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2 nucleotide sequences
1 Institut für Tierzucht und Genetik, Veterinärmedizinische Universität Wien, A-1210 Wien, Austria
2 ApoGene Biotechnologie, D-86567 Hilgertshausen, Germany
3 Lehrstuhl für Molekulare Tierzucht und Biotechnologie, Ludwig-Maximilians-Universität München, D-85764 Oberschleißheim, Germany
Correspondence
Nikolai Klymiuk
n.klymiuk{at}gen.vetmed.uni-muenchen.de
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ABSTRACT |
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sequences described to date have been classified into several families. The known infectious, human-tropic PERVs have been assigned to the PERV 1 subfamilies A, B and C. High copy numbers and full-length clones have also been observed for an additional family, designated PERV 2. The aim of this study was to examine the PERV 2 family by analysis of retroviral pro/pol gene sequences. The proviral load was observed to be similar among various pig breeds. Although clones harbouring an open reading frame in the examined region were found, analysis of published large PERV 2 clones revealed multiple deleterious mutations in each of the retroviral genes. Various recombination events between 2 genomes were revealed. In contrast to PERV 1, phylogenetic analyses did not distinguish defined subfamilies, but indicated the independent evolution of the proviruses after a single event of retroviral amplification. Expression analysis showed large PERV 2 transcripts and variable transcription in several tissues. Analysis of the two published 2 env gene sequences observed the partial lack of the receptor-binding domain. Overall, this study indicated the low infectious potential for PERV 2. The GenBank/EMBL/DDBJ accession numbers for the sequences determined in this work are DQ084470 (clone g63), DQ084471 (g620), DQ084472 (g61), DQ084473 (g68x), DQ084474 (g611), DQ084475 (g617), DQ084476 (g619), DQ084477 (m2116), DQ084478 (m2118) and DQ084479 (g618x).
Present address: Lehrstuhl für Molekulare Tierzucht und Biotechnologie, Moorversuchsgut, Hackerstraße 27, D-85764 Oberschleißheim, Germany. 
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Phylogenetic analyses have classified ERVs into the retroviral 
(B- or D-type) and (C-type) genera, which consist of various families (van Regenmortel et al., 2000). The PERV sequences described to date have been classified into ten families, 1 to 10. The well-characterized PERV 1 family and an additional family originally named PERV E are present at about 50 copies per haploid genome, whereas the other families are present with less than ten copies each (Klymiuk et al., 2002; Mang et al., 2001; Patience et al., 2001). Breed-specific and/or individual variations have been revealed for the PERV 1 load (Magre et al., 2003).
The known infectious, human-tropic PERVs have been assigned to the PERV 1 family consisting of the subfamilies A, B and C. Differences in the env genes of the three subfamilies explain their different host tropisms (Patience et al., 2001). Functional hybrid viruses derived from recombination events of PERV 1 genomes have been found (Klymiuk et al., 2003a; Lee et al., 2002; Oldmixon et al., 2002).
PERV E was named after its phylogenetic relationship to the human ERV (HERV) E sequences (Mang et al., 2001). Independently, sequences of this family were also designated PERV 2 and 6 (Klymiuk et al., 2002; Patience et al., 2001). Following the nomenclature of PERV sequences proposed by Patience et al. (2001), in this study we have classified the sequences of this family as PERV 2.
Few PERV 2 sequences have been published to date. Two clones have been derived from a domestic pig genomic DNA phage 
library. One clone (GenBank no. AF356697) showed a complete 8 kb retroviral sequence, whereas the other 7 kb clone (GenBank no. AF356698) lacked the 5' LTR and gag untranslated regions. Multiple mutations have been observed in both sequences (Mang et al., 2001). In another study, a non-functional 1 kb pro/pol gene sequence (GenBank no. AF274706) was found (Patience et al., 2001). Recently, we described 17 additional pro/pol clones (including GenBank nos AF511100–AF511110) derived from at least 11 loci amplified from the Landrace genome. None of the clones was found to contain an open reading frame (ORF) (Klymiuk et al., 2002).
