细胞自噬对HTLV-1在HeLa细胞中复制动力学的影响

Influence of cellular autophagy on replication dynamics of human T cell leukemia virus type I （HTLV-1） in HeLa cells

目的探讨成人T淋巴细胞白血病病毒1型（HTLV-1）感染后的复制动力学及其对宿主细胞状态的影响。方法将HTLV-1病毒阳性细胞系MT2与HeLa细胞进行共培养，获得病毒早期感染模型，免疫印迹检测病毒核心蛋白p19、凋亡相关蛋白caspase-3/9、自噬相关蛋白LC3；real-time PCR检测病毒载量；荧光素酶活性检测3′长末端重复序列（3′-LTR）转录活性；免疫荧光检测自噬体数目。结果通过细胞共培养成功模拟了早期病毒感染；共培养后4 h、8 h、16 h、24 h、36 h，病毒核酸载量及长末端重复序列3′-LTR转录活性逐渐增加（P〈0.01），24-36 h达到平台期；而核心蛋白p19的表达4 h可以检测到，但8 h后降低，16 h后又升高，24 h和36 h差异无统计学意义；病毒感染后宿主细胞中caspase-3/7/9的活性及蛋白没有明显变化（P〉0.05），但是自噬相关蛋白LC3B/LC3A比例逐渐上升，伴有胞内自噬体数目的增加（P〈0.01）。结论HTLV-1病毒复制动力学与宿主细胞的自噬密切相关。

Objective To investigate the replication dynamics and cellular states of host cells with human T cell leukemia virus type 1 （ HTLV-1 ） infection. Methods HTLV-1 positive cell line MT2 was co-cultured with HeLa cells for 4 to 36 hours to acquire the virus early infection model. Immunoblotting was used to detect HTLV-1 core protein p19, apoptosis-related proteins easpase-3/9 and autophagy-related pro- tein LC3 at different time points. Real-time PCR was used to measure viral loads. Luciferase reporter gene assay was used to detect the enzymatic activities of caspase-3/7/9 and the transcriptional activities of 3 ＇-LTR at different time points after co-cuhuring. Autophagosomes were analyzed by immunofluorescence after co- culturing. The HTLV-1 long terminal repeat reporter gene （pHTLV-1-LTR-luc） was transfected into HeLa ceils by lipidosome 2000-mediated transfection and the luciferase activities at different time points were detected. Results The HTLV-1 infection model was successfully constructed by co-culturing MT2 cells with HeLa cells in vitro. Viral load and transcriptional activity of the 3＇-LTR gradually increased at 4 to 16 h after virus infection （ P〈0.01 ） and the viral load reached a steady state at 24 h. The expression of core protein p19 could be detected at 4 h after infection, decreased at 8 h and increased at 16 h, followed by reaching a steady state at 24 h. The enzymatic activity and cleaved protein of caspase-3/7/9 had no significant change at different time points after infection （P〉0.05） , but the proportion of LC3B/LC3A showed a gradual in- creasing trend with the persistent increase of autophagosomes （P〈0.01）. Conclusion Viral replication dynamics of HTLV-1 is closely related to autophagy in host ceils.