral and cellular proteomes,have been applied to define viruses and their interactions with their hosts.Here,we review the major areas of viral proteomics,including virion proteomics,structural proteomics,viral protein interactomics,and changes to the host cell proteome upon viral infection.
文摘Dengue virus type 2 ThNH7/93 retained infectious activity after purification by ceramic hydroxyapatite chromatogra-phy. Dengue virus type 2 culture fluid was loaded onto the ceramic hydroxyapatite column and eluted with a linear gradient of sodium phosphate buffer. Culture fluid and protein contaminants derived from host cells were eluted initially, followed by elutions of dsDNA, and then dengue virus type 2. The recoveries of dengue virus type 2 were 64 ± 14% (n = 11) in the hemagglutination (HA) test and 60% (n = 2) determined by focus assay for viral infectivity. This protocol was highly reproducible, simple, rapid, and appears applicable to other virus species such as influenza virus, Japanese encephalitis virus and adenovirus.
基金Supported by The NIH-AREA Grant,No.1R15GM52696-01the NSF-RUI Grant,No.DMB-8608480+2 种基金the Howard Hughes Medical Research Institute(two grants to the PLNU Biology Department)Research Associates of PLNU(a 300 member alumni support group)the PLNU Administration
文摘AIM: To probe the organizational structure of the adsorption apparatus of bacteriophage epsilon 15(E15) using genetic and biochemical methodology METHODS: Hydroxylamine was used to create nonsense mutants of bacteriophage E15. The mutants were then screened for defects in their adsorption apparatus proteins, initially by measuring the concentrations of free tail spike proteins in lysates of cells that had been infected by the phage mutants under nonpermissive growth conditions. Phage strains whose infected cell lysates contained above-average levels of free tail spike protein under non-permissive growth conditions were assumed to contain nonsense mutations in genes coding for adsorption apparatus proteins.These mutants were characterized by classical genetic mapping methods as well as automated sequencing of several of their genes. Finally, sodium dodecyl sulfatepolyacrylamide gel electrophoresis and autoradiography were used to examine the protein compositions of the radioactive particles produced when the various mutants were grown on a non-permissive host cell in the presence of 35S-methionine and co-purified along with E15 wt phage on Cs Cl block gradients.RESULTS: Our results are consistent with gp4 forming the portal ring structure of E15. In addition, they show that proteins gp15 and gp17 likely comprise the central tube portion of the E15 adsorption apparatus, with gp17 being more distally positioned than gp15 and dependent upon both gp15 and gp16 for its attachment. Finally, our data indicates that tail spike proteins comprised of gp20 can assemble onto nascent virions that contain gp7, gp10, gp4 and packaged DNA, but which lack both gp15 and gp17, thereby forming particles that are of sufficient stability to survive Cs Cl buoyant density centrifugation.CONCLUSION: The portal ring(gp4) of E15 is bound to tail spikes(gp20) and the tail tube(gp15 and gp17); gp17's attachment requires both gp15 and gp16.
文摘It is suggested that the isolates of Oryctes Nudivirus (OrNV), cultured for decades in cells of Heteronychus arator (F.) (HA), be checked to verify genomic changes have not occurred which adapt them to culture but reduce or cancel their ability to infect the target pest, the coconut rhinoceros beetle (CRB), Oryctes rhinoceros (L.). Full genomes of field-caught OrNV isolates, and their infectivity against larvae and adults, could be compared with those of HA-cultured isolates. Further data to correlate OrNV dosage indices with doses in number of virions/ml could be advantageous so as to explore if CRB larvae or adults may resist infection by a sub-threshold dose. Also the possibility of changes in the HA culture cells which alter the outer coat of the resulting virion, hence perhaps its infectivity towards CRB cells, could be checked. Might it be possible to move beyond HA-culture and develop tissue culture of Oryctes rhinoceros cells for mass production of OrNV as this beetle species is the target? Nuclear genomes of OrNV-resistant and OrNV-susceptible strains of the CRB could be examined for changes perhaps correlated with resistance. The possibility of endosymbiotic bacteria affecting CRB susceptibility to OrNV might be checked.
基金Beijing Natural Science Foundation,No.7161006Beijing Municipal Administration of Hospitals’ Youth Program,No.QML20161801 and No.QML20171801
文摘AIM To explore hepatitis C virus(HCV) adaptive mutations or combinations thereof responsible for enhanced viral production and investigate the underlying mechanisms.METHODS A series of plasmids with adaptive mutations were constructed. After the plasmids were transfected into Huh7.5 cells, we determined the infectious HCV particle titers by NS5 A immunofluorescence assays, and detected HCV RNA replication by real-time PCR and protein expression by Western blot. Then we carried out immunoblotting of supernatants and celllysates with anti-NS3 to analyze the virus release level. In addition, co-localization of lipid droplets(LDs) with NS5 A was measured using confocal laser scanning microscopy. The ratio between the p56 and p58 phosphoforms of NS5 A was analyzed further.RESULTS The plasmids named JFH1-m E2, JFH1-mp7, JFH1-m NS4 B, JFH1-m NS5 A, JFH1-m E2/NS5 A, JFH1-mp7/NS5 A, JFH1-m NS4 B/NS5 A, JFH1-m E2/p7/NS5 A, and m JFH1 were constructed successfully. This study generated infectious HCV particles with a robust titer of 1.61 × 106 focus-forming units(FFUs)/m L. All of the six adaptive mutations increased the HCV particle production at varying levels. The NS5 A(C2274 R, I2340 T, and V2440 L) and p7(H781 Y) were critical adaptive mutations. The effect of NS5 A(C2274 R, I2340 T, and V2440 L), p7(H781 Y), and NS4 B(N1931 S) on infectious HCV titers was investigated by measuring the HCV RNA replication, protein expression, and virion release. However, the six adaptive mutations were not required for the LD localization of NS5 A proteins or the phosphorylation of NS5 A.CONCLUSION In this study, we generated infectious HCV particles with a robust titer of 1.61 × 106 FFUs/m L, and found that the viral replication and release levels could be enhanced by some of the adaptive mutations.
