Evidence and consequence of porcine endogenous retrovirus recombination

Abstract

The genetic nature and biological effects of recombination between porcine endogenous retroviruses (PERV) were studied. An infectious molecular clone was generated from a high-titer, human-tropic PERV isolate, PERV-A 14/220 (B. A. Oldmixon, et al. J. Virol. 76:3045-3048, 2002; T. A. Ericsson et al. Proc. Natl. Acad. Sci. USA 100:6759-6764, 2003). To analyze this sequence and 15 available full-length PERV nucleotide sequences, we developed a sequence comparison program, LOHA(TM) to calculate local sequence homology between two sequences. This analysis determined that PERV-A 14/220 arose by homologous recombination of a PERV-C genome replacing an 850-bp region around the pol-env junction with that of a PERV-A sequence. This 850-bp PERV-A sequence encompasses the env receptor binding domain, thereby conferring a wide host range including human cells. In addition, we determined that multiple regions derived from PERV-C are responsible for the increased infectious titer of PERV-A 14/220. Thus, a single recombination event may be a fast and effective way to generate high-titer, potentially harmful PERV. Further, local homology and phylogenetic analyses between 16 full-length sequences revealed evidence for other recombination events in the past that give rise to other PERV genomes that possess the PERV-A, but not the PERV-B, env gene. These results indicate that PERV-A env is more prone to recombination with heterogeneous backbone genomes than PERV-B env. Such recombination events that generate more active PERV-A appear to occur in pigs rather frequently, which increases the potential risk of zoonotic PERV transmission. In this context, pigs lacking non-human-tropic PERV-C would be more suitable as donor animals for clinical xenotransplantation.

FIG. 1.

PERV recombination profiles shown by local homology analysis. (Top) Four full-length nucleotide sequences, A14/220 (8,188 nt), Cmsl (8,144 nt), Ap60 (8,261 nt), and A130 (8,299 nt), were analyzed by LOHA with 41-nt windows. The alignment length was 8,325 nt positions and a percent match plot for A14/220 versus Cmsl was overlaid on that for A14/220 versus Ap60. (Middle) Ap60, A130, and B192 (8,255 nt) were analyzed, and a percent match plot for Ap60 versus B192 was overlaid on that for Ap60 versus A130. The alignment size was 8,372 positions. (Bottom) A463 (8,204 nt), A130, and B192 were aligned: A463 versus B192 overlaid on A463 versus A130 for 8453 positions. A schema of a PERV provirus is displayed on top to indicate the positions of the viral genes and domains. The apparent recombination crossover points are indicated by arrows.

FIG. 2.

Genetic variability of PERV-A and -B proviruses. (Top) The three germ line proviral sequences A130, A151 (8,291 nt), and A463 as well as the prototype cell line-derived PERV-A sequence Ap60 were analyzed by LOHA (aligned into 8,356 positions). (Bottom) The three germ line proviral sequences B192, B213 (8,215 nt), and Bimut (8,217 nt) as well as the prototype cell line-derived PERV-B sequence Bp17 (8,256 nt) were analyzed (aligned into 8,253 nt positions). All six possible homology plots (0, 20, 40, 60, 80, or 100% was subtracted from percent match values as indicated [y axis]) for each set of sequences are shown.

FIG. 3.

Phylogenetic trees for PERV genome regions of leader, Gag, PRO, RT, IN, SU, TM, and U3R. Phylogram trees were obtained for 16 different full-length PERV nucleotide sequences rooted against sequence A151. Scales show substitution(s) per site. Bootstrap values higher than >90% are shown.

FIG. 4.

Recombination crossover points in PERV-A/C hybrid env genes. Six nucleotide sequences of env genes (NIH, accession no. AF130444 [55]; T2A3, AF417230; T2A5, AF417231; T6E5, AF417232; T12C9, AF417229; T1428, AY364236 [28]) and three env coding sequences extracted from A14/220, Cmsl, and A130 (their lengths are between 1,917 nt of Cmsl and 1,983 nt of A130), were analyzed by LOHA_TM with a window size of 21 nt. Homology plots of Cmsl against seven A/C recombinant sequences are shown (1,984 nt positions in the alignment [x axis]; 0, 10, 20, 30, 40, 50, or 60% was subtracted from percent match values as indicated [y axis]). A plot of Cmsl versus A130 is also shown (percent match, 100 [y axis]). The apparent crossover points from PERV-A- to PERV-C-like sequence are indicated by arrows.

FIG. 5.

Infectious titers of A14/220 and Ap60 chimeras. (A) Full-length proviral PERV chimeras consisting of A14/220 (gray bars) or Ap60 (white bars) sequences were constructed by using four restriction sites: two conserved sites (NruI and NheI¹ sites), an NheI² site (created in Ap60 by mutagenesis), and an NotI site outside of the provirus genome. Infectious titer (infectious units per milliliter) of replicating PERV following establishment of persistently infected cultures by DNA transfection was measured on human 293 cells by an immunocytological focus assay (2). A data set of a representative of two independent experiments is shown by bars as well as numbers. (B) Chimeric PERV containing a mix-and-match set of protein coding region (fragments B and C in panel A) between A14/220 (gray bars) and Ap60 (white bars) sequences as well as the rest of the genome derived from A14/220 were constructed. Fragments B were divided to subfragments b1 and b2 by XbaI and fragments C were divided into to c1 and c2 by StuI. Results for chimeras containing three subfragments from A14/220 (three A14/220 plus one Ap60) as well as one subfragment from A14/220 (one A14/220 plus three Ap60) are shown. Infectious titer (infectious units per milliliter) of replicating PERV harvested 3 days after DNA transfection was measured on human 293 cells by an immunocytological focus assay. A data set of a representative of two independent experiments (transfection followed by titration) is shown.