Kinnetz Michaela, Alghamdi Faris, Racz Michael, Hu Wenwei, Shi Binshan
Department of Basic and Clinical Sciences, Albany College of Pharmacy and Health Sciences, 106 New Scotland Ave, Albany, NY, 12208, USA.
Rutgers Cancer Institute of New Jersey, Rutgers the State University of New Jersey, New Brunswick, NJ, 08903, USA.
Virol J. 2017 Aug 9;14(1):151. doi: 10.1186/s12985-017-0820-7.
The function of p53 in cancer biology has been studied extensively, but its role in anti-retrovirus infection has been elusive for many years. The restriction of retrovirus early stage replication by p53 was investigated in this study.
VSV-G pseudotyped retrovirus with GFP reporter gene was used to infect both HCT116 p53 cells and its isogenic p53 knockout HCT116 p53 cells. The infection was detected by flow cytometry. Reverse transcription products were quantified by real time PCR. Mutation analysis was performed after 1-LTR cycle and 2-LTR cycle DNA were amplified and PCR products were sequenced. Transcription and translation of cyclin-dependent kinase inhibitor 1 (p21) and SAM domain and HD domain-containing protein 1 (SAMHD1) were analyzed by TaqMan PCR and Western blot experiments. siRNA experiment was applied to study the role of p53 downstream gene p21 in the restriction of retrovirus infection.
It was found that the block of retrovirus infection in non-cycling cells was significantly attenuated in HCT116 p53 cells when compared to HCT116 p53 cells. It was found that both late reverse transcription products and viral 2-LTR cycle DNA were significantly increased in infected non-cycling HCT116 p53 cells. Furthermore, the mutation frequency detected in 1-LTR DNA from HCT116 p53 cells were significantly decreased in comparison to HCT116 p53 cells. A higher number of insertion and deletion mutations were detected in the joint region of 2-LTR cycle DNA in infected p53 cells. Cell cycle analysis showed retrovirus infection promoted host cell replication. Higher levels of mRNA and protein of p21 were found in HCT116 p53 cells in comparison to the HCT116 p53 cells. Furthermore, knockdown of p21 in non-cycling HCT116 p53 cells significantly increased the infection.
The results of this study showed that p53 is an important restriction factor that interferes with retrovirus infection in its early stage of replication. Our results suggested that p53 mediates the inhibition of retrovirus infection in non-cycling cells through it downstream gene p21, and p53 also functions to influence formation of 1-LTR cycle and 2-LTR cycle DNA.
p53在癌症生物学中的功能已得到广泛研究，但其在抗逆转录病毒感染中的作用多年来一直难以捉摸。本研究调查了p53对逆转录病毒早期复制的限制作用。
使用带有绿色荧光蛋白（GFP）报告基因的水泡性口炎病毒糖蛋白（VSV-G）假型逆转录病毒感染HCT116 p53细胞及其同基因p53基因敲除的HCT116 p53细胞。通过流式细胞术检测感染情况。通过实时聚合酶链反应（PCR）对逆转录产物进行定量。在1-长末端重复序列（1-LTR）循环和2-LTR循环DNA扩增后进行突变分析，并对PCR产物进行测序。通过TaqMan PCR和蛋白质免疫印迹实验分析细胞周期蛋白依赖性激酶抑制剂1（p21）和含SAM结构域和HD结构域蛋白1（SAMHD1）的转录和翻译情况。应用小干扰RNA（siRNA）实验研究p53下游基因p21在限制逆转录病毒感染中的作用。
发现与HCT116 p53细胞相比，HCT116 p53细胞中静止细胞的逆转录病毒感染阻滞明显减弱。发现在受感染的静止HCT116 p53细胞中，晚期逆转录产物和病毒2-LTR循环DNA均显著增加。此外，与HCT116 p53细胞相比，HCT116 p53细胞1-LTR DNA中检测到的突变频率显著降低。在受感染的p53细胞中，在2-LTR循环DNA的接头区域检测到更多的插入和缺失突变。细胞周期分析表明，逆转录病毒感染促进宿主细胞复制。与HCT116 p53细胞相比，HCT116 p53细胞中p21的mRNA和蛋白质水平更高。此外，在静止的HCT116 p53细胞中敲低p21可显著增加感染。
本研究结果表明，p53是一种重要的限制因子，可在逆转录病毒复制早期干扰其感染。我们的结果表明，p53通过其下游基因p21介导对静止细胞中逆转录病毒感染的抑制，并且p53还影响1-LTR循环和2-LTR循环DNA的形成。