Here, we examined PERV 2 in various pig breeds at the genomic and expression level and carried out phylogenetic analyses by using pro/pol and env gene sequences.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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2 pro/pol sequences were examined in three animals each of the six pig breeds Duroc, Landrace, Large White, Mangaliza, Pietrain and Turopolja. The genomic organization of the PERV 2 sequences was analysed in Duroc, Landrace and Mangaliza animals, as well as in Munich Troll mini pigs and Schwaebisch–Haellisch pigs. Expression analysis was carried out in Landrace animals.
Detection of PERV 2 sequences.
Genomic pig DNA was used to amplify PERV 2 pro/pol sequences according to published protocols (Klymiuk et al., 2002). Briefly, five degenerate primers (two 5' primers and three 3' primers) were mixed in a PCR with an annealing temperature of 38 °C. PCR products were separated by agarose-gel electrophoresis and 0·5–1·2 kb fragments were isolated and cloned into the pGEM-T Easy vector (Promega). After bacterial transformation into TOP10F' cells (Invitrogen) and cultivation on agar plates, bacterial colonies were transferred to Hybond-N+ membranes (Amersham Biosciences). After treatment with bacterial cell-lysis solution (0·5 M NaOH, 1·5 M NaCl), the filters were neutralized [0·5 M Tris/HCl (pH 7·5), 1·5 M NaCl] and washed (5x SSC). Fixation of DNA was done by UV cross-linking.
Radiolabelling of the probe and hybridization were carried out according to standard protocols. The 941 nt PERV 2-specific PCR product from clone m211 (GenBank no. AF511105) showed no cross-hybridization to the closest relative, PERV 10 (Klymiuk et al., 2002), and was used as a probe. Plasmid DNA from positive clones was prepared and sequenced with an Amersham DNA sequencing kit and an ABI PRISM 377 automated DNA sequencer. Clones were sequenced bidirectionally by using additional primers that annealed within the sequences.
Bioinformatic tools.
Sequences were analysed for homology, intact ORFs and endonuclease restriction sites by using Gene Jockey II (Biosoft). Additional PERV 2 sequences were obtained from GenBank by using a BLAST search. For multiple alignments, sequences were prepared in SeqApp (http://ftp.bio.indiana.edu/soft/molbio/seqapp/) or BioEdit (Hall, 1999), aligned by CLUSTAL_W (Jeanmougin et al., 1998) and post-edited manually. MacClade (http://phylogeny.arizona.edu/macclade/macclade.html) and PAUP* (http://www.paup.csit.fsu.edu/downl.html) were used to prepare the data for the phylogenetic studies. Most-parsimony, neighbour-joining and maximum-likelihood phylogenetic trees were calculated with the PHYLIP package (http://evolution.genetics.washington.edu/phylip.html) and trees were generated by TreeView (http://taxonomy.zoology.gla.ac.uk/rod/rod.html).
Southern blot hybridization.
For analysis of the genomic organization of PERV 2, porcine DNA was isolated from tissue samples by standard protocols. Ten micrograms of genomic DNA was digested with EcoRV or PstI and separated on a 1 % agarose gel. After transfer of the DNA to Hybond-N+ membranes (Amersham Pharmacia Biotech), hybridization was carried out using the 941 nt PERV 2-specific probe described for the colony hybridization (see above).
PERV 2 expression analysis.
