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. 2018 Jan 30;92(4):e01610-17.
doi: 10.1128/JVI.01610-17. Print 2018 Feb 15.

Merkel Cell Polyomavirus Infection of Animal Dermal Fibroblasts

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Free PMC article

Merkel Cell Polyomavirus Infection of Animal Dermal Fibroblasts

Wei Liu et al. J Virol. .
Free PMC article

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Abstract

Merkel cell polyomavirus (MCPyV) is the first polyomavirus to be associated with human cancer. Mechanistic studies attempting to fully elucidate MCPyV's oncogenic mechanisms have been hampered by the lack of animal models for MCPyV infection. In this study, we examined the ability of MCPyV-GFP pseudovirus (containing a green fluorescent protein [GFP] reporter construct), MCPyV recombinant virions, and several MCPyV chimeric viruses to infect dermal fibroblasts isolated from various model animals, including mouse (Mus musculus), rabbit (Oryctolagus cuniculus), rat (Rattus norvegicus), chimpanzee (Pan troglodytes), rhesus macaque (Macaca mulatta), patas monkey (Erythrocebus patas), common woolly monkey (Lagothrix lagotricha), red-chested mustached tamarin (Saguinus labiatus), and tree shrew (Tupaia belangeri). We found that MCPyV-GFP pseudovirus was able to enter the dermal fibroblasts of all species tested. Chimpanzee dermal fibroblasts were the only type that supported vigorous MCPyV gene expression and viral replication, and they did so to a level beyond that of human dermal fibroblasts. We further demonstrated that both human and chimpanzee dermal fibroblasts produce infectious MCPyV virions that can successfully infect new cells. In addition, rat dermal fibroblasts supported robust MCPyV large T antigen expression after infection with an MCPyV chimeric virus in which the entire enhancer region of the MCPyV early promoter has been replaced with the simian virus 40 (SV40) analog. Our results suggest that viral transcription and/or replication events represent the major hurdle for MCPyV cross-species transmission. The capacity of rat dermal fibroblasts to support MCPyV early gene expression suggests that the rat is a candidate model organism for studying viral oncogene function during Merkel cell carcinoma (MCC) oncogenic progression.IMPORTANCE MCPyV plays an important role in the development of a highly aggressive form of skin cancer, Merkel cell carcinoma (MCC). With the increasing number of MCC diagnoses, there is a need to better understand the virus and its oncogenic potential. However, studies attempting to fully elucidate MCPyV's oncogenic mechanisms have been hampered by the lack of animal models for MCPyV infection. To pinpoint the best candidate for developing an MCPyV infection animal model, we examined MCPyV's ability to infect dermal fibroblasts isolated from various established model animals. Of the animal cell types we tested, chimpanzee dermal fibroblasts were the only isolates that supported the full MCPyV infectious cycle. To overcome the infection blockade in the other model animals, we constructed chimeric viruses that achieved robust MCPyV entry and oncogene expression in rat fibroblasts. Our results suggest that the rat may serve as an in vivo model to study MCV oncogenesis.

Keywords: Merkel cell polyomavirus; animal dermal fibroblasts; viral life cycle.

