Replication Kinetics for a Reporter Merkel Cell Polyomavirus |
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| Authors: | Bizunesh Abere Hongzhao Zhou Masahiro Shuda Donna B Stolz Kyle Rapchak Patrick S Moore Yuan Chang |
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| Institution: | 1.Cancer Virology Program, Hillman Cancer Center, University of Pittsburgh, Pittsburgh, PA 15213, USA; (B.A.); (H.Z.); (M.S.); (K.R.);2.Department of Microbiology and Molecular Genetics, University of Pittsburgh, Pittsburgh, PA 15213, USA;3.Department Cell Biology, Center for Biologic Imaging, University of Pittsburgh, Pittsburgh, PA 15213, USA;4.Department of Pathology, University of Pittsburgh, Pittsburgh, PA 15213, USA |
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| Abstract: | 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. |
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| Keywords: | Merkel cell polyomavirus minicircle replication |
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