Department of Biochemistry and Molecular Biology, Sichuan Cancer Institute, Chengdu, 610041, People's Republic of China.
Department of Gynecologic Oncology, Sichuan Cancer Hospital, School of Medicine, University of Electronic Science and Technology of China, Chengdu, 610041, People's Republic of China.
Virol J. 2022 May 26;19(1):90. doi: 10.1186/s12985-022-01826-x.
Persistent HPV16 infection is the leading risk factor for developing cervical cancer. Anti-L1 antibodies against HPV16 produced in HPV16 infections play diverse roles in the clearance of virus infection and prevention of persistence. It has been implicated that the cervicovaginal squamous epithelial cells actually express TRIM21 and that some HPV16 particles could escape leaky endosomal compartment into the cytosol and that Fc receptor TRIM21 directly neutralize infection by targeting antibody-opsonized viruses for proteasomal degradation. We explored whether anti-L1 antibody opsonized HPV16 pseudovirus (PsV) entered into the cytosol could be neutralized by TRIM21-mediated activation of a proteasomal pathway to reduce the chance of persistent HPV16 infection.
HPV16 PsV were generated and extracted in HEK 293FT cells co-transfected with pcDNA3.1-eGFP and p16sheLL plasmids according to the standard protocol. The HPV16 PsV with capsid protein L1 was characterized by fluorescence microscopy and western blot, and the HPV16 PsV titer and anti-L1-bound PsV entry efficiency were detected by flow cytometry. The expressions of transcription factors (TF) and cytokines elicited by the TRIM21-activated proteasomal pathway were confirmed by dual-luciferase reporter assay and RT-qPCR. The changes in HPV16 PsV load with or without inhibitors in the infected HEK 293FT cells were determinated by qPCR.
Simultaneous transfection with pcDNA3.1-eGFP and p16sheLL plasmids into the HEK 293FT cells resulted in the self-assembly of HPV16 PsV with capsid protein L1. Both HPV16 PsV and anti-L1-bound HPV16 PsV could infect HEK 293FT cells. Anti-L1-bound PsV up-regulated TRIM21 mediated-activation of proteasome and increased expressions of TF and cytokines in the infected cells where HPV16 PsV load reduced by ~ 1000-fold in the presence of anti-L1 antibody, but inhibition of proteasomal activity increased HPV16 PsV load.
Our preliminary results indicate that anti-L1 antibody entered with HPV16 PsV into the cells could mediate degradation of HPV16 PsV by TRIM21-activated proteasomal pathway intracellularly, giving anti-capsid protein L1 antibody a role in host defense of persistent HPV16 infection.
持续性 HPV16 感染是导致宫颈癌的主要危险因素。HPV16 感染中产生的针对 HPV16 L1 的抗体在清除病毒感染和防止持续性感染方面发挥着多种作用。已经表明，宫颈阴道鳞状上皮细胞实际上表达 TRIM21，并且一些 HPV16 颗粒可以从渗漏的内体隔间逃逸到细胞质中，并且 Fc 受体 TRIM21 通过靶向抗体调理的病毒进行蛋白酶体降解来直接中和感染。我们探讨了抗 L1 抗体调理的 HPV16 假病毒（PsV）进入细胞质后是否可以通过 TRIM21 介导的蛋白酶体途径激活来中和，以降低持续性 HPV16 感染的机会。
根据标准方案，在共转染 pcDNA3.1-eGFP 和 p16sheLL 质粒的 HEK 293FT 细胞中生成和提取 HPV16 PsV。通过荧光显微镜和 Western blot 对带有衣壳蛋白 L1 的 HPV16 PsV 进行表征，并通过流式细胞术检测 HPV16 PsV 的效价和抗 L1 结合的 PsV 进入效率。通过双荧光素酶报告基因测定和 RT-qPCR 确认 TRIM21 激活的蛋白酶体途径引发的转录因子（TF）和细胞因子的表达变化。通过 qPCR 确定感染的 HEK 293FT 细胞中有无抑制剂时 HPV16 PsV 载量的变化。
同时转染 pcDNA3.1-eGFP 和 p16sheLL 质粒到 HEK 293FT 细胞中导致带有衣壳蛋白 L1 的 HPV16 PsV 自组装。HPV16 PsV 和抗 L1 结合的 HPV16 PsV 均可感染 HEK 293FT 细胞。抗 L1 结合的 PsV 上调了 TRIM21 介导的蛋白酶体激活，并增加了感染细胞中 TF 和细胞因子的表达，其中 HPV16 PsV 载量在存在抗 L1 抗体的情况下降低了约 1000 倍，但蛋白酶体活性的抑制增加了 HPV16 PsV 载量。
我们的初步结果表明，与 HPV16 PsV 一起进入细胞的抗 L1 抗体可以通过 TRIM21 激活的蛋白酶体途径在细胞内降解 HPV16 PsV，从而使抗衣壳蛋白 L1 抗体在宿主防御持续性 HPV16 感染中发挥作用。
