Characterization of chromosomally assigned replication-competent gamma porcine endogenous retroviruses derived from a large white pig and expression in human cells

Abstract

Vertically transmitted endogenous retroviruses pose an infectious risk in the course of pig-to-human transplantation of cells, tissues, and organs. Two classes of polytropic type C porcine endogenous retroviruses (PERV) productively infect human cells in vitro. The cloning and characterization of replication-competent PERV-B sequences from infected human cells (F. Czauderna, N. Fischer, K. Boller, R. Kurth, and R. R. Tönjes, J. Virol. 74:4028-4038, 2000) as well as the cloning of functional PERV-A and -B sequences from porcine cell line PK15 (U. Krach, N. Fischer, F. Czauderna, and R. R. Tönjes, J. Virol. 75:5465-5472, 2001) have been previously described. Here we report the isolation of four full-length proviral sequences from a porcine bacterial artificial chromosome (BAC) library that comprises chromosomally assigned PERV. Clones Bac-PERV-A(130A12) and Bac-PERV-A(151B10) map to pig chromosome 1 and demonstrate close homology to PK15-PERV-A(58) in env and to PERV-MSL in long terminal repeat (LTR), gag, and pro/pol sequences. Clone Bac-PERV-A(463H12) is located on pig chromosome 3 and demonstrates close homology to PK15-PERV-A(58) in env and to 293-PERV-B(43) in LTR, gag, and pro/pol (Czauderna et al.; R. R. Tönjes, F. Czauderna, N. Fischer, U. Krach, K. Boller, P. Chardon, C. Rogel-Gailard, M. Niebert, G. Scheef, A. Werner, and R. Kurth, Transplant Proc. 32:1158-1161, 2000). Clone Bac-PERV-B(192B9) is located on pig chromosome 7 in the swine leukocyte antigen region and is highly homologous with but distinct from the previously described functional clone 293-PERV-B(43) and bears the number of repeats initially observed in the LTRs of clone 293-PERV-A(42) (Czauderna et al.; Krach et al.). Clones Bac-PERV-A(130A12), Bac-PERV-A(151B10), and Bac-PERV-A(463H12) were replication competent upon transfection into susceptible 293 and HeLa cells. Bac-PERV-B(192B9), however, bears two stop codons in pro/pol preventing this clone from being replication competent in some individual pigs, but initial screenings indicate that this provirus might be intact in others. The data suggest that the porcine genome harbors a limited number of infectious PERV sequences, allowing for specific screening in different pig breeds.

FIG. 1.

Schematic representation of replication-competent PERV proviral structure and LTR organization. (A) Proviral sequences of Bac-PERV-A(130A12), Bac-PERV-A(151B10), Bac-PERV-A(463H12), and Bac-PERV-B(192B9) are 8,918, 8,882, 8,754, and 8,840 bp in length, respectively. Genes are shown as boxes and the first and last nucleotide of LTR and structural genes are shown in bold letters (A to I) which are explained in Table 2. Arrowheads mark the transcriptional start site (cap), the primer binding site (PBS), the splice donor site (SD), the splice acceptor site (SA), the poly(A) addition site [p(A)], and the polypurine tract (ppt). Crosses show the positions of two in-frame stop codons in pol of Bac-PERV-B(192B9) at nt 4687 and 5251. (B) Organization of repetitive sequences in U3 of PERV LTR modulating the transcriptional activity shown for clones Bac-PERV-A(130A12) (I), Bac-PERV-A(151B10) (II), Bac-PERV-A(463H12) (III), and Bac-PERV-B(192B9) (IV) (24). Statistical analysis reveals several binding motifs for transcription factors within the repeat structure. The hatched boxes in sequences I and II indicate homologous but different sequences (24).

FIG. 2.

Detection of PERV Gag expression. Indirect immunofluorescence assay at different time points after transfection and infection of BAC DNA into HeLa cells using an antibody against Gag p10 (11). Expression of clones Bac-PERV-A(130A12) (A to D), Bac-PERV-A(151B10) (E to H), Bac-PERV-A(463H12) (I to L), and Bac-PERV-B(192B9) (M to P). The top three rows show protein expression investigated at 3, 20, and 31 d.p.t. (from top to bottom). The bottom row shows protein expression of HeLa cells at 22 d.p.i. with cell-free supernatant harvested from cells at 32 d.p.t. Scale bar, 50 μm (same magnification for all panels).

FIG. 3.

Replicative properties of PERV. (A) Detection of RT activity in cell-free culture supernatants of 293 cells upon transfection of BAC DNA. Results are average values from three independent experiments. (B) Detection of RT activity in cell-free culture supernatants of 293 cells upon infection with cell-free supernatant from respective clones harvested at 32 d.p.t. Results are average values from two independent experiments. Gray bars, Bac-PERV-A(130A12); white bars, Bac-PERV-A(151B10); hatched bars, Bac-PERV-A(463H12); black bars, Bac-PERV-B(192B9).

FIG. 4.

Proviral integration of functional PERV. (A) Genomic DNA of HeLa cells transfected with DNA of BAC clones was isolated at 40 d.p.t. by CsCl gradient centrifugation to avoid amplification of episomal proviral sequences. This DNA was tested for integration of PERV by PCR (for details, see text). The PCR amplification product was calculated to be 729 bp in length. (B) Same experiment as for panel A conducted with genomic DNA isolated from HeLa cells at 40 d.p.i. that had been treated with cell-free supernatant of HeLa cells harvested at 32 d.p.t. Genomic DNA of uninfected HeLa cells was prepared from the same stock used to perform the transfection experiment. Lane 1, Bac-PERV-A(130A12); lane 2, Bac-PERV-A(151B10); lane 3, Bac-PERV-A(463H12); lane 4, Bac-PERV-B(192B9); lane 5, uninfected HeLa cells; M, molecular size standard (Smart ladder; Eurogentec, Brussels, Belgium).