人3型副流感病毒(Human Parainfluenza Virus type 3,HPIV-3)是引起婴幼儿严重细支气管炎及肺炎等下呼吸道疾病的主要病原体,其在发达国家和发展中国家都造成了沉重的疾病负担。迄今,对HPIV-3感染的预防和治疗都还没有有效的疫苗和药物...人3型副流感病毒(Human Parainfluenza Virus type 3,HPIV-3)是引起婴幼儿严重细支气管炎及肺炎等下呼吸道疾病的主要病原体,其在发达国家和发展中国家都造成了沉重的疾病负担。迄今,对HPIV-3感染的预防和治疗都还没有有效的疫苗和药物,因此WHO将HPIV-3疫苗列为未来重点研发的疫苗。近年来,随着重组技术和反向遗传学的发展,HPIV-3疫苗的研制取得了重要进展,部分疫苗已进入临床评价阶段。就HPIV-3的生物学特性如病毒结构特征、复制过程、流行病学特征,以及近年来传统冷适应减毒活疫苗、亚单位疫苗、以反向遗传学为基础的新型减毒活疫苗的研制成果及临床试验进展作简要综述。展开更多
为确定人副流感病毒3型(Human parainfluenza virus type 3,hPIV3)病毒包膜表面血凝素神经氨酸酶(Hemagglutinin-neuraminidase,HN)糖蛋白茎部区十一肽重复序列中保守氨基酸中具有关键性作用的位点,进一步探讨HN蛋白茎部区在融合机制中...为确定人副流感病毒3型(Human parainfluenza virus type 3,hPIV3)病毒包膜表面血凝素神经氨酸酶(Hemagglutinin-neuraminidase,HN)糖蛋白茎部区十一肽重复序列中保守氨基酸中具有关键性作用的位点,进一步探讨HN蛋白茎部区在融合机制中的重要作用。结合定点突变和同源重组技术将HN蛋白茎部区十一肽重复序列中5个保守氨基酸位点(I102、P111、L114、S119、I125)突变为丙氨酸(Alanine,A),通过痘苗病毒-T7聚合酶系统在BHK-21细胞中表达突变蛋白,定性定量检测各突变体蛋白的促细胞融合活性、受体结合活性、神经氨酸酶活性和半融合活性。突变体蛋白I102A、P111A、L114A、S119A、I125A的促细胞融合活性均有不同程度下降,依次为野生型的6%、16%、14%、87%和4%,除S119A外其余4个突变型与野生型相比差别均具有统计学意义(P<0.01);突变体蛋白I102A、P111A、L114A、S119A、I125A的受体结合活性也出现不同程度下降,依次分别为野生型的32.2%、77.4%、74.2%、83.9%和38.7%,其中I102A和I125A的受体结合活性与野生型相比差别具有统计学意义(P<0.01);突变体蛋白I102A、P111A、L114A、S119A、I125A的神经氨酸酶活性分别为野生型的66.5%、73.1%、69.1%、76.1%和72.8%,与野生型相比差别无统计学意义(P>0.05)。结果表明:茎部区十一肽重复序列对hPIV3HN蛋白的促细胞融合活性和受体结合活性具有重要意义。该区域氨基酸I102、P111、L114、I125具有关键作用,推测其能通过影响头部区受体结合活性或是与融合蛋白的相互作用等不同方式导致HN蛋白结构功能发生改变。展开更多
Human parainfluenza virus type 3(HPIV3), a member of the Paramyxoviridae family, can cause lower respiratory disease in infants and young children. The phosphoprotein(P) of HPIV3 is an essential cofactor of the viral ...Human parainfluenza virus type 3(HPIV3), a member of the Paramyxoviridae family, can cause lower respiratory disease in infants and young children. The phosphoprotein(P) of HPIV3 is an essential cofactor of the viral RNA-dependent RNA polymerase large protein(L). P connects nucleocapsid protein(N) with L to initiate genome transcription and replication.Sumoylation influences many important pathways of the target proteins, and many viral proteins are also themselves sumoylated. In this study, we found that the P of HPIV3 could be sumoylated, and mutation of K492 and K532 to arginine(PK492 R/K532 R) failed to be sumoylated within P, which enhances HPIV3 minigenome activity. Biochemical studies showed that PK492 R/K532 Rhad no effect on its interactions with N, formation of homo-tetramers and formation of inclusion bodies.Finally, we found that incorporation of K492 R/K532 R into a recombinant HPIV3(rHPIV3-PK492 R/K532 R) increased viral production in culture cells, suggesting that sumoylation attenuates functions of P and down-regulates viral replication.展开更多
