Isolation of an Ecotropic Porcine Endogenous Retrovirus PERV-C from a Yucatan SLAD/D Inbred Miniature Swine

Abstract

Xenotransplantation may compensate the limited number of human allografts for transplantation using pigs as organ donors. Porcine endogenous retroviruses inherit infectious potential if pig cells, tissues, or organs were transplanted to immunosuppressed human recipients. Particularly, ecotropic PERV-C that could recombine with PERV-A to highly replication-competent human-tropic PERV-A/C should be excluded from pig breeds designed for xenotransplantation. Because of their low proviral background, SLA^D/D (SLA, swine leukocyte antigen) haplotype pigs are potential candidates as organ donors as they do not bear replication-competent PERV-A and -B, even if they carry PERV-C. In this work, we characterized their PERV-C background isolating a full-length PERV-C proviral clone number 561 from a SLA^D/D haplotype pig genome displayed in a bacteriophage lambda library. The provirus truncated in env due to cloning in lambda was complemented by PCR, and the recombinants were functionally characterized, confirming an increased infectivity in vitro compared to other PERV-C. Recombinant clone PERV-C(561) was chromosomally mapped by its 5'-proviral flanking sequences. Full-length PCR using 5'-and 3'-flanking primers specific to the PERV-C(561) locus verified that this specific SLA^D/D haplotype pig harbors at least one full-length PERV-C provirus. The chromosomal location is different from that of the previously described PERV-C(1312) provirus, which was derived from the porcine cell-line MAX-T. The sequence data presented here provide further knowledge about PERV-C infectivity and contribute to targeted knockout in order to generate PERV-C-free founder animals. IMPORTANCE Yucatan SLA^D/D haplotype miniature swine are candidates as organ donors for xenotransplantation. A full-length replication-competent PERV-C provirus was characterized. The provirus was chromosomally mapped in the pig genome. In vitro, the virus showed increased infectivity compared to other functional PERV-C isolates. Data may be used for targeted knockout to generate PERV-C free founder animals.

Keywords: PERV; SLAD/D haplotype; Xenotransplantation.

FIG 1

Genomic organization of full-length PERV-C clones isolated from the genome of a SLA^D/D haplotype miniature sow. (A) Chromosomal 5′-flanking sequences (F) were digested with SpeI so that 281 bp out of 5.607 bp remained. 5′-LTR as well as gag and pro/pol genes were components of bacteriophage clone 561. The env gene containing the envC-specific region (envC) was partially restricted by Sau3AI due to the cloning strategy and complemented by a PCR-generated env/3′-LTR amplicon subcloned in pGEM-T Easy. This plasmid was cut with SpeI and KpnI to release the missing part. Three-point ligation of the bacteriophage backbone with the PCR amplicon at the KpnI restriction site and SpeI-restricted pBlueScript vector finally resulted in full-length PERV-C(5634) and PERV-C(5683) clones. (B) Genomic positions of the restored PERV-C are indicated by numbers. F, 5′-flanking region; LTR, long terminal repeat; CAP, cap site; PBS, primer binding site; SD, splice donor; gag, group-specific antigen; pro/pol, protease/polymerase; env, envelope; SA, splice acceptor; SU/TM, surface unit/transmembrane cleavage site; PPT, polypurine tract; p(A), polyadenylation site.

FIG 2

Analysis of provirus functionality in susceptible cells in vitro. ST-IOWA cells were subjected to transfection with plasmids containing a copy of PERV-C(5634) and PERV-C(5683), respectively. RT activity of transfected cells was monitored in cell-free supernatants weekly. PERV-C(1312) served as positive control and nontransfected cells as negative control. Mean values and standard deviation of biological duplicates are depicted.

FIG 3

Examination of replication competence of full-length PERV-C clones. Twenty days p.t. viral supernatants of transfected ST-IOWA cells were cell-free filtered and used to challenge naive ST-IOWA cells in vitro. (A) Expression of RT activity was monitored semiweekly using C-type RT activity assay. PERV-C(1312) served as positive control and uninfected cells as negative control. (B) PERV-C env vRNA copy numbers were determined 7, 28, and 56 days p.i. by qRT-PCR. Experiments were conducted as biological triplicates, and each assay was performed with samples in triplicate. Mean values as well as standard deviation are shown.

FIG 4

Provirus detection in infected ST-IOWA cells. Nested PCR was performed to detect PERV-C provirus in infected ST-IOWA cells 7, 28, and 56 days p.i. Employing envC-specific primer amplicons of 208-bp size were generated using genomic DNA extracted from infected ST-IOWA cells with PERV-C(5634) (lines 1, 5, 9), PERV-C(5683) (lines 2, 6, 10), and PERV-C(1312) (lines 3, 7, 11) as positive control. Genomic DNA of naive ST-IOWA cells served as negative control over the different time points (lines 4, 8, 12). Amplification of the porcine GAPDH gene (poGAPDH) confirmed integrity and quality of DNA samples used to generate 150 bp amplicons. No-template control (line 13) excluded putative PCR contamination.

FIG 5

Full-length PCR with 5′- and 3′-flanking primers specific to the PERV-C(561) locus. (i) p13t_5f2 (5′-TTGGTAGGCACTCTAAGGAAAG-3′) and p13t_3r3 (5′-GCGTGCCTCTTAGGAATGTTA-3′); (ii) p13t_5f3 (5′-GGAATCACCCAAGTTCCCGT-3′) and p13t_3r3. PCRs were performed in duplicate, and each includes a no-template control. M, 1 kb DNA Ladder (NEB, Germany).