AP2M1在狂犬病病毒侵入中的作用研究
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摘要
本研究前期利用RNA干扰技术在人类全基因组水平上筛选到了与狂犬病病毒(RABV)生命周期有关的宿主基因AP2M1。为进一步研究AP2M1影响RABV感染的机制,本实验在人胚胎肾细胞(HEK-293)中沉默或过表达AP2M1,采用高内涵筛选、病毒滴度检测等方法分析AP2M1对RABV感染率的影响。结果显示:敲低AP2M1,RABV感染率显著下降;过表达AP2M1,RABV感染率上升。酸绕过实验显示AP2M1在RABV感染过程中发挥作用。激光共聚焦观察显示RABV G蛋白与AP2M1无共定位。免疫共沉淀实验显示AP2M1与RABV G蛋白无直接相互作用。本研究结果表明,AP2M1基因在RABV感染过程中发挥作用,但不是通过与RABV G蛋白的直接作用影响病毒的感染。本研究为阐明AP2M1在RABV生命周期中具体作用奠定了基础。
We have screened host genes related to the rabies virus(RABV) life cycle in the human genome using RNA interference(RNAi), and demonstrated that the RABV infection rate was decreased when AP2 M1 gene expression was knockdown. To explore the mechanism how AP2 M1 affects RABV infection, the infection rate of rabies virus in HEK-293 cells were analyzed by high-content system and virus titer detection when AP2 M1 gene expression was knocked-down or over-expressed. We proved that AP2 M1 gene played a role in the early stage of RABV infection by acid bypass experiments. The interaction between AP2 M1 and RABV G protein was investigated by co-immunoprecipitation assay and laser scanning confocal microscopy examination. The results showed that the RABV infection rate was decreased when AP2 M1 gene expression was knockdown. We found that AP2 M1 protein was not co-located with G protein by co-immunoprecipitation assays, which illustrated that there was no interaction between G protein and AP2 M1 protein. This study provides a basis for the specific role of AP2 M1 in the RABV life cycle.
We have screened host genes related to the rabies virus(RABV) life cycle in the human genome using RNA interference(RNAi), and demonstrated that the RABV infection rate was decreased when AP2 M1 gene expression was knockdown. To explore the mechanism how AP2 M1 affects RABV infection, the infection rate of rabies virus in HEK-293 cells were analyzed by high-content system and virus titer detection when AP2 M1 gene expression was knocked-down or over-expressed. We proved that AP2 M1 gene played a role in the early stage of RABV infection by acid bypass experiments. The interaction between AP2 M1 and RABV G protein was investigated by co-immunoprecipitation assay and laser scanning confocal microscopy examination. The results showed that the RABV infection rate was decreased when AP2 M1 gene expression was knockdown. We found that AP2 M1 protein was not co-located with G protein by co-immunoprecipitation assays, which illustrated that there was no interaction between G protein and AP2 M1 protein. This study provides a basis for the specific role of AP2 M1 in the RABV life cycle.
引文
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[11]Suja M S,Mahadevan A,Madhusudana S N,et al.Role of apoptosis in rabies viral encephalitis:A comparative study in mice,canine,and human brain with a review of literature[J].Patholog Res Int,2011,2011:374286.
[12]Marston D A,Mcelhinney L M,Banyard A C,et al.Interspecies protein substitution to investigate the role of the lyssavirus glycoprotein[J].J Gen Virol,2013,94(Pt 2):284-292.
[13]Phan T,Beran R K,Peters C,et al.Hepatitis C virus NS2 protein contributes to virus particle assembly via opposing epistatic interactions with the E1-E2 glycoprotein and NS3-NS4A enzyme complexes[J].J Virol,2009,83(17):8379-8395.
[14]Piccinotti S,Kirchhausen T,Whelan S P J.Uptake of rabies virus into epithelial cells by clathrin-mediated endocytosis depends upon actin[J].J Virol,2013,87(21):11637-11645.