Total RNA was extracted from eight tissues (blood, heart, kidney, liver, lung, placenta, spleen and thymus) by using TRIzol reagent (Invitrogen). Two pregnant Landrace sows and two or three of their fetuses each were examined in the last third of the pregnancy. Potential DNA contamination was removed by DNase I treatment [2 U (µg RNA)–1; Roche] in the presence of RNasin ribonuclease inhibitor [1 U (µg RNA)–1; Promega]. Reverse transcription was carried out with Superscript II RNase H– reverse transcriptase (Invitrogen) and oligo(dT)12–18 primers (Invitrogen), the primer rna-tag [5'-(GA)7-CTCGAGCGGCCGC(T)16V-3'] or random-primer hexamers (Promega) following the protocol supplied by the manufacturer.
RNA integrity was assessed with the primers act1 (5'-ACCACACCTTCTACAACGAGC-3') and act2 (5'-TAGTTTCGTGAATGCCGCAGG-3'), producing a 573 nt fragment from -actin cDNA. PERV 2-specific pro/pol sequences were amplified with the primers f37 (5'-CAACCTGKGGCTCCCCTCTYG-3') and r840 (5'-TTYCTTTGTCCCTGTATGTGK-3') resulting in an 804 nt product, and the primers f191 (5'-TRATGGGAARAGAYCTGCTGG-3') and r840 giving rise to a 650 nt product. Absence of genomic DNA contamination was shown by using the PERV 2-specific primers and RNA that had not been reverse-transcribed as template.
RNA integrity, DNA contamination and PERV 2 expression were analysed by PCR with an initial denaturation step at 95 °C for 2 min, followed by 35 cycles of 95 °C for 30 s, 56 °C for 30 s and 72 °C for 1 min. For quantification of PERV 2 expression, the DNA concentration of the 941 nt PERV 2-specific probe m211 (GenBank no. AF511105) was measured by spectrophotometry at 260 nm and also determined visually on an agarose gel. Dilutions of the probe were used as controls in the semi-quantitative RT-PCR.
The amplification products of the RT-PCR were purified, cloned into the pGEM-T Easy vector and sequenced as described above to confirm their PERV 2 origin. Long-range PCR was done on cDNA from liver and thymus generated with the primer rna-tag. The primers f37 and cdna-tag [5'-(GA)7-CTCGAGCGGCCGCTT-3'] were used with an initial denaturation step of 95 °C for 4 min, followed by 20 cycles of 95 °C for 1 min, 56 °C for 1 min and 72 °C for 6 min, and 15 cycles of 95 °C for 1 min, 56 °C for 1 min and 72 °C for 9 min. The PCR products were diluted 1 : 10 000 and used as a template for the semi-nested PCR with the primers f191 and cdna-tag. The PCR products were separated on a 1 % agarose gel and transferred to membranes. PERV 2-specific hybridization was carried out as described above.
Nucleotide sequence accession numbers.
The sequences determined in this study have been deposited in GenBank with accession numbers DQ084470 (clone g63), DQ084471 (g620), DQ084472 (g61), DQ084473 (g68x), DQ084474 (g611), DQ084475 (g617), DQ084476 (g619), DQ084477 (m2116), DQ084478 (m2118) and DQ084479 (g618x). ORF-containing clones are indicated with an ‘x’.
Our recently described non-functional PERV 2 sequences (Klymiuk et al., 2002) were included in the phylogenetic analysis of this study and have been published with GenBank accession numbers AF511100 (m116), AF511101 (m2110), AF511102 (m2119), AF511103 (m2113), AF511104 (m218), AF511105 (m211), AF511106 (m2117), AF511107 (m217), AF511108 (m1116), AF511109 (p1117) and AF511110 (m216).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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2 pro/pol clones2 proviruses. In previous studies, analysis of three pigs of the Landrace, and LandracexDuroc breeds and an unspecified breed observed no intact pro/pol sequences (Klymiuk et al., 2002; Mang et al., 2001; Patience et al., 2001). For evaluation of PERV 2 diversity, genomic DNA was pooled from 18 animals of six different breeds to obtain a broad spectrum of 2 proviruses. After amplification and cloning of the 1 kb fragments, PERV 2-specific colony hybridization revealed 19 positive clones. Sequence analysis of these clones found that 18 showed >90 % identity to published 2 sequences and had a length of 810–950 nt. Five of the 18 clones revealed an ORF [g67x, g68x (GenBank no. DQ084473), g69x, g612x and g618x (GenBank no. DQ084479)] and four [g67x, g69x, g612x, g618x (GenBank no. DQ084479)] differed in not more than four out of 941 nt (0·4 %). One clone (g610) had a 132 nt gap, corresponding to five previously described clones [m216 (GenBank no. AF511110), m2111, m2118, p118, p1117 (GenBank no. AF511109)].
