Identification of receptors for pig endogenous retrovirus

Thomas Ericsson

Yasuhiro Takeuchi

Christian Templin

Gary Quinn

Jennifer Ishii+5 authors

^{Immerge BioTherapeutics, Inc., 300 Technology Square, Cambridge, MA 02139}

^{Wohl Virion Centre, Windeyer Institute, University College London, 46 Cleveland Street, London W1T 4JF, United Kingdom}

^{Department of Molecular and Experimental Medicine, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037}

^{Research Center for Emerging Infectious Diseases, Research Institute for Microbial Diseases, Osaka University, 3-1 Yamadaoka, Suita, Osaka 565-0871, Japan}

^{Host and Defence, PRESTO, Japan Science and Technology Corporation, Tachikawa City, Tokyo 565-0871, Japan}

^{To whom correspondence may be addressed. E-mail:}moc.tbegremmi@ecneitap.evilc or ku.ca.lcu@ssiew.r.

^{Present address: Leibniz Research Laboratories for Biotechnology and Artificial Organs, Hannover Medical School, D-30659 Hannover, Germany.}

Edited by Peter K. Vogt, The Scripps Research Institute, La Jolla, CA

Received 2003 Jan 2; Accepted 2003 Apr 1.

Abstract

Xenotransplantation of porcine tissues has the potential to treat a wide variety of major health problems including organ failure and diabetes. Balanced against the potential benefits of xenotransplantation, however, is the risk of human infection with a porcine microorganism. In particular, the transmission of porcine endogenous retrovirus (PERV) is a major concern [Chapman, L. E. & Bloom, E. T. (2001) J. Am. Med. Assoc. 285, 2304–2306]. Here we report the identification of two, sequence-related, human proteins that act as receptors for PERV-A, encoded by genes located on chromosomes 8 and 17. We also describe homologs from baboon and porcine cells that also are active as receptors. Conversely, activity could not be demonstrated with a syntenic murine receptor homolog. Sequence analysis indicates that PERV-A receptors [human PERV-A receptor (HuPAR)-1, HuPAR-2, baboon PERV-A receptor 2, and porcine PERV-A receptor] are multiple membrane-spanning proteins similar to receptors for other gammaretroviruses. Expression is widespread in human tissues including peripheral blood mononuclear cells, but their biological functions are unknown. The identification of the PERV-A receptors opens avenues of research necessary for a more complete assessment of the retroviral risks of pig to human xenotransplantation.

Abstract

A serious donor-tissue shortage is a major barrier to clinical therapies that might be used to treat severe illness such as end-stage organ disease and diabetes mellitus. In this context, appropriate animal donors have the potential to be a renewable and unlimited source of tissues for transplantation that could also be transgenically engineered to enhance their efficacy and safety. However, the promise of clinical xenotransplantation is offset at the present time by the potential of a public health risk due to the cross-species transmission of pathogens from animal donors to human patients (1, 2).

Particular attention has been focused on the risks associated with porcine endogenous retroviruses (PERVs). This attention reflects the current opinion that the pig is a suitable species to engineer as the source for many types of organs and tissues. In the context of a possible infectious disease risk, we have reported previously that multiple copies of PERV are contained in all pig genomes (3, 4).

Two subgroups of PERV (PERV-A and PERV-B) can infect human cells in vitro, and these subgroups use cellular receptors that are distinct from those used by each other and other retroviruses (3–5). We sought to identify the receptor used by PERV-A, because all human-tropic PERV isolates obtained from primary pig cells have been PERV-A (6, 7), and this subgroup of virus is present in porcine DNA at a higher copy number than PERV-B (4). Thus, it is likely that PERV-A represents the primary PERV subgroup for which humans are at risk in the context of pig-tissue xenotransplantation.

The viruses related most closely to PERV [gibbon ape leukemia virus, feline leukemia virus, and murine leukemia virus (MLV)] are associated with hematopoietic cell malignancies (8, 9). Therefore, if PERV transmission were to occur, the risk to the transplant recipient and possibly the general populace might be real, especially if the initial infection were to occur under conditions of intensive immunosuppression that might allow time for the virus to adapt to infection of human tissues. Accordingly, to determine the nature of the risk and maximize safety in the design of clinical xenotransplantation trials, it is critical that the biology of PERV be understood. The identification of the molecules that PERV uses as receptors to infect human cells represents a significant step toward this goal.

Interference studies show that PERV-A 14/220 uses the same receptor as prototype PERV-A PK isolate. 293 target cells, either uninfected or infected with replication-competent PERV-A or PERV-B, were exposed to LacZ pseudotypes of PERV-A and PERV-B. A >10-fold reduction in titer indicates receptor blocking.

HuPAR-1, HuPAR-2, BaPAR-2, and PoPAR are functional PERV-A receptors when expressed in SIRC and, for HuPAR-1, in NIH3T3 cells. NT, not tested.

The HuPAR-1/HIS and HuPAR-2/EGFP chimeric constructs possess N-terminal 6×-His and C-terminal EGFP tags, respectively. PERV-A infection was measured by plating of PERV-A 14/220/LacZ pseudotype virus and by quantitative real-time PCR, with PERV DNA copy numbers being determined from standard curves (correlation coefficient, 0.998). These data are representative of the results of three similar experiments. UD, undetectable (i.e., copy number values were below the confidence limits of the standard curve); NT, not tested.

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Acknowledgments

We thank Jay Fishman for expert discussion, Richard Myers and John Hogenesch for help in sequence analyses, and David Ryan for assistance in the preparation of the manuscript. This work was supported by Immerge BioTherapeutics, Inc., the British Medical Research Council, National Institutes of Health Grant R01 AI45494, Japan Science and Technology Corporation, and Herbert Quandt Stiftung-VARTA AG (Hannover, Germany).

Acknowledgments

Notes

This paper was submitted directly (Track II) to the PNAS office.

Abbreviations: PERV, porcine endogenous retrovirus; RT, reverse transcriptase; PBMC, peripheral blood mononuclear cell; MLV, murine leukemia virus; HuPAR, human PERV-A receptor; BaPAR-2, baboon PERV-A receptor 2; EGFP, enhanced GFP; PoPAR, porcine PERV-A receptor.

Data deposition: The sequences reported in this paper have been deposited in the GenBank database [accession nos. AY070774 (HuPAR-1), AY070775 (HuPAR-2), AY070776 (BaPAR-2), and AY134475 (PoPAR)].

Notes

This paper was submitted directly (Track II) to the PNAS office.

Data deposition: The sequences reported in this paper have been deposited in the GenBank database [accession nos. AY070774 (HuPAR-1), AY070775 (HuPAR-2), AY070776 (BaPAR-2), and AY134475 (PoPAR)].

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