Replication Kinetics for a Reporter Merkel Cell Polyomavirus

Overview

Journal Viruses

Publisher MDPI

Specialty Microbiology

Date 2022 Mar 26

PMID 35336880

Authors

Bizunesh Abere

Hongzhao Zhou

Masahiro Shuda

Donna B Stolz

Kyle Rapchak

Patrick S Moore

Yuan Chang

Affiliations

Soon will be listed here.

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.

Citing Articles

Membrane-bound Merkel cell polyomavirus middle T protein constitutively activates PLCγ1 signaling through Src-family kinases.

Peng W, Abere B, Shi H, Toland S, Smithgall T, Moore P Proc Natl Acad Sci U S A. 2023; 120(51):e2316467120.

PMID: 38079542 PMC: 10740393. DOI: 10.1073/pnas.2316467120.

Characterization of molecular mechanisms driving Merkel cell polyomavirus oncogene transcription and tumorigenic potential.

Yang J, Liu W, You J PLoS Pathog. 2023; 19(8):e1011598.

PMID: 37647312 PMC: 10468096. DOI: 10.1371/journal.ppat.1011598.

Unlicensed origin DNA melting by MCV and SV40 polyomavirus LT proteins is independent of ATP-dependent helicase activity.

Wan L, Toland S, Robinson-McCarthy L, Lee N, Schaich M, Hengel S Proc Natl Acad Sci U S A. 2023; 120(30):e2308010120.

PMID: 37459531 PMC: 10372695. DOI: 10.1073/pnas.2308010120.

Merkel cell polyomavirus small T antigen is a viral transcription activator that is essential for viral genome maintenance.

Rapchak K, Yagobian S, Moore J, Khattri M, Shuda M PLoS Pathog. 2022; 18(12):e1011039.

PMID: 36574443 PMC: 9829177. DOI: 10.1371/journal.ppat.1011039.

Current In Vitro and In Vivo Models to Study MCPyV-Associated MCC.

Loke A, Lambert P, Spurgeon M Viruses. 2022; 14(10).

PMID: 36298759 PMC: 9607385. DOI: 10.3390/v14102204.

References

Bayer N, Januliene D, Zocher G, Stehle T, Moeller A, Blaum B . Structure of Merkel Cell Polyomavirus Capsid and Interaction with Its Glycosaminoglycan Attachment Receptor. J Virol. 2020; 94(20). PMC: 7527053. DOI: 10.1128/JVI.01664-19. View

Kim J, Lee S, Li L, Park H, Park J, Lee K . High cleavage efficiency of a 2A peptide derived from porcine teschovirus-1 in human cell lines, zebrafish and mice. PLoS One. 2011; 6(4):e18556. PMC: 3084703. DOI: 10.1371/journal.pone.0018556. View

Henno L, Tombak E, Geimanen J, Orav M, Ustav E, Ustav M . Analysis of Human Papillomavirus Genome Replication Using Two- and Three-Dimensional Agarose Gel Electrophoresis. Curr Protoc Microbiol. 2017; 45:14B.10.1-14B.10.37. DOI: 10.1002/cpmc.28. View

Kwun H, Shuda M, Feng H, Camacho C, Moore P, Chang Y . Merkel cell polyomavirus small T antigen controls viral replication and oncoprotein expression by targeting the cellular ubiquitin ligase SCFFbw7. Cell Host Microbe. 2013; 14(2):125-35. PMC: 3764649. DOI: 10.1016/j.chom.2013.06.008. View

Scheller A, Prives C . Simian virus 40 and polyomavirus large tumor antigens have different requirements for high-affinity sequence-specific DNA binding. J Virol. 1985; 54(2):532-45. PMC: 254826. DOI: 10.1128/JVI.54.2.532-545.1985. View

Starrett G, Thakuria M, Chen T, Marcelus C, Cheng J, Nomburg J . Clinical and molecular characterization of virus-positive and virus-negative Merkel cell carcinoma. Genome Med. 2020; 12(1):30. PMC: 7081548. DOI: 10.1186/s13073-020-00727-4. View

