Replication Kinetics for a Reporter Merkel Cell Polyomavirus

Viruses. 2022 Feb 25;14(3):473. doi: 10.3390/v14030473.

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.

Keywords: Merkel cell polyomavirus; minicircle; replication.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Antigens, Viral, Tumor / genetics
  • Humans
  • Kinetics
  • Merkel cell polyomavirus* / genetics
  • Merkel cell polyomavirus* / metabolism
  • Polyomavirus Infections*
  • Polyomavirus* / genetics
  • Polyomavirus* / metabolism
  • Recombinases / metabolism
  • Tumor Virus Infections*
  • Virus Replication / genetics

Substances

  • Antigens, Viral, Tumor
  • Recombinases