Identification of receptors for pig endogenous retrovirus

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.

Figures

Fig. 1.

Sequence comparison of the PERV-A receptors. (A) Amino acid alignment of the PERV-A receptors and murine homolog. Transmembrane domains predicted by tmpred based on statistical analysis of TMbase (www.ch.embnet.org/software/TMPRED_form.html) are highlighted by overscores. Ten transmembrane domains were predicted in this model, whereas an alternative model using TMbase had 11 including the 10 domains shown. The amino acid residue number of the consensus sequence is indicated. (B) Amino acid sequence identity of the PERV-A receptors and the murine homolog AK008081.

Fig. 2.

Analysis of PERV receptor activity in vitro. (A) SIRC cells expressing HuPAR-1, HuPAR-2, or BaPAR-2 support productive replication of PERV. Cells were exposed to a PERV-A 14/220, and virus replication was assessed by measuring RT activity in the culture supernatants. Similar results were obtained with NIH 3T3 cells as well as for SIRC/PoPAR cells in separate experiments (results not shown). (B) HuPAR-2/EGFP protein is expressed at the plasma membrane of transduced SIRC cells. Intracellular protein, particularly in the perinuclear endoplasmic reticulum region, is also evident. (C) PERV-A but not PERV-B envelope binding is enhanced on SIRC cells by the expression of HuPAR-2 proteins. Envelope binding for parental SIRC (gray-filled histogram) and SIRC/HuPAR-2 (line without fill) cells. Specific binding of PERV-A Env to SIRC/PoPAR was observed (data not shown).

Fig. 3.

In vivo expression and cellular localization of PERV-A receptors. (A) PBMCs express HuPAR-1 and HuPAR-2 RNA. RT-PCR products from total cellular RNA were electrophoresed. Lanes 1 and 2, human PBMCs; lane 3, SIRC control; lane 4, SIRC HuPAR-1; lane 5, SIRC HuPAR-2. Results from two typical volunteers are presented. All 11 volunteers expressed both HuPAR-1 and HuPAR-2 RNA. The presence of two products in the HuPAR-2 RT-PCR is due to the presence of a nonspecific amplicon. (B) Infection of primary human PBMCs by PERV-A 14/220. After exposure to PERV-A 14/220, PERV-A sequences were detectable by PCR for pol (data not shown) and env (shown) sequences. Lanes 1 and 6, marker; lanes 2 and 3, PBMCs on days 8 and 14 postinfection, respectively; lane 4, uninfected PBMCs; lane 5, 293 cells chronically infected with PERV-A 14/220. (C) Expression of PERV-A receptor RNA was determined in multiple human primary tissue samples by Northern blot with a probe that detects both HuPAR-1 and HuPAR-2.