Figures

FIG 1
FIG 1
MCPyV-GFP and MCPyV infection of dermal fibroblasts isolated from mouse and human skin samples. (A) Dermal fibroblasts isolated from mouse and human skin samples were treated with MCPyV-GFP pseudovirions in DMEM–F-12 medium containing EGF, bFGF, CHIR99021, and 1 mg/ml collagenase IV for 2 days. After switching to fresh DMEM–F-12 medium containing 20% FBS for three more days, cells were counterstained with Hoechst 33458. Bar, 10 μm. (B) Percentages of GFP+ cells in the experiment whose results are shown in panel A. (C) Dermal fibroblasts isolated from mouse and human skin samples were treated with MCPyV virions in DMEM–F-12 medium containing EGF, bFGF, CHIR99021, and 1 mg/ml collagenase IV for 2 days. After switching to fresh DMEM–F-12 medium containing 20% FBS for three more days, cells were immunostained using the indicated antibodies and counterstained with DAPI. Bar, 10 μm. (D) Percentages of LT+ cells in the experiment whose results are shown in panel C. Error bars represent standard errors of the means (SEM) of the results of at least three independent experiments.
FIG 2
FIG 2
MCPyV-GFP and MCPyV infection of dermal fibroblasts isolated from rabbit and rat dermal fibroblasts. (A) Dermal fibroblasts isolated from rabbit and rat skin samples were treated with MCPyV-GFP pseudovirions as described in the legend to Fig. 1A and examined under a fluorescence microscope. Bar, 10 μm. (B) Percentages of GFP+ cells in the experiment whose results are shown in panel A. (C) Dermal fibroblasts isolated from rabbit and rat skin samples were treated with MCPyV virions and stained as described for Fig. 1C. Bar, 10 μm. (D) Percentages of LT+ cells in the experiment whose results are shown in panel C. Error bars represent SEM of the results of at least three independent experiments.
FIG 3
FIG 3
MCPyV-GFP and MCPyV infection of dermal fibroblasts isolated from nonhuman primate skin samples. (A) Dermal fibroblasts isolated from nonhuman primate skin samples were treated with MCPyV-GFP pseudovirions as described in the legend to Fig. 1A, and the cells' images were captured on day 5. (B) Percentages of GFP+ cells in the experiment whose results are shown in panel A. Bar, 10 μm. (C) Dermal fibroblasts isolated from nonhuman primates were treated with MCPyV virions and stained as described in the legend to Fig. 1C. Bar, 10 μm. (D) Percentages of LT+ cells in the experiment whose results are shown in panel C. (E) Chimpanzee fibroblast cell lines S033665 (female, sampled at 1 day old), S008861 (male, sampled at 16 years old), and S008933 (female, sampled at 10 years old) were treated with MCPyV virions and stained as described in the legend to Fig. 1C, and percentages of LT+ cells were determined. (F) Chimpanzee and human dermal fibroblasts can produce infectious MCPyV virions. The flow chart shows MCPyV's initial infection and reinfection of dermal fibroblasts. (G) Chimpanzee dermal fibroblasts were treated with MCPyV virions in DMEM–F-12 medium containing EGF, bFGF, CHIR99021, and 1 mg/ml collagenase IV or DMEM–F-12 medium containing 20% FBS to block viral entry (Control) for 2 days. After switching to fresh DMEM–F-12 medium containing 20% FBS for 9 more days, the medium was harvested and centrifuged at 12,000 rpm at 4°C for 10 min. The supernatants were used to treat fresh human dermal fibroblasts in DMEM–F-12 medium containing EGF, bFGF, CHIR99021, and 4 mg/ml collagenase IV for 2 days. After switching to fresh DMEM–F-12 medium containing 20% FBS for 3 more days, cells were analyzed by qPCR. (H) Human dermal fibroblasts treated with MCPyV virions as described in the legend to panel G were analyzed by qPCR. Error bars represent SEM of the results of at least three independent experiments.
FIG 4
FIG 4
Construction of MCPyV-MuPyV, MCPyV-SV40 A, and MCPyV-SV40 B chimeric viruses. (A) The enhancer region of the MCPyV early promoter was replaced with the analogous region from the MuPyV genome. (B) Dermal fibroblasts isolated from human and rhesus macaque skin samples were transfected with the religated SV40 genome. After 5 days, all cells were immunostained using the indicated antibodies and counterstained with DAPI. Bar, 10 μm. (C) The enhancer region of the MCPyV early promoter was replaced with the analogous region of the SV40 genome. (D) The MCPyV late promoter region was deleted from the MCPyV-SV40 A genome. (E) The virus titers of MCPyV and the chimeric virions as represented by the genome copy numbers were determined by qPCR.
FIG 5
FIG 5
MCPyV-MuPyV, MCPyV-SV40 A, and MCPyV-SV40 B infection of dermal fibroblasts isolated from human skin samples. (A) Dermal fibroblasts isolated from human skin samples were infected with MCPyV-MuPyV, MCPyV-SV40 A, and MCPyV-SV40 B chimeric virions and stained as described in the legend to Fig. 1C. Bar, 10 μm. (B) Percentages of LT+ cells in the experiment whose results are shown in panel A. Error bars represent SEM of the results of at least three independent experiments.
FIG 6
FIG 6
MCPyV chimeric virus infection of primary rhesus macaque dermal fibroblasts. (A) Primary rhesus macaque dermal fibroblasts were treated with MCPyV-MuPyV, MCPyV-SV40 A, and MCPyV-SV40 B chimeric virions and stained as described in the legend to Fig. 1C. Bar, 10 μm. (B) Percentages of LT+ cells in the experiment whose results are shown in panel A. Error bars represent SEM of the results of at least three independent experiments.
FIG 7
FIG 7
MCPyV-MuPyV, MCPyV-SV40 A, and MCPyV-SV40 B infection of dermal fibroblasts isolated from rat skin samples. (A) Dermal fibroblasts isolated from rat skin samples were treated with MCPyV-MuPyV, MCPyV-SV40 A, and MCPyV-SV40 B chimeric virions and stained as described in the legend to Fig. 1C. Bar, 10 μm. (B) Percentages of LT+ cells in the experiment whose results are shown in panel A. (C) Rat dermal fibroblasts were treated with MCPyV or MCPyV-SV40 B virions as described in the legend to Fig. 1C. After switching to fresh DMEM–F-12 medium containing FBS, the cells were harvest at 54 h and 120 h postinfection. The cells were analyzed by qPCR. Error bars represent SEM of the results of at least three independent experiments.

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