Replication Kinetics for a Reporter Merkel Cell Polyomavirus

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 M...

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Bibliographic Details
Published in:Viruses 2022-02, Vol.14 (3), p.473
Main Authors: Abere, Bizunesh, Zhou, Hongzhao, Shuda, Masahiro, Stolz, Donna B, Rapchak, Kyle, Moore, Patrick S, Chang, Yuan
Format: Article
Language:English
Subjects:
Antigens, Viral, Tumor - genetics
C-Terminus
Cancer
Cloning
Endoplasmic reticulum
Enzymes
Fusion protein
Gene expression
Genomes
Humans
Infections
Kinetics
Latency
Merkel cell polyomavirus
Merkel cell polyomavirus - genetics
Merkel cell polyomavirus - metabolism
MicroRNAs
minicircle
miRNA
Mutagenesis
Mutation
Plasmids
Point mutation
Polyomavirus - genetics
Polyomavirus - metabolism
Polyomavirus Infections
Proteins
Recombinase
Recombinases - metabolism
Replication
Tumor Virus Infections
Virus Replication - genetics
Virus-like particles
Viruses
VP1 protein
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Summary: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.
ISSN:1999-4915
1999-4915
DOI:10.3390/v14030473