Cancer Virology Program, Hillman Cancer Center, University of Pittsburgh, Pittsburgh, PA 15213, USA.
Department of Microbiology and Molecular Genetics, University of Pittsburgh, Pittsburgh, PA 15213, USA.
Viruses. 2022 Feb 25;14(3):473. doi: 10.3390/v14030473.
Merkel cell polyomavirus (MCV) causes one of the most aggressive human skin cancers, but laboratory studies on MCV replication have proven technically difficult. We report the first recombinase-mediated MCV minicircle (MCVmc) system that generates high levels of circularized virus, allowing facile MCV genetic manipulation and characterization of viral gene expression kinetics during replication. Mutations to Fbw7, Skp2, β-TrCP and hVam6p interaction sites, or to the stem loop sequence for the MCV-encoded miRNA precursor, markedly increase viral replication, whereas point mutation to an origin-binding site eliminates active virus replication. To further increase the utility of this system, an mScarlet fusion protein was inserted into the VP1 c-terminus to generate a non-infectious reporter virus for studies on virus kinetics. When this reporter virus genome is heterologously expressed together with MCV VP1 and VP2, virus-like particles are generated. The reporter virus genome is encapsidated and can be used at lower biosafety levels for one-round infection studies. Our findings reveal that MCV has multiple, self-encoded viral restriction mechanisms to promote viral latency over lytic replication, and these mechanisms are now amenable to examination using a recombinase technology.
Merkel 细胞多瘤病毒（MCV）可导致最具侵袭性的人类皮肤癌之一，但 MCV 复制的实验室研究证明技术上具有挑战性。我们报告了第一个重组酶介导的 MCV 微环（MCVmc）系统，该系统可产生高水平的环状病毒，从而便于进行 MCV 遗传操作，并在复制过程中对病毒基因表达动力学进行特征分析。Fbw7、Skp2、β-TrCP 和 hVam6p 相互作用位点的突变，或对 MCV 编码的 miRNA 前体的茎环序列的突变，可显著增加病毒复制，而对一个起源结合位点的点突变则消除了活跃的病毒复制。为了进一步提高该系统的实用性，将 mScarlet 融合蛋白插入 VP1 羧基末端，以生成用于病毒动力学研究的非感染性报告病毒。当将这种报告病毒基因组与 MCV VP1 和 VP2 异源表达时，会产生病毒样颗粒。报告病毒基因组被包裹，并可在较低的生物安全水平下用于一轮感染研究。我们的研究结果表明，MCV 具有多种自我编码的病毒限制机制，以促进病毒潜伏而非裂解复制，并且这些机制现在可以使用重组酶技术进行检查。
