Expression of porcine endogenous retroviruses (PERVs) in melanomas of Munich miniature swine (MMS) Troll

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Abstract

Porcine endogenous retroviruses (PERVs) are integrated in the genome of all pig breeds. Since some of them are able to infect human cells, they might represent a risk for xenotransplantation using pig cells or organs. However, the expression and biological role of PERVs in healthy pigs as well as in porcine tumours is largely unknown. Since we and others have recently shown overexpression of a human endogenous retrovirus, HERV-K, in human melanomas, we studied the expression of PERVs in melanomas of selectively bred Munich miniature swine (MMS) Troll. This breeding herd of MMS Troll is characterised by a high prevalence of melanomas, which histologically resemble various types of cutaneous melanomas in humans. Several genetic factors have been defined when studying inheritance of melanomas and melanocytic nevi in MMS Troll. Here we show that the polytropic PERV-A and PERV-B as well as the ecotropic PERV-C are present in the genome of all melanoma bearing MMS Troll investigated. Most interestingly, in the spleen, but not in other organs, recombinant PERV-A/C proviruses were found. PERV expression was found elevated in melanomas when compared to normal skin and viral proteins were expressed in melanomas and pulmonary metastasis-derived melanoma cell cultures. During passaging of these cells in vitro the expression of PERV mRNA and protein increased and virus particles were released as shown by RT activity in the supernatant and by electron microscopy. Genomic RNA of PERV-A, -B and -C were found in pelleted virus particles. Although PERV expression was elevated in melanomas and pulmonary metastasis-derived cell cultures, the function of the virus in tumour development is still unclear.

Introduction

Porcine endogenous retroviruses (PERVs) are an integrated part of the genome of all pig breeds (Ericsson et al., 2001, Patience et al., 2001) and were found to be released as infectious particles from normal pig cells (Martin et al., 1998a; Tacke et al., 2000, Tacke et al., 2003, McIntyre et al., 2003). Three subtypes of PERV, which differ in the env region encoding for the receptor binding site, are known (Le Tissier et al., 1997, Patience et al., 2001, Patience et al., 1997, Takeuchi et al., 1998). Two of them, PERV-A and PERV-B, are polytropic viruses and able to infect human cells in vitro (Martin et al., 2000, Patience et al., 1997, Specke et al., 2001, Takeuchi et al., 1998, Wilson et al., 1998, Wilson et al., 2000). Therefore they may represent a risk for xenotransplantation when pig cells or organs will be used. PERV-C is an ecotropic virus, infecting only porcine cells. Recombinant PERVs with the long terminal repeat (LTR) of PERV-C and the receptor-binding domain (tropism) of PERV-A are able to adapt to human cells and this adaptation is associated with higher titres and an increase in the length of the LTR (Denner et al., 2003). PERV-A, -B and -C belong to the genera of the gammaretroviruses and are closely related to the murine and feline leukaemia viruses which are able to induce tumours and immunodeficiencies in the infected host (Denner, 1998). The tumour development by gammaretroviruses depends on high level replication, insertional mutagenesis, and alterations in the viral LTR (Athas et al., 1995). The expression of PERV and their biological role in normal pigs as well as in pig tumours is insufficiently analysed.

We (Buscher et al., 2005, Buscher et al., 2006) and others (Muster et al., 2003) have recently shown that a human endogenous retrovirus, HERV-K, is highly expressed in human melanomas. Malignant melanomas commonly arise from pigment-producing cells in the epidermis and take an aggressive course of disease in man. In order to establish an animal model, a breeding herd of MMS Troll with a high spontaneous rate of both congenital and postnatally occurring cutaneous melanocytic lesions, including invasive cutaneous melanomas with metastases, were established at the University of Munich and maintained as closed colony since 1986 (Wanke et al., 1998). Founder animals were derived from stock MMS Troll originally developed from Hanford and Columbian miniature swine at the Medical Service Munich (Sambraus, 1987). The overall prevalence of cutaneous melanoma in this breeding colony of MMS Troll is almost 50% (Wanke et al., 1998). Studies of the inheritance of melanocytic lesions suggested different modes for nevi and melanomas. For melanoma, a major gene model did not fit and therefore a two or three locus model was supposed (Muller et al., 1995). Melanomas of MMS Troll histologically resemble various types of cutaneous melanomas in humans but with a less aggressive course. MMS Troll represent a suitable animal model to study melanomagenesis, metastasis and melanoma regression, associated with vitiligo (Wanke et al., 1998) and provide the opportunity to study a possible participation of endogenous retroviruses in tumour development.

