Human Papillomavirus 16 replication converts SAMHD1 into a homologous recombination factor and promotes its recruitment to replicating viral DNA.
Human Papillomavirus 16 replication converts SAMHD1 into a homologous recombination factor and promotes its recruitment to replicating viral DNA.
复制标题
人乳头瘤病毒 16 复制将 SAMHD1 转化为同源重组因子，并促进其募集到复制病毒 DNA 中。
DOI：
10.1101/2023.11.13.566899
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发表时间：
2023
期刊：
影响因子：
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通讯作者：
Morgan,IainM
中科院分区：
文献类型：
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作者：
James,ClaireD;Youssef,Aya;Prabhakar,ApurvaT;Otoa,Raymonde;Witt,Austin;Lewis,RachelL;Bristol,MollyL;Wang,Xu;Zhang,Kun;Li,Renfeng;Morgan,IainM
我们已经证明 SAMHD1（包含无菌 α 基序和组氨酸天冬氨酸结构域 HD 的蛋白 1）是人乳头瘤病毒 16 (HPV16) 生命周期的限制因素。在这里，我们证明，在 HPV 阴性宫颈癌 C33a 细胞和由 HPV16（HFK+HPV16）永生化的人包皮角质形成细胞中，SAMHD1 被招募到 E1-E2 复制 DNA 中。 HPV16 复制需要同源重组 (HR) 因子，病毒复制会促进 SAMHD1 磷酸化，从而将其从 dNTPase 转化为独立于 E6/E7 表达的 HR 因子。 SAMHD1 磷酸模拟物 (SAMHD1 T592D) 可减少 C33a 细胞中 E1-E2 介导的 DNA 复制，并增强复制 DNA 的募集。在 HFK+HPV16 细胞中，SAMHD1 T592D 被招募到病毒 DNA 中并减弱细胞生长，但不会减弱仅通过 E6/E7 永生化的同基因 HFK 细胞的生长。 SAMHD1 T592D 还可减弱角质形成细胞分化后病毒复制灶的发展。结果表明，增强的 SAMHD1 磷酸化可能对具有 HPV16 复制基因组的细胞具有治疗作用。蛋白磷酸酶2A（PP2A）可以使SAMHD1去磷酸化，并且PP2A功能可以被endothall抑制。我们证明 endothall 减少 E1-E2 复制并促进 SAMHD1 募集到 E1-E2 复制 DNA，模仿 SAMHD1 T592D 表型。最后，我们证明在具有 HPV16 附加型基因组的头颈癌细胞系中，endothall 会减弱其生长并促进 SAMHD1 招募到病毒基因组中。结果表明，针对细胞磷酸酶具有治疗 HPV 感染和癌症的潜力。 重要性 人乳头瘤病毒 (HPV) 是约 5% 的人类癌症的病原体。抗病毒疗法的发展取决于对病毒生命周期的进一步了解。在这里，我们证明 HPV16 复制通过磷酸化将不育 α 基序和含组氨酸天冬氨酸结构域 HD 的蛋白 1 (SAMHD1) 转化为同源重组 (HR) 因子。这种磷酸化促进 SAMHD1 募集到病毒 DNA 中以协助复制。模拟磷酸化的 SAMHD1 突变体被病毒 DNA 过度招募并减弱病毒复制。该突变体在 HPV16 永生化细胞中的表达会减弱这些细胞的生长，但不会减弱仅由病毒癌基因 E6/E7 永生化的细胞的生长。最后，我们证明磷酸酶抑制剂 endothall 促进内源性 SAMHD1 过度招募到 HPV16 复制 DNA，并可以减弱 HPV16 永生化人包皮角质形成细胞 (HFK) 和 HPV16 阳性头颈癌细胞系的生长。我们认为磷酸酶抑制剂是对抗 HPV 感染和疾病的一种新工具。
We have demonstrated that SAMHD1 (sterile alpha motif and histidine-aspartic domain HD-containing protein 1) is a restriction factor for the human papillomavirus 16 (HPV16) life cycle. Here, we demonstrate that in HPV-negative cervical cancer C33a cells and human foreskin keratinocytes immortalized by HPV16 (HFK+HPV16), SAMHD1 is recruited to E1-E2 replicating DNA. Homologous recombination (HR) factors are required for HPV16 replication, and viral replication promotes phosphorylation of SAMHD1, which converts it from a dNTPase to an HR factor independent from E6/E7 expression. A SAMHD1 phospho-mimic (SAMHD1 T592D) reduces E1-E2-mediated DNA replication in C33a cells and has enhanced recruitment to the replicating DNA. In HFK+HPV16 cells, SAMHD1 T592D is recruited to the viral DNA and attenuates cellular growth, but does not attenuate growth in isogenic HFK cells immortalized by E6/E7 alone. SAMHD1 T592D also attenuates the development of viral replication foci following keratinocyte differentiation. The results indicated that enhanced SAMHD1 phosphorylation could be therapeutically beneficial in cells with HPV16 replicating genomes. Protein phosphatase 2A (PP2A) can dephosphorylate SAMHD1, and PP2A function can be inhibited by endothall. We demonstrate that endothall reduces E1-E2 replication and promotes SAMHD1 recruitment to E1-E2 replicating DNA, mimicking the SAMHD1 T592D phenotypes. Finally, we demonstrate that in head and neck cancer cell lines with HPV16 episomal genomes, endothall attenuates their growth and promotes recruitment of SAMHD1 to the viral genome. The results suggest that targeting cellular phosphatases has therapeutic potential for the treatment of HPV infections and cancers.IMPORTANCEHuman papillomaviruses (HPVs) are causative agents in around 5% of all human cancers. The development of anti-viral therapeutics depends upon an increased understanding of the viral life cycle. Here, we demonstrate that HPV16 replication converts sterile alpha motif and histidine-aspartic domain HD-containing protein 1 (SAMHD1) into a homologous recombination (HR) factor via phosphorylation. This phosphorylation promotes recruitment of SAMHD1 to viral DNA to assist with replication. A SAMHD1 mutant that mimics phosphorylation is hyper-recruited to viral DNA and attenuates viral replication. Expression of this mutant in HPV16-immortalized cells attenuates the growth of these cells, but not cells immortalized by the viral oncogenes E6/E7 alone. Finally, we demonstrate that the phosphatase inhibitor endothall promotes hyper-recruitment of endogenous SAMHD1 to HPV16 replicating DNA and can attenuate the growth of both HPV16-immortalized human foreskin keratinocytes (HFKs) and HPV16-positive head and neck cancer cell lines. We propose that phosphatase inhibitors represent a novel tool for combating HPV infections and disease.