Merkel Cell Polyomavirus Infection and Detection

Summary

Here, we present a protocol to infect primary human dermal fibroblast with MCPyV. The protocol includes isolation of dermal fibroblasts, preparation of MCPyV virions, virus infection, immunofluorescence staining, and fluorescence in situ hybridization. This protocol can be extended for characterizing MCPyV-host interactions and discovering other cell types infectable by MCPyV.

Abstract

Merkel cell polyomavirus (MCPyV) infection can lead to Merkel cell carcinoma (MCC), a highly aggressive form of skin cancer. Mechanistic studies to fully investigate MCPyV molecular biology and oncogenic mechanisms have been hampered by a lack of adequate cell culture models. Here, we describe a set of protocols for performing and detecting MCPyV infection of primary human skin cells. The protocols describe the isolation of human dermal fibroblasts, preparation of recombinant MCPyV virions, and detection of virus infection by both immunofluorescent (IF) staining and in situ DNA-hybridization chain reaction (HCR), which is a highly sensitive fluorescence in situ hybridization (FISH) approach. The protocols herein can be adapted by interested researchers to identify other cell types or cell lines that support MCPyV infection. The described FISH approach could also be adapted for detecting low levels of viral DNAs present in the infected human skin.

Introduction

Merkel cell polyomavirus (MCPyV) is a small, double-stranded DNA virus that has been associated with a rare but aggressive skin cancer, Merkel cell carcinoma (MCC)^1,2. The mortality rate of MCC, around 33%, exceeds that of melanoma^3,4. MCPyV has a circular genome of ~5 kb^1,5 bisected by a non-coding regulatory region (NCRR) into early and late coding regions¹. The NCRR contains the viral origin of replication (Ori) and bidirectional promoters for viral transcription

Protocol

Human neonatal foreskins were obtained from Penn Skin Disease Research Center. Adult human fibroblasts were obtained from discarded normal skin after surgery. All the protocols were approved by the University of Pennsylvania Institutional Review Board.

1. Isolation of human dermal fibroblasts

1. Use a pair of scissors to trim off fat and subcutaneous tissue from the human neonatal foreskin and cut the skin sample in halves or quarters.
2. Incubate the tissue in 5 mL of 10 mg/mL.......

Discussion

The methods described above , including isolation of dermal fibroblasts from human skin tissue, preparation of recombinant MCPyV virions, infection of cultured cells, immunofluorescent staining, and a sensitive FISH method adapted from HCR technology, which should enable researchers to analyze MCPyV infection²⁷. One of the most critical steps to achieving MCPyV infection in vitro is the production of high-titer virion preparations. Using the protocol for preparation of recombinant MCPyV virions de.......

Materials

Name	Company	Catalog Number	Comments
Fetal calf serum	HyClone	SH30071.03
MEM Non-Essential Amino Acids Solution, 100X	Thermo Fisher Scientific	11140050
GLUTAMAX I, 100X	Thermo Fisher Scientific	35050061	L-Glutamine
DPBS, no calcium, no magnesium	Thermo Fisher Scientific	14190136
0.05% Trypsin-EDTA	Thermo Fisher Scientific	25300-054
DMEM/F12 medium	Thermo Fisher Scientific	11330-032
Recombinant Human EGF Protein, CF	R&D systems	236-EG-200	Store at -80 degree celsius
CHIR99021	Cayman Chemical	13122	Store at -80 degree celsius
CHIR99021	Sigma	SML1046	Store at -80 degree celsius
Collagenase type IV	Thermo Fisher Scientific	17104019
Dispase II	Roche	4942078001
Antibiotic-Antimycotic	Thermo Fisher Scientific	15240-062	Protect from light
DMEM medium	Thermo Fisher Scientific	11965084
Alexa Fluor 594 goat anti-mouse IgG	Thermo Fisher Scientific	A11032	Protect from light
Alexa Fluor 488 goat anti-rabbit IgG	Thermo Fisher Scientific	A11034	Protect from light
OptiPrep Density Gradient Medium	Sigma	D1556	Protect from light
Paraformaldehyde	Sigma	P6148
anti-MCPyV LT (CM2B4)	Santa Cruz	sc-136172	Lot # B2717
MCV VP1 rabbit		Rabbit polyclonal serum #10965	https://home.ccr.cancer.gov/lco/BuckLabAntibodies.htm
Hygromycin	Roche	10843555001
Basic Fibroblast Growth Factors (bFGF), Human Recombinant	Corning	354060	Store at -80 degree celsius
Benzonase Nuclease	Sigma	E8263
Plasmid-Safe ATP-Dependent DNase	EPICENTRE	E3101K
Probe hybridization buffer	Molecular technologies
Probe wash buffer	Molecular technologies
Amplification buffer	Molecular technologies
Alexa 594-labeled hairpins	Molecular technologies	B4	Protect from light
Triton X-100	Sigma	X100
Quant-iT PicoGreen dsDNA Reagent	Thermo Fisher Scientific	P7581
BamHI-HF	NEB	R3136
Buffer PB	Qiagen	19066
blue miniprep spin column	Qiagen	27104
50mL Conical Centrifuge Tubes	Corning	352070
T4 ligase	NEB	M0202T
MagicMark XP	Thermo Fisher Scientific	LC5602