Phylogeny and recombination analysis of the PERV 2 pro/pol clones
In addition to our clones and the published 2 sequences AF356697, AF356698 (Mang et al., 2001) and AF274706 (Patience et al., 2001), a BLAST search revealed five 7–10 kb retroviral sequences (GenBank nos AX111980, AX175459, AX175460, AX175461 and NC_003059) and the 120 kb BAC clone PigE-108A11 (GenBank no. CR450381) that were highly homologous to the PERV 2 family. Thus, a total dataset of 44 pro/pol fragments (808–950 nt) was used in the phylogeny and recombination analysis (Table 1).
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a). The genetic distances of between 0·000 and 0·005 were proposed to occur within a given haplotype, whereas genetic distances between different haplotypes resulted in higher values. Thus, we defined 22 haplotypes and chose representative clones for each haplotype by the lowest number of single nucleotide exchanges in an alignment of all 44 sequences (not shown). Subsequently, phylogenetic trees were generated from the 22 haplotypes and showed weak resolutions (Fig. 1b). Eleven haplotypes representing 16 sequences were separated by bootstrap values higher than 50 %. All 16 sequences were analysed in a separate nucleotide alignment, which was condensed by PAUP* as described previously (Klymiuk & Aigner, 2005). The alignment showed 198 polymorphic nucleotides (20 %) and clearly revealed four different recombination patterns (Fig. 1c). The recombined sequences were separated in homologous and different fragments in the alignment, and phylogenetic trees were generated from this manipulated dataset. The trees revealed a bush-like evolution of the PERV 2 family with low bootstrap values (Fig. 1d). Independent evolution of the 2 proviruses was confirmed by their nucleotide alignment, as 273 out of 282 polymorphic sites (97 %) were either single nucleotide exchanges or patterns occurring only once in the alignment and therefore did not contribute to the evaluation of the phylogenetic relationship between the haplotypes (data not shown).
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2 proviral load in different pig breeds2 proviral load was examined by Southern blot analysis and was estimated to be 50 copies per haploid genome for the five breeds Duroc, Landrace, Large White, Mangaliza and Pietrain, and about 25 copies in the Turopolja breed (Klymiuk et al., 2002). BamHI restriction analysis resulted in a 2-specific 3 kb fragment for most of the 2 loci due to conserved restriction sites among the proviruses (e.g. in GenBank no. AF356697 at nt 926 and 3943). To evaluate the genomic distribution of the proviruses among the breeds, we produced 2 locus-specific signal patterns. PERV 2 env and LTR sequences have not been well defined to date and therefore the 2-specific pro/pol fragment was chosen as a probe. EcoRV and PstI showed different restriction sites in AF356697, AX111980 and CR4503812 and were used for the digestion of genomic DNA. Analysis of the five breeds Duroc, Landrace, Mangaliza, Munich Troll and Schwaebisch–Haellisch showed similar signal patterns among the breeds (not shown).
