不同马传染性贫血病毒株编码的S2蛋白拮抗SERINC5分子机理的研究

Molecular mechanism for SERINC5 antagonism by different strain of EIAV S2

为研究不同马传染性贫血病毒(EIAV)编码的S2蛋白拮抗宿主限制因子丝氨酸整合蛋白5(Ser5)的机制,本研究利用HIV-1野生型(WT)和HIV-1ΔNef前病毒质粒包装两种HIV-1假病毒,通过测定细胞上清中病毒粒子含量和靶细胞中荧光素酶活性检测病毒相对感染性,分析pSPEIAV19编码的S2蛋白(S2)和中国EIAV弱毒疫苗编码的S2蛋白(HRB-S2)对Ser5抗病毒活性的影响,结果显示S2和HRB-S2蛋白均能拮抗宿主Ser5的抗病毒活性,且S2拮抗Ser5的能力比HRB-S2强。将表达Ser5和野生型HRB-S2及其突变体的质粒共转染293T细胞,检测HRB-S2对Ser5表达的影响,结果显示HRB-S2能显著降解Ser5蛋白,并且HRB-S2降解Ser5的关键氨基酸位点是L^(26)。将表达Ser5和野生型HRB-S2及其突变体的质粒共转染Hela细胞,通过激光共聚焦试验检测HRB-S2对细胞内Ser5定位的影响,结果显示HRB-S2将位于细胞膜表面的Ser5表达下调并内化至细胞浆内,而L^(26)突变后HRB-S2下调Ser5的能力降低。将表达Ser5、HRB-S2、AP-2、Rab7和Rab11的质粒共转染293T细胞,检测AP-2、Rab7和Rab11在HRB-S2降解Ser5中的作用,结果发现三者在HRB-S2降解Ser5中均发挥重要的作用。将与VC或者VN融合表达的Ser5、HRB-S2、S2的质粒共转染Hela细胞,检测三者在细胞内的共定位情况,结果发现HRB-S2存在二聚体,而S2则不会形成二聚体。将表达Ser5、HRB-S2、S2的质粒共转染293T细胞,转染24 h后加入不同降解通路的抑制剂,检测S2降解Ser5的不同通路,结果显示HRB-S2可以通过蛋白酶体通路和溶酶体通路降解Ser5;而S2蛋白只通过溶酶体途径降解Ser5。本研究结果首次表明不同EIAV编码的S2蛋白均能通过降解Ser5蛋白而拮抗其抗病毒活性,其中S2拮抗Ser5的能力比HRB-S2强,HRB-S2蛋白的二聚体化可能会影响其对Ser5的敏感性。本研究阐明了不同EIAV S2蛋白拮抗Ser5的差异及机制,为抗逆转录病毒如HIV-1抗病毒制剂的开发提供参考依据。

To evaluate the difference and mechanism of the S2 protein from different equine infectious anemia virus(EIAV) to antagonize host restriction factor Serine incorporator 5(Ser5),HIV-1 WT and ΔNef proviral plasmids were used to produce two kinds of HIV-1 pseudoviruses.Viral production in supernatant and luciferase activities in target cells were detected and then relative infectivity were calculated.The influence of S2 protein encoded by pSPEIAV19(S2) and attenuated Chinese EIAV strain(HRB-S2) on antiviral activity of Ser5 were determined according to the relative infectivity.The results showed that S2 antagonizes Ser5 more strong than HRB-S2.The plasmids of Ser5 and wide type HRB-S2 or its mutants were co-transfected in 293 T cells to detect the effect of HRB-S2 on Ser5 expression,the results showed that Ser5 expression was effectively degraded by HRB-S2,and L^(26) residue was required for HRB-S2 degradation of Ser5.The plasmids of Ser5 and wide type HRB-S2 or its mutants were cotransfected in Hela cells to determine Ser5 subcellular localization by confocal microscopy,the results showed that HRB-S2 downregulate Ser5 from the plasma membrane to perinuclear compartments but L^(26) mutant did poorly.The plasmids of Ser5,HRB-S2,AP-2,Rab7 and Rab11 were co-transfected in 293 T cells to detect the effect of HRB-S2 of Ser5 degradation,the results showed that AP-2,Rab7 and Rab11 were required for Ser5 down-regulation by HRB-S2.The C terminus of Ser5,HRB-S2 and S2 were fused to VC or VN and then were co-transfected in Hela cells to detect the co-localization,the results showed that HRB-S2 was forming dimer but S2 was not.The plasmids of Ser5,HRB-S2 and S2 were co-transfected in 293 T cells for 24 h,and then the inhibitor of different degradation pathways were added to detect the degradation pathway of S2 to degrade Ser5.The results at first time showed that HRB-S2 targeted Ser5 to lysosomes and proteasome for degradation,but S2 only targeted Ser5 to lysosomes.Although both EIAV S2 can antagonize Ser5 by down-regulating its expression,S2 was more strong than HRB-S2.However,dimerization of HRB-S2 may be affect its sensitivity to Ser5.These mechanistic understandings might provide a scientific basis for designing antiviral strategies of retroviral such as HIV-1.