Characterization of two porcine endogenous retrovirus integration loci and variability in pigs

The pig (Sus scrofa) is a potential organ donor for man but porcine endogenous retroviruses (PERVs) represent an important concern for patients, and identification or engineering of PERV-free pigs suitable for xenotransplantation is a major undertaking. Consequently, studies of variability in pigs for the presence of PERVs at specific loci are a prerequisite. We identified genomic flanking sequences of two PERVs cloned in bacterial artificial chromosomes, a replication-competent PERV-A at locus 1q2.4 and a defective PERV-B at locus 7p1.1–2. PERV-A is embedded in the second repeat of a tandem of eight 190 bp repeats. A short duplicated 4 bp cellular motif, AGAC, was found at each flank of PERV-A and a degenerate 4 bp motif was found for PERV-B. At each locus, the PERV flanks matched expressed sequence tags available in public databases. Primer pairs were designed to amplify either genomic flanks or PERV-genomic junctions. Polymerase chain reaction screening was performed on pigs from 11 distinct Chinese breeds and from the European Large White breed. PERV-B at locus 7p1.1–2 was detected in all animals whereas the presence of PERV-A at locus 1q2.4 was variable. Our results suggest that a genetic selection can be designed to identify animals lacking a potentially active PERV at a specific locus and that Chinese and European pig breeds represent large biodiversity reservoirs to explore. Our results point also to the existence of PERVs that might be fixed in the pig genome, and that might not be eliminated by classical genetic selection.Accession numbers: Nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank databases under Accession numbers AY160111–AY160114     

Analysis of natural recombination in porcine endogenous retrovirus envelope genes

Human tropic Porcine Endogenous Retroviruses (PERVs) are the major concern in zoonosis for xenotransplantation because PERVs cannot be eliminated by specific pathogen-free breeding. Recently, a PERV A/C recombinant with PERV-C bearing PERV-A gp70 showed a higher infectivity (approximately 500-fold) to human cells than PERV-A. Additionally, the chance of recombination between PERVs and HERVs is frequently stated as another risk of xenografting. Overcoming zoonotic barriers in xenotransplantation is more complicated by recombination. To achieve successful xenotransplantation, studies on the recombination in PERVs are important. Here, we cloned and sequenced proviral PERV env sequences from pig gDNAs to analyze natural recombination. The envelope is the most important element in retroviruses as a pivotal determinant of host tropisms. As a result, a total of 164 PERV envelope genes were cloned from pigs (four conventional pigs and two miniature pigs). Distribution analysis and recombination analysis of PERVs were performed. Among them, five A/B recombinant clones were identified. Based on our analysis, we determined the minimum natural recombination frequency among PERVs to be 3%. Although a functional recombinant envelope clone was not found, our data evidently show that the recombination event among PERVs may occur naturally in pigs with a rather high possibility.     

Characterizing and mapping porcine endogenous retroviruses in Westran pigs

Since porcine endogenous retroviruses (PERVs) can infect cultured human cells, they are a potential hazard to xenotransplantation. For this reason, endogenous retroviruses from the Westran (Westmead Hospital transplantation) inbred line of pigs were analyzed by using consensus primers for the type A and type B viruses to amplify 1.8-kb envelope gene fragments. After preliminary analysis with restriction enzymes KpnI and MboI, 31 clones were sequenced. Between types A and B, five recombinant clones were identified. Fifty-five percent of clones (17 of 31) had premature stop codons within the envelope protein-encoding region. Endogenous retroviruses in Westran pigs were physically mapped by fluorescence in situ hybridization (FISH) using PERV-A and PERV-B envelope clones as probes to identify at least 32 integration sites (19 PERV-A sites and 13 PERV-B sites). The chromosomal sites of integration in the Westran strain are quite different from those in the European Large White pig. The recombinant clones suggest that defective PERVs could become infective through recombination and further that PERVs might recombine with human endogenous retroviruses in xenotransplants.     

Comparison of PERV genomic locations between Asian and European pigs

Xenotransplantation from pigs provides a possible solution to the shortage of human organs for allotransplantation. Porcine endogenous retroviruses (PERVs) are a possible obstacle to using porcine organs in addition to the immunological barriers. Three main types of PERVs (A, B and C) have been previously investigated in diverse pig breeds. To examine the copy numbers of PERVs and their genomic locations in the Korean native pig genome, we screened a BAC (Bacterial Artificial Chromosome) library with PERV-specific protease primers for initial recognition of PERV-positive clones and three sets of envelope-specific primers for the identification of PERV types. A total of 45 PERV-positive clones, nine PERV-A and 36 PERV-B, have been identified from the library screening and the BAC contigs were constructed using the primers designed from BAC end sequences (BESs). These primers were also used for SCH (Somatic Cell Hybrid) and RH (Radiation Hybrid) mapping of the PERV-positive clones. The results indicate that 45 PERV-positive BAC clones belong to nine contigs and a singleton. SCH and IMpRH (INRA-Minnesota Porcine Radiation Hybrid) mapping results indicated that there are at least eight separate PERV genomic locations, consisting of three PERV-A and five PERV-B. One contig could not be mapped, and two contigs are closely located on SSC7. Southern blotting indicates there may be up to 15 additional sites. Further investigation of these clones will contribute to a general strategy to generate PERV-free lines of pigs suitable for xenotransplantation.     

Determinants of high titer in recombinant porcine endogenous retroviruses

Porcine endogenous retroviruses (PERVs) pose a potential stumbling block for therapeutic xenotransplantation, with the greatest threat coming from viruses generated by recombination between members of the PERV subgroup A (PERV-A) and PERV-C families (PERV-A/C recombinants). PERV-A and PERV-B have been shown to infect human cells in culture, albeit with low titers. PERV-C has a more restricted host range and cannot infect human cells. A recombinant PERV-A/C virus (PERV-A14/220) contains the PERV-A sequence between the end of pol and the middle of the SU region in env. The remaining sequence is derived from PERV-C. PERV-A14/220 is approximately 500-fold more infectious than PERV-A. To determine the molecular basis for the increased infectivity of PERV-A14/220, we have made a series of vector constructs. The primary determinant for the enhanced replicative potential of the recombinant virus appeared to be the env gene. Using a series of chimeric env genes, we could identify two determinants of high infectivity; one was an isoleucine to valine substitution at position 140 between variable regions A and B, and the other lies within the proline rich region. Taken together, these results show that the novel juxtaposition of env gene sequences enhanced the infectivity of PERV-A14/220 for human cells, perhaps by stabilization of the envelope glycoprotein or increased receptor binding.     

Enhancer Activity of Solitary Long Terminal Repeat of Human Endogenous Retrovirus K

The transient expression of the luciferase reporter gene was used to detect the tissue-specific enhancer activity of the solitary extraviral long terminal repeat (LTR) of the human endogenous retrovirus K (HERV-K). The LTR was previously mapped to the 19q13.2 locus. It contains a number of potential regulatory elements including TATA box, binding sites for some nuclear factors, and a polyadenylation signal. However, an analysis of the genomic sequences close to the LTR did not reveal any known genes or the expressed sequences (EST), whose functioning could be regulated by this LTR. The enhancer activity can be preserved in the solitary LTR due to its involvement in the long-range control of genome functioning or by the absence of functional disruptive mutations within the human-specific LTR, because it is of a relatively young evolutionary age.