PERV 2 expression
Expression analysis of PERV 2 pro/pol sequences was carried out in various tissues of pregnant Landrace animals and their fetuses. PERV 2-specific primers were designed to amplify the cDNA of the ORF containing sequences g67x, g68x (GenBank no. DQ084473), g69x, g612x and g618x (GenBank no. DQ084479). Use of the primer pair f37 and r840 resulted in detection of the expected 804 nt product (Fig. 2). Sequencing of 24 RT-PCR clones confirmed the PERV 2 origin. In addition, smaller fragments were obtained in several tissues. These 682 nt transcripts were found to be identical to p118 by sequence analysis. Use of the primer pair f191 and r840 gave analogous results (not shown). The results of the semi-quantitative PCR were validated by repeating the cDNA preparation three times.
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2 expression was examined in various tissues of seven animals. Differences were observed among non-related as well as among related animals. Compared with fetal tissues, lower expression levels were observed in pregnant pigs. No tissue was found to be negative in all animals; however, low abundance generally occurred in the heart and blood. A more diverse expression pattern was found in other organs, where the expression level was not consistent in the tested animals. The maximum expression level of about 10 000 copies (µg total RNA)–1 was found in the spleen and liver (Table 2).
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2 expression. The primer pairs f37/cdna-tag and f191/cdna-tag failed to produce a visible product. However, a semi-nested PCR using dilutions of the f37/cdna-tag PCR products as template for a subsequent PCR with the primers f191 and cdna-tag resulted in multiple bands (Fig. 3a). The separated PCR products were blotted on to a nylon membrane and hybridized with the PERV 2-specific probe used for colony and Southern blot hybridization. In the liver and thymus, several signals occurred. In addition to strong 1–2 kb signals, a faint 5 kb signal was observed (Fig. 3b).
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2 full-length sequences2 sequences outside the investigated pro/pol region. In the phylogenetic analysis of the 1 kb pro/pol fragment, four (GenBank nos AF356698, AX111980, AX175459 and AX175460), three (GenBank nos AF356697, AX175461 and NC_003059) and one (GenBank no. CR450381) of these were classified as haplotypes. In the adjoining parts of the sequence, they differed in not more than 3 nt out of 7 kb within a haplotype. Compared with GenBank no. AF356698, the 10 753 nt sequence AX111980 had two mismatches in the overlapping 7153 nt region and extended the PERV 2 sequence at the 5' end by 84 nt. The upstream sequence was not of PERV 2 origin; thus, the AX111980 provirus lacked 967 nt at the 5' end. The 120 kb clone PigE-108A11 (GenBank no. CR450381) was derived from a Large WhitexMeishan offspring. The 6298 nt 5' end of CR450381 appeared to be a PERV 2 sequence in reverse orientation and was therefore designated CR4503812. The ORF containing the 941 nt clone g618x was identical to the corresponding CR4503812 sequence. The BAC clone contained the 5' LTR and the gag and pro/pol genes, but lacked most of the env gene. Thus, we found three different haplotypes of PERV 2 revealing large parts of the respective provirus. The alignment of the three sequences with GenBank nos AF356697, AX111980 and CR4503812 showed deleterious mutations (gaps, insertions and stop codons) in each sequence, but indicated a lower number of mutations in the gag and pro/pol genes of CR4503812. The largest ORF-containing fragment included the region from nt 2683 to 3900 (Fig. 4).
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2 provirus containing both LTRs differing in 2 out of 434 nt. In the absence of selective pressure, the age of proviruses depends on the neutral mutation rate in the genome and the generation time. For PERV 1, a mutation rate of 2·3x10–9–5·0x10–9 substitutions nt–1 year–1 has been calculated (Tönjes & Niebert, 2003). This proposed mutation rate resulted in an age of 100 000–200 000 years for the sequence with GenBank no. AF356697.