[15]Cureton D K,Massol R H,Saffaricn S,et al.Vesicular stomatitis virus enters cells through vesicles incompletely coated with clathrin that depend upon actin for internalization[J].PLoSPathog,2009,5(4):e1000394.
[16]Velandiaromero M L,Castellanos J E,Martínezgutiérrez M.In vivo differential susceptibility of sensory neurons to rabies virus infection[J].J Neurovirol,2013,19(4):367-375.
[17]Yamaoka S,Ito N,Ohka S,et al.Involvement of the rabies virus phosphoprotein gene in neuroinvasiveness[J].J Virol,2013,87(22):12327-12338.
[2]Rupprecht C,Kuzmin I,Meslin F.Lyssaviruses and rabies:Current conundrums,concerns,contradictions and controversies[J].F1000 Res,2017,6(184):184.
[3]尹伟,徐洁萍,张金阳,等.狂犬病病毒核蛋白在Bac-ToBac/AcMNPV杆状病毒系统的表达[J].中国预防兽医学报,2010,32(2):86-89.
[4]Basu R,Munteanu E L,Chang F.Role of turgor pressure in endocytosis in fission yeast[J].Mol Biol Cell,2014,25(5):679.
[5]Kadlecova Z,Spielman S J,Loerke D,et al.Regulation of clathrin-mediated endocytosis by hierarchical allostenc activation of AP2[J].J Cell Biol,2017,216(1):167-179.
[6]Beier K T,Saunders A B,Oldenburg I A,et al.Vesicular stomatitis virus with the rabies virus glycoprotein directs retrograde transsynaptic transport among neurons in vivo[J].Front Neural Circuits,2013,7(7):11.
[7]Davis A D,Gordy P A,Bowen R A.Unique characteristics of bat rabies viruses in big brown bats(Eptesicus fuscus)[J].Arch Virol,2013,158(4):809-820.
[8]Motley A,Bright N A,Seaman M N,et al.Clathrin-mediated endocytosis in AP-2-depleted cells[J].J Cell Biol,2003,162(5):909-918.
[9]Stapleford K A,Lindenbach B D.Hepatitis C virus ns2 coordinates virus particle assembly through physical interactions with the E1-E2 glycoprotein and NS3-NS4A enzyme complexes[J].JVirol,2011,85(4):1706-1717.
[10]朱士茂,李慧,王春华,等.狂犬病病毒及其与宿主之间的相互作用[J].中国预防兽医学报,2015,37(6):486-490.
[11]Suja M S,Mahadevan A,Madhusudana S N,et al.Role of apoptosis in rabies viral encephalitis:A comparative study in mice,canine,and human brain with a review of literature[J].Patholog Res Int,2011,2011:374286.
[12]Marston D A,Mcelhinney L M,Banyard A C,et al.Interspecies protein substitution to investigate the role of the lyssavirus glycoprotein[J].J Gen Virol,2013,94(Pt 2):284-292.
[13]Phan T,Beran R K,Peters C,et al.Hepatitis C virus NS2 protein contributes to virus particle assembly via opposing epistatic interactions with the E1-E2 glycoprotein and NS3-NS4A enzyme complexes[J].J Virol,2009,83(17):8379-8395.
[14]Piccinotti S,Kirchhausen T,Whelan S P J.Uptake of rabies virus into epithelial cells by clathrin-mediated endocytosis depends upon actin[J].J Virol,2013,87(21):11637-11645.
[15]Cureton D K,Massol R H,Saffaricn S,et al.Vesicular stomatitis virus enters cells through vesicles incompletely coated with clathrin that depend upon actin for internalization[J].PLoSPathog,2009,5(4):e1000394.
[16]Velandiaromero M L,Castellanos J E,Martínezgutiérrez M.In vivo differential susceptibility of sensory neurons to rabies virus infection[J].J Neurovirol,2013,19(4):367-375.
[17]Yamaoka S,Ito N,Ohka S,et al.Involvement of the rabies virus phosphoprotein gene in neuroinvasiveness[J].J Virol,2013,87(22):12327-12338.