文摘人3型副流感病毒(Human Parainfluenza Virus type 3,HPIV-3)是引起婴幼儿严重细支气管炎及肺炎等下呼吸道疾病的主要病原体,其在发达国家和发展中国家都造成了沉重的疾病负担。迄今,对HPIV-3感染的预防和治疗都还没有有效的疫苗和药物,因此WHO将HPIV-3疫苗列为未来重点研发的疫苗。近年来,随着重组技术和反向遗传学的发展,HPIV-3疫苗的研制取得了重要进展,部分疫苗已进入临床评价阶段。就HPIV-3的生物学特性如病毒结构特征、复制过程、流行病学特征,以及近年来传统冷适应减毒活疫苗、亚单位疫苗、以反向遗传学为基础的新型减毒活疫苗的研制成果及临床试验进展作简要综述。
文摘为确定人副流感病毒3型(Human parainfluenza virus type 3,hPIV3)病毒包膜表面血凝素神经氨酸酶(Hemagglutinin-neuraminidase,HN)糖蛋白茎部区十一肽重复序列中保守氨基酸中具有关键性作用的位点,进一步探讨HN蛋白茎部区在融合机制中的重要作用。结合定点突变和同源重组技术将HN蛋白茎部区十一肽重复序列中5个保守氨基酸位点(I102、P111、L114、S119、I125)突变为丙氨酸(Alanine,A),通过痘苗病毒-T7聚合酶系统在BHK-21细胞中表达突变蛋白,定性定量检测各突变体蛋白的促细胞融合活性、受体结合活性、神经氨酸酶活性和半融合活性。突变体蛋白I102A、P111A、L114A、S119A、I125A的促细胞融合活性均有不同程度下降,依次为野生型的6%、16%、14%、87%和4%,除S119A外其余4个突变型与野生型相比差别均具有统计学意义(P<0.01);突变体蛋白I102A、P111A、L114A、S119A、I125A的受体结合活性也出现不同程度下降,依次分别为野生型的32.2%、77.4%、74.2%、83.9%和38.7%,其中I102A和I125A的受体结合活性与野生型相比差别具有统计学意义(P<0.01);突变体蛋白I102A、P111A、L114A、S119A、I125A的神经氨酸酶活性分别为野生型的66.5%、73.1%、69.1%、76.1%和72.8%,与野生型相比差别无统计学意义(P>0.05)。结果表明:茎部区十一肽重复序列对hPIV3HN蛋白的促细胞融合活性和受体结合活性具有重要意义。该区域氨基酸I102、P111、L114、I125具有关键作用,推测其能通过影响头部区受体结合活性或是与融合蛋白的相互作用等不同方式导致HN蛋白结构功能发生改变。
基金supported by grants from National Key R&D Program of China (2017YFA0505801)the National Natural Science Foundation of China (81825015, 81871650 and 31630086)+2 种基金National Science and Technology Major Project (2018ZX10101004)the Natural Science Foundation of Hubei Province Innovation Group (2017CFA022)Advanced Customer Cultivation Project of Wuhan National Biosafety Laboratory (2019ACCP-MS06)。
文摘Human parainfluenza virus type 3(HPIV3), a member of the Paramyxoviridae family, can cause lower respiratory disease in infants and young children. The phosphoprotein(P) of HPIV3 is an essential cofactor of the viral RNA-dependent RNA polymerase large protein(L). P connects nucleocapsid protein(N) with L to initiate genome transcription and replication.Sumoylation influences many important pathways of the target proteins, and many viral proteins are also themselves sumoylated. In this study, we found that the P of HPIV3 could be sumoylated, and mutation of K492 and K532 to arginine(PK492 R/K532 R) failed to be sumoylated within P, which enhances HPIV3 minigenome activity. Biochemical studies showed that PK492 R/K532 Rhad no effect on its interactions with N, formation of homo-tetramers and formation of inclusion bodies.Finally, we found that incorporation of K492 R/K532 R into a recombinant HPIV3(rHPIV3-PK492 R/K532 R) increased viral production in culture cells, suggesting that sumoylation attenuates functions of P and down-regulates viral replication.