Analysis of PERV 2 Env sequences
GenBank sequences AF356697 and AX111980 contained the complete env gene, which is crucial for retroviral host tropism. We compared the published PERV 2 env sequences with other retroviral env genes at the amino acid level. A BLAST conserved-domain database search (Marchler-Bauer et al., 2005) revealed the pfam00429.11 database (Bateman et al., 2004), which contains the most diverse retroviral Env proteins. We included the Env protein of the PERV 2 proviruses AF356697 and AX111980, as well as those from representatives of the three subfamilies A, B and C of PERV 1 (GenBank nos AJ279056, AY099324 and AF038600; Klymiuk et al., 2003a) in the alignment of the dataset. Furthermore, the Env protein of HERV E (GenBank no. M10976), HERV R (GenBank no. M12140), human syncytin (GenBank no. NG_004112), the endogenous human chronic myeloid leukemia virus (HCML; GenBank no. AF499232) and the murine leukemia virus (MLV; GenBank no. NC_001501) were included. M10976, a multiple defective HERV E (HERV 4-1), was used for phylogenetic analysis by Mang et al. (2001). M12140 was obtained from a disrupted provirus named HERV R or ERV3 in different studies (Andersson et al., 2002; de Parseval et al., 2003) and NG_004112 encodes human syncytin, a gene of proviral origin also named HERV W, which is involved in placental morphogenesis (Blond et al., 2000; Mi et al., 2000). The intact HERV sequence AF499232 was found to be the closest relative of PERV 2 in the BLAST search and MLV (NC_001501) has been used previously to assign the Env domains (Bénit et al., 2001; Rothenberg et al., 2001).
The 19 sequences were aligned and resulted in an 806 aa alignment. Phylogenetic trees were based on all conserved regions spanning aa 70–107, 194–220, 327–357, 398–451 and 537–782 and revealed the PERV 2 sequences to be related most closely to HCML, HERV E and HERV R. Three other branches contained the other retroviruses. They were clearly separated from the 2-containing branch by the most-parsimony method, as well as by the genetic-distance method. In addition, five separate phylogenetic trees based on defined highly conserved regions (aa 70–107, 327–357, 612–642, 656–702 and 754–782) of the Env protein confirmed the same branching (Fig. 5a). However, the 2 sequences had lengths of 489 and 511 aa, whereas the HERV sequences contained 664–672 aa. To understand this difference, we aligned 2 Env with its closest relatives. Alignment of the five sequences resulted in a dataset of 686 positions. Identity as well as similarity analysis of the amino acids was carried out by defining similarity as positive scores in the PAM120 scoring matrix. A large 123 aa gap from aa 136 to 258 in the alignment explained the different lengths, whereas the rest of the alignment was conserved among the sequences (Fig. 5b). The additional alignment of the thoroughly investigated MLV Env (Rothenberg et al., 2001) localized the gap in the receptor-binding domain (RBD) of the PERV 2 env gene.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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2 pro/pol sequences revealed a number of ORF-containing clones, as well as recombined clones. We found five ORF-containing clones, which could be divided into two 2 haplotypes. One haplotype matched the 6298 nt sequence CR4503812, showing multiple mutations outside the investigated pro/pol region. In total, we identified 21 haplotypes as defective, which was almost half of the number of PERV 2 loci in the porcine genome. Thus, the presence of intact PERV 2 proviruses in the pig genome cannot be ruled out, although their number certainly is low.
With regard to retroviral recombination, the observation of hybrid PERV 2 pro/pol clones (14 %, n=5) was in accordance with our findings in BAC clones of the PERV 1 family (Klymiuk et al., 2003a). Our previous study on ovine ERVs using the same PCR amplification technique failed to detect retroviral recombination in sheep, showing that the method does not result in artificial recombination per se (Klymiuk et al., 2003b). Retroviral recombination occurs favourably between similar sequences, but has also been found between distantly related sequences (Mikkelsen & Pedersen, 2000; Negroni & Buc, 2001). Chimeric ERVs consisting of B/D-type pro/pol and C-type env sequences have been described (Huder et al., 2002).