PERV particles have been found in the blood of leukaemic pigs, some of them treated by radiation (Frazier, 1985). PERVs were also isolated from pig lymphomas (Suzuka et al., 1985, Suzuka et al., 1986) and from transformed porcine lymphocytes treated with phytohemagglutinin and 5-azacytidine (Kaeffer et al., 1990), but it is still unclear whether they played a role in the induction or progression of these tumours.

In the present study, we analysed the genetic distribution of PERV-A, PERV-B and PERV-C in MMS Troll and their expression in melanomas in comparison to normal skin of the same individual. An enhanced expression of PERV in porcine melanomas was found by real-time PCR and virus particles were found released from melanoma pulmonary metastasis-derived cell cultures. This is consistent with findings showing high-level expression of HERV-K in human melanomas and further supports similarities between both entities.

Section snippets

Animals

Munich miniature swine (MMS) of the strain Troll were kept under spf conditions at the Friedrich-Loeffler Institute (FLI), Tübingen, and as a closed colony at the Institute of Veterinary Pathology, University of Munich. Four melanoma-bearing MMS Troll ranging from 4 to 8 weeks of age were included in the study. Animals were sacrificed by barbiturate overdose and complete necropsies were performed.

Cell culture

A melanoma cell culture was established from an aseptically taken pulmonary metastasis (animal

Morphology, histological and immunohistochemical characterisation of the melanomas

The MMS Troll included in this study exhibited single or multiple black skin tumours, which were frequently ulcerated (Fig. 1A). Melanomatous lesions were found in regional lymph nodes and a variety of internal organs, including lungs, liver and spleen (Fig. 1B and C, Table 1). Histologically, the skin tumours were composed of heavily pigmented melanoma cells, invading the dermis and subcutaneous tissue as well as pigment laden macrophages (Fig. 2A). Regional lymph nodes were heavily

Discussion

The results of this study provide first evidence for an elevated expression of PERVs in melanomas of selectively bred MMS Troll characterised by a high prevalence of cutaneous melanomas. In addition, a cell line derived from a pulmonary metastasis showed enhanced expression as well as release of PERV. Melanomas in these animals and in other pig breeds (Perez et al., 2002) represent an excellent model system to study the genetic background, the involvement of endogenous retroviruses and

Acknowledgments

We wish to thank Debora Mihica, Martina Lau, and Lisa Pichl for technical assistance, Prof. Thomas Mettenleiter and Prof. Lothar Stitz for providing the opportunity to keep MMS Troll at FLI Tübingen, and Dr. Schierack for the cell line IPEC-J2.

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      The expression of PERV in different organs, especially in organs relevant for xenotransplantation, was not yet studied in detail. Only a limited number of organs was analyzed and differences in the expression in different organs were observed with highest expression in the lung and the thymus of miniature swines (Akiyoshi et al., 1998), in the kidneys and the liver of a Large White pig (Clemenceau et al., 1999), in the spleen and the lung of miniature pigs with melanomas (Dieckhoff et al., 2007) or of transgenic German landrace pigs (Dieckhoff et al., 2008; Semaan et al., 2012). To study a larger number of tissues and organs and to compare expression at the mRNA and protein level, the Yucatan miniature pig 385, characterized by high expression of PERV, was analyzed.

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