Kwun H, Chang Y, Moore P . Protein-mediated viral latency is a novel mechanism for Merkel cell polyomavirus persistence. Proc Natl Acad Sci U S A. 2017; 114(20):E4040-E4047. PMC: 5441811. DOI: 10.1073/pnas.1703879114. View

Deb S, DeLucia A, Koff A, Tsui S, Tegtmeyer P . The adenine-thymine domain of the simian virus 40 core origin directs DNA bending and coordinately regulates DNA replication. Mol Cell Biol. 1986; 6(12):4578-84. PMC: 367242. DOI: 10.1128/mcb.6.12.4578-4584.1986. View

Guo X, Chen P, Hou X, Xu W, Wang D, Wang T . The recombined cccDNA produced using minicircle technology mimicked HBV genome in structure and function closely. Sci Rep. 2016; 6:25552. PMC: 4865889. DOI: 10.1038/srep25552. View

10.

Prives C, Murakami Y, Kern F, Folk W, Basilico C, Hurwitz J . DNA sequence requirements for replication of polyomavirus DNA in vivo and in vitro. Mol Cell Biol. 1987; 7(10):3694-704. PMC: 368025. DOI: 10.1128/mcb.7.10.3694-3704.1987. View

11.

Dobson S, Anene A, Boyne J, Mankouri J, MacDonald A, Whitehouse A . Merkel cell polyomavirus small tumour antigen activates the p38 MAPK pathway to enhance cellular motility. Biochem J. 2020; 477(14):2721-2733. PMC: 7398664. DOI: 10.1042/BCJ20200399. View

12.

Lee S, Paulson K, Murchison E, Afanasiev O, Alkan C, Leonard J . Identification and validation of a novel mature microRNA encoded by the Merkel cell polyomavirus in human Merkel cell carcinomas. J Clin Virol. 2011; 52(3):272-5. PMC: 3196837. DOI: 10.1016/j.jcv.2011.08.012. View

13.

Kean J, Rao S, Wang M, Garcea R . Seroepidemiology of human polyomaviruses. PLoS Pathog. 2009; 5(3):e1000363. PMC: 2655709. DOI: 10.1371/journal.ppat.1000363. View

14.

Lee M, Yuan H, Jeon H, Zhu Y, Yoo S, Shi S . Human Mesenchymal Stem Cells of Diverse Origins Support Persistent Infection with Kaposi's Sarcoma-Associated Herpesvirus and Manifest Distinct Angiogenic, Invasive, and Transforming Phenotypes. mBio. 2016; 7(1):e02109-15. PMC: 4742711. DOI: 10.1128/mBio.02109-15. View

15.

Shuda M, Feng H, Kwun H, Rosen S, Gjoerup O, Moore P . T antigen mutations are a human tumor-specific signature for Merkel cell polyomavirus. Proc Natl Acad Sci U S A. 2008; 105(42):16272-7. PMC: 2551627. DOI: 10.1073/pnas.0806526105. View

16.

Feng H, Kwun H, Liu X, Gjoerup O, Stolz D, Chang Y . Cellular and viral factors regulating Merkel cell polyomavirus replication. PLoS One. 2011; 6(7):e22468. PMC: 3142164. DOI: 10.1371/journal.pone.0022468. View

17.

Revie D, Tseng B, Grafstrom R, Goulian M . Covalent association of protein with replicative form DNA of parvovirus H-1. Proc Natl Acad Sci U S A. 1979; 76(11):5539-43. PMC: 411684. DOI: 10.1073/pnas.76.11.5539. View

18.

Carter J, Daugherty M, Qi X, Bheda-Malge A, Wipf G, Robinson K . Identification of an overprinting gene in Merkel cell polyomavirus provides evolutionary insight into the birth of viral genes. Proc Natl Acad Sci U S A. 2013; 110(31):12744-9. PMC: 3732942. DOI: 10.1073/pnas.1303526110. View

19.

Feng H, Shuda M, Chang Y, Moore P . Clonal integration of a polyomavirus in human Merkel cell carcinoma. Science. 2008; 319(5866):1096-100. PMC: 2740911. DOI: 10.1126/science.1152586. View

20.

Rio D, Tjian R . SV40 T antigen binding site mutations that affect autoregulation. Cell. 1983; 32(4):1227-40. DOI: 10.1016/0092-8674(83)90305-7. View