Contrary to the establishment of the three subfamilies A, B and C in the PERV 1 family, which are discriminated by the phylogeny of env as well as of the pro/pol gene (Klymiuk et al., 2003a), PERV 2 pro/pol revealed a bush-like evolution without defined subfamilies. Whether the PERV 2 env genes encode different host tropisms will need to be addressed in further analyses. Comparison of the two published PERV 2 Env protein sequences with related retroviral Env sequences showed the absence in both clones of large parts of the RBD, which is essential for host-cell infection. These results strongly indicate diminished infectious potential of PERV 2, although transcriptional activity was shown for several proviral loci.
Phylogenetic analysis of PERV 2 included the definition of retroviral haplotypes and the identification of recombined sequences. This method may also help our understanding of the evolution of other multi-copy ERV families, such as the HERV families, which are present in the human genome at up to 1000 copies each (de Parseval et al., 2003; Tristem, 2000). The phylogeny of the PERV 2 pro/pol sequences supported the propagation of the proviruses in the porcine genome by a single round of (re)infection. A significantly lower 2 copy number has been revealed in the ancestor of the domestic pig, the wild boar (Mang et al., 2001). The age of 2 was estimated to be 100 000–200 000 years by using the LTR of GenBank accession no. AF356697 and a similar 2 signal pattern was found in the tested breeds. Therefore, the data indicate an accumulation event in the time period following pig domestication, but before the establishment of modern pig breeds. Based on comparisons of the LTRs, integration of PERV 2 presumably took place after the establishment of the PERV 1 family. However, this is in contrast to the occurrence of multiple mutations in the proviruses. The additional finding of an identical 132 nt gap in three independent haplotypes, as well as the partial absence of the RBD in the two env genes examined, may suggest the hypothesis of co-infection, but leaves the mechanism of the accumulation of PERV 2 in the porcine genome unclear.
Expression analysis of PERV 2 in seven individuals showed an inconsistent pattern. Under the influence of environmental factors, the diverse genetic background of the animals may explain the varying presence of 2 transcripts in our samples. This aspect also needs to be examined further in PERV 1. Overall, our analysis of the PERV 2 family at the genetic and expression levels revealed novel findings for PERV biology. Our data did not indicate an obvious infectious risk of PERV 2 for the implementation of xenotransplantation.
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REFERENCES |
|---|
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
Bateman, A., Coin, L., Durbin, R. & 10 other authors (2004). The Pfam protein families database. Nucleic Acids Res 32, D138–D141.
Beckmann, J. P., Brem, G., Eigler, F. W., Günzburg, W., Hammer, C., Müller-Ruchholtz, W., Neumann-Held, E. M. & Schreiber, H.-L. (2000). Xenotransplantation von Zellen, Geweben oder Organen. Berlin: Springer (in German).
Bénit, L., Dessen, P. & Heidmann, T. (2001). Identification, phylogeny, and evolution of retroviral elements based on their envelope genes. J Virol 75, 11709–11719.
Blond, J.-L., Lavillette, D., Cheynet, V., Bouton, O., Oriol, G., Chapel-Fernandes, S., Mandrand, B., Mallet, F. & Cosset, F.-L. (2000). An envelope glycoprotein of the human endogenous retrovirus HERV-W is expressed in the human placenta and fuses cells expressing the type D mammalian retrovirus receptor. J Virol 74, 3321–3329.
de Parseval, N., Lazar, V., Casella, J.-F., Benit, L. & Heidmann, T. (2003). Survey of human genes of retroviral origin: identification and transcriptome of the genes with coding capacity for complete envelope proteins. J Virol 77, 10414–10422.
Hall, T. A. (1999). BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symp Ser 41, 95–98.
Huder, J. B., Böni, J., Hatt, J.-M., Soldati, G., Lutz, H. & Schüpbach, J. (2002). Identification and characterization of two closely related unclassifiable endogenous retroviruses in pythons (Python molurus and Python curtus). J Virol 76, 7607–7615.
