Human Papillomavirus Genome Copy Number Is Maintained by S-Phase Amplification, Genome Loss to the Cytosol during Mitosis, and Degradation in G1 Phase
Human Papillomavirus Genome Copy Number Is Maintained by S-Phase Amplification, Genome Loss to the Cytosol during Mitosis, and Degradation in G1 Phase
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DOI：
10.1128/jvi.01879-22
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发表时间：
2023-02-07
影响因子：
5.4
通讯作者：
Sapp, Martin
中科院分区：
文献类型：
--
作者：
Bienkowska-Haba, Malgorzata;Zwolinska, Katarzyna;Sapp, Martin
HPV基因组的维持目前被认为是通过调节病毒复制因子E1和E2的表达和活性来实现的。此外，E8E2抑制子通过与E1和E2竞争结合到病毒复制起点和招募抑制子复合体来限制基因组拷贝数。目前的人乳头状瘤病毒(HPV)复制模型由三种复制模式组成。在感染性传播之后，病毒基因组在建立阶段被放大，达到每个细胞高达数百个拷贝。在维持阶段，HPV基因组拷贝数保持不变。感染细胞的分化诱导HPV基因组扩增。应用高灵敏的原位杂交技术和新鲜感染HPV16的细胞株以及已建立的HPV16阳性细胞系，我们观察到病毒基因组在培养成单层的未分化角质形成细胞的每个S期都有扩增。在有丝分裂过程中，核病毒基因组拷贝数被重置到S之前的水平。大多数病毒基因组无法与宿主染色体相连，并丢失到细胞质中。在细胞周期进程中，胞质病毒基因组逐渐减少。在NH4Cl或其他干扰溶酶体酸化的药物存在下，胞质基因组的丢失被阻止，这表明自噬参与了病毒基因组的降解。这些观察也是对从患者样本中获得的HPV31细胞株进行的。在携带整合HPV16DNA的UMSCC47细胞中未检测到胞浆病毒基因组。对含有上体HPV16的角质形成细胞的器官型RAFT培养的分析也揭示了胞浆病毒基因组的存在。我们得出结论，HPV通过平衡S期的病毒基因组扩增和有丝分裂期间病毒基因组到胞浆中的丢失来维持病毒基因组的拷贝数。限制病毒基因组与有丝分裂染色体的连接似乎可以重置每个细胞周期的基因组拷贝数。重要的是，目前认为HPV基因组的维持是通过调节病毒复制因子E1和E2的表达和活性来实现的。此外，E8E2抑制子已被证明是限制基因组拷贝数的重要因素，它与E1和E2竞争结合到病毒复制的起点，并招募阻遏子复合体。在这里，我们证明了人乳头瘤病毒基因组在每个S阶段都被扩增了。在有丝分裂过程中，由于大多数基因组未能与有丝分裂染色体捆绑在一起，核基因组拷贝数被重置。相反，HPV基因组积累在新分裂细胞的细胞质中。胞质病毒DNA在G(1)中以溶酶体依赖的方式降解，有助于基因组复制重置。我们的数据表明，在建立和维持期间的复制模式是相同的，并进一步表明，对基因组拴系的限制显著有助于病毒基因组的维持。
HPV genome maintenance is currently thought to be achieved by regulating the expression and activity of the viral replication factors E1 and E2. In addition, the E8E2 repressor has been shown to be important for restricting genome copy numbers by competing with E1 and E2 for binding to the viral origin of replication and by recruiting repressor complexes.The current model of human papillomavirus (HPV) replication is comprised of three modes of replication. Following infectious delivery, the viral genome is amplified during the establishment phase to reach up to some hundred copies per cell. The HPV genome copy number remains constant during the maintenance stage. The differentiation of infected cells induces HPV genome amplification. Using highly sensitive in situ hybridization (DNAscope) and freshly HPV16-infected as well as established HPV16-positive cell lines, we observed that the viral genome is amplified in each S phase of undifferentiated keratinocytes cultured as monolayers. The nuclear viral genome copy number is reset to pre-S-phase levels during mitosis. The majority of the viral genome fails to tether to host chromosomes and is lost to the cytosol. Cytosolic viral genomes gradually decrease during cell cycle progression. The loss of cytosolic genomes is blocked in the presence of NH4Cl or other drugs that interfere with lysosomal acidification, suggesting the involvement of autophagy in viral genome degradation. These observations were also made with HPV31 cell lines obtained from patient samples. Cytosolic viral genomes were not detected in UMSCC47 cells carrying integrated HPV16 DNA. Analyses of organotypic raft cultures derived from keratinocytes harboring episomal HPV16 revealed the presence of cytosolic viral genomes as well. We conclude that HPV maintains viral genome copy numbers by balancing viral genome amplification during S phase with the loss of viral genomes to the cytosol during mitosis. It seems plausible that restrictions to viral genome tethering to mitotic chromosomes reset genome copy numbers in each cell cycle.IMPORTANCE HPV genome maintenance is currently thought to be achieved by regulating the expression and activity of the viral replication factors E1 and E2. In addition, the E8E2 repressor has been shown to be important for restricting genome copy numbers by competing with E1 and E2 for binding to the viral origin of replication and by recruiting repressor complexes. Here, we demonstrate that the HPV genome is amplified in each S phase. The nuclear genome copy number is reset during mitosis by a failure of the majority of the genomes to tether to mitotic chromosomes. Rather, HPV genomes accumulate in the cytoplasm of freshly divided cells. Cytosolic viral DNA is degraded in G(1) in a lysosome-dependent manner, contributing to the genome copy reset. Our data imply that the mode of replication during establishment and maintenance is the same and further suggest that restrictions to genome tethering significantly contribute to viral genome maintenance.