犬Serinc5对HIV-1复制能力影响的研究

摘要

丝氨酸整合因子5 (Serinc5)是最新报道能够被携带到病毒粒子中的宿主限制性因子,具有抵抗人类免疫缺陷病毒Ⅰ型(HIV-1)感染性的作用.与人Serinc5 (hSerinc5)具有高度同源性的犬Serinc5存在两种形式,一种为全长基因组编码的犬Serinc5 (cSerinc5),另一种为转录剪接体(cSerinc5X2),该剪接体在N端缺少1～37位氨基酸.为鉴定这两种犬Serinc5是否具有抑制HIV-1的复制能力,本研究从犬肾细胞系MDCK中提取犬总RNA,经RT-PCR分别扩增两种犬Serinc5的cDNA,并克隆到pcDNA3.1载体中,构建编码两种犬Serinc5的重组质粒.将这两种重组质粒分别与编码HIV-1野生型或Nef缺失型的质粒共转染293T细胞,收获所产生的HIV-1病毒粒子,通过ELISA方法检测到这两种犬Serinc5对HIV-1病毒粒子的产量无影响.进一步将病毒粒子分别感染TZM-bl细胞,通过荧光强度检测结果显示,与对照组相比,cSerinc5X2对HIV-1感染性无显著抑制作用,而携带cSerinc5的HIV-1感染性与携带cSerinc5X2的病毒和对照组相比降低约9倍(p＜0.01),表明cSefinc5的N端对于病毒感染活性的抑制具有关键作用.本研究为鉴定Serinc5抗病毒的功能区提供实验依据.%Serine incorporator 5 (Serinc5) is a novel host restrictive factor that inhibits HIV-1 replication by reduce viral infectivity.Serinc5 is highly conserved among mammalian species.Here we investigated whether canis Serinc5 (cSerinc5) inhibits HIV-1 infectivity.cSerinc5 has a transcript variant (cSerinc5X2) which lack 37aa at the N-terminus compared to cSerinc5.We first constructed recombinant plasmids encoding cSerinc5 and cSerinc5X2,respectively.The cSerinc5 and cSerinc5X2 cDNA were prepared from the total RNA extracted from canis kidney cell line MDCK,and cloned into pcDNA3.1 expression vector to construct pcDNA-cSerinc5 and pcDNAcSerinc5X2.The HIV-1 viral particles were produced by transfecting molecular clone of HIV-1 wild-type or Nef-deleted full-length genome into 293T cells,in the presence of exogenous cSerinc5 or cSerinc5X2.These viral particles were collected and further infected to TZM-bl cells to detect virus infection efficiency.The results showed that cSerinc5X2 had no effect on HIV-1 infectivity,while cSerinc5 had a strong impact on virus infectivity,and the infectivity was about 10-fold lower than that of the cSerinc5X2 or blank control group.The results demonstrated that the N-terminus of cSerinc5 plays an important role in the inhibition of H1V-1 virus infectivity by cSerinc5.The study provides an evidence for the identification of antiviral functional regions in cSerinc5.