Jeanmougin, F., Thompson, J. D., Gouy, M., Higgins, D. G. & Gibson, T. J. (1998). Multiple sequence alignment with Clustal X. Trends Biochem Sci 23, 403–405.[CrossRef][Medline]
Klymiuk, N. & Aigner, B. (2005). Reliable classification and recombination analysis of porcine endogenous retroviruses. Virus Genes 30, 357–362.[Medline]
Klymiuk, N., Müller, M., Brem, G. & Aigner, B. (2002). Characterization of porcine endogenous retrovirus pro-pol nucleotide sequences. J Virol 76, 11738–11743.
Klymiuk, N., Müller, M., Brem, G. & Aigner, B. (2003a). Recombination analysis of human-tropic porcine endogenous retroviruses. J Gen Virol 84, 2729–2734.
Klymiuk, N., Müller, M., Brem, G. & Aigner, B. (2003b). Characterization of endogenous retroviruses in sheep. J Virol 77, 11268–11273.
Lee, J.-H., Webb, G. C., Allen, R. D. M. & Moran, C. (2002). Characterizing and mapping porcine endogenous retroviruses in Westran pigs. J Virol 76, 5548–5556.
Magre, S., Takeuchi, Y. & Bartosch, B. (2003). Xenotransplantation and pig endogenous retroviruses. Rev Med Virol 13, 311–329.[CrossRef][Medline]
Mang, R., Maas, J., Chen, X., Goudsmit, J. & van der Kuyl, A. C. (2001). Identification of a novel type C porcine endogenous retrovirus: evidence that copy number of endogenous retroviruses increases during host inbreeding. J Gen Virol 82, 1829–1834.
Marchler-Bauer, A., Anderson, J. B., Cherukuri, P. F. & 21 other authors (2005). CDD: a Conserved Domain Database for protein classification. Nucleic Acids Res 33, D192–D196.
Mi, S., Lee, X., Li, X.-P. & 9 other authors (2000). Syncytin is a captive retroviral envelope protein involved in human placental morphogenesis. Nature 403, 785–789.[CrossRef][Medline]
Mikkelsen, J. G. & Pedersen, F. S. (2000). Genetic reassortment and patch repair by recombination in retroviruses. J Biomed Sci 7, 77–99.[Medline]
Negroni, M. & Buc, H. (2001). Mechanisms of retroviral recombination. Annu Rev Genet 35, 275–302.[CrossRef][Medline]
Oldmixon, B. A., Wood, J. C., Ericsson, T. A., Wilson, C. A., White-Scharf, M. E., Andersson, G., Greenstein, J. L., Schuurman, H.-J. & Patience, C. (2002). Porcine endogenous retrovirus transmission characteristics of an inbred herd of miniature swine. J Virol 76, 3045–3048.
Patience, C., Switzer, W. M., Takeuchi, Y., Griffiths, D. J., Goward, M. E., Heneine, W., Stoye, J. P. & Weiss, R. A. (2001). Multiple groups of novel retroviral genomes in pigs and related species. J Virol 75, 2771–2775.
Rothenberg, S. M., Olsen, M. N., Laurent, L. C., Crowley, R. A. & Brown, P. O. (2001). Comprehensive mutational analysis of the Moloney murine leukemia virus envelope protein. J Virol 75, 11851–11862.
Tönjes, R. R. & Niebert, M. (2003). Relative age of proviral porcine endogenous retrovirus sequences in Sus scrofa based on the molecular clock hypothesis. J Virol 77, 12363–12368.
Tristem, M. (2000). Identification and characterization of novel human endogenous retrovirus families by phylogenetic screening of the human genome mapping project database. J Virol 74, 3715–3730.
van Regenmortel, M. H. V., Fauquet, C. M., Bishop, D. H. L. & 8 other editors (2000). Virus Taxonomy: the Classification and Nomenclature of Viruses. Seventh Report of the International Committee on Taxonomy of Viruses. San Diego: Academic Press.
Received 23 September 2005;
accepted 25 November 2005.
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