| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
1 ApoGene Biotechnologie, D-86567 Hilgertshausen, Germany
2 Institut für Tierzucht und Genetik, Veterinärmedizinische Universität Wien, A-1210 Vienna, Austria
3 Ludwig-Boltzmann-Institut für Immuno-, Zyto- und Molekulargenetische Forschung Wien, A-1210 Vienna, Austria
Correspondence
Bernhard Aigner
b.aigner{at}gen.vetmed.uni-muenchen.de
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
|---|
1 clones and on the complete envelope (env) gene sequences published to date. Several recombined full-length clones and a high number of different recombination patterns in the env gene were identified. In addition, recombinations with retroviral genomes not yet known were found. Thus, the potential risk of infection also exists for recombination products, including defective PERV loci.
Present address: Lehrstuhl für Molekulare Tierzucht und Biotechnologie, Moorversuchsgut, Hackerstr. 27, D-85764 Oberschleißheim, Germany 
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
|---|
). Production and cross-species infection of functional PERV have been observed in in vivo experiments (Deng et al., 2000; van der Laan et al., 2000). In addition, PERV have been shown to infect human cells in vitro (Boneva et al., 2001). Xenotransplantation of tissues derived from pigs that have been genetically modified to reduce rejection of the donor organ has been suggested to enhance the risk of infection with PERV (Weiss, 1998).
PERV are classified into the retroviral 
(B- or D-type) and (C-type) genera (van Regenmortel et al., 2000). All known human-tropic infectious PERV have been assigned to the PERV 1 family, consisting of the subfamilies A, B and C (Patience et al., 2001). Examination of porcine cell lines and pig breeds resulted in the detection of about 50 PERV 1 sequences, including several intact copies (Akiyoshi et al., 1998; Bartosch et al., 2002; Czauderna et al., 2000; Krach et al., 2001; Niebert et al., 2002). PERV 1A, -B and -C are highly homologous in their gag and pro/pol retroviral genes, whereas significant differences in the envelope (env) gene explain their different host tropism (Akiyoshi et al., 1998; Le Tissier et al., 1997; Takeuchi et al., 1998). Recently, chimeric PERV 1 sequences have been observed (Klymiuk et al., 2002; Lee et al., 2002; Oldmixon et al., 2002; Wilson et al., 2000).
To assign proviral genomic sequences to different host tropism and to evaluate the potential infectious risk of recombinant clones in xenotransplantation, we analysed full-length PERV 1 genomes as well as complete PERV 1 env gene sequences.
|
METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
|---|
1 genomes and 82 complete PERV 1 env gene sequences were identified in GenBank using BLAST searches. The GenBank accession numbers of the sequences are given in the legend to Fig. 2. Comparative sequence analysis was done using CLUSTALW (Jeanmougin et al., 1998), MACCLADE (http://phylogeny.arizona.edu/macclade/macclade.html) and SEQAPP (http://ftp.bio.indiana.edu/soft/molbio/seqapp/). Complete polymorphism pattern analysis was carried out by the alignment of the full-length PERV 1 gag, pro/pol and env genes. In the data set, the gag, pro/pol and env genes span nt 1–1577, nt 1578–5167 and nt 5040–7074, respectively. After the removal of invariant nucleotide positions, potential recombination sites were identified in individual sequences by a change in the pattern of nucleotide polymorphism. Phylogenetic trees of the respective genome fragments were created using PHYLIP (http://evolution.genetics.washington.edu/phylip.html). Recombination analysis subsequently included the nucleotide positions where only one single clone differed from the other proviruses.
|
1 env gene analysis started with the assignment of the different host tropisms to the specific nucleotide sequences representing the three subfamilies A, B and C. Subsequently, chimeric sequences were compared to these subfamilies. env gene fragments that showed significant sequence diversity to PERV 1A, -B and -C were not classified to the known subfamilies. |
RESULTS AND DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
|---|
1 full-length sequences1 nucleotide sequences harbouring the complete gag, pro/pol and env genes were taken from GenBank (Fig. 1). Alignments started with the first ATG codon of gag and ended with the env stop codon at nt 7059 and nt 7074 for PERV 1A and PERV 1B and -C, respectively. For the detection of similarities between the 16 sequences, common nucleotides were deleted and nucleotide positions where only one of the sequences showed a polymorphism (n=108) remained unconsidered. As a result, we obtained 972 polymorphic nucleotide positions (13·7 %) for further analysis. Of these, 904 (representing 83·7 % of all polymorphic nucleotides) were found to be involved in the definition of three distinct subfamilies. Patterns were assigned to PERV 1A, -B and -C, which were defined by their different host tropism. Subsequent comparison of the polymorphic nucleotide patterns revealed the appearance of recombination events in individual sequences. Four obvious recombination sites were observed in the alignment between nt 4052–4113, nt 4475–5026, nt 5059–5073 and nt 6754–6764 (Fig. 1A). Separate phylogenetic analyses were carried out for the five fragments between the four recombination sites by the most parsimony method and strictly confirmed the classification of the clones (Fig. 1B). In addition, the same result was found in genetic distance trees showing slightly lower bootstrap values (data not shown).
|
; Czauderna et al., 2000). AY099323 was derived from overlapping PCR fragments; however, the recombination sites observed matched within single PCR fragments (Bartosch et al., 2002). The origins of A66552 and A66553 were not investigated further. All five recombinant proviruses were classified to PERV 1B, in both the 5' and the 3' end, whereas the intermediate sequences were PERV 1A. The recombinant PERV 1A fragments included the partial pro/pol gene (A66552 and A66553) as well as the 3' end of pro/pol and the major part of the env gene (AF038601, AJ133817 and AY099323), resulting in the 1A host tropism for the replication-competent clones AJ133817 and AY099323. AJ133817 showed the same nucleotide polymorphism pattern as AF038601 and AY099323 3' of nt 5027. However, the exact assignment of the recombination site of the 5' end was not possible due to sequence variations in nt 4498–4975. Two of the five recombined proviruses (AJ133817 and AY099323) have been shown to be human tropic and replication competent (Bartosch et al., 2002; Krach et al., 2001), whereas another recombined locus (A66553) harboured intact ORFs for all three genes; this was not tested for its infectivity to human cells. Inclusion of the unique nucleotide positions in the recombination analysis revealed that AJ279056 showed 12 nucleotide differences in the fragment of nt 1802–1895 to the closest relative AJ293656 (data not shown). Due to the absence of a potential ‘donor’ sequence, a recombination could not be confirmed confidently as cause for this divergence.
Two additional full-length PERV 1A sequences (AF435966 and AF435967), which were derived from BAC clones of Large White pig genomic DNA and described previously to be replication competent upon transfection in human cells (Niebert et al., 2002), were not included in Fig. 1
due to multiple nucleotide polymorphisms in the gag and/or pro/pol genes. In addition to the high number of unique nucleotide positions (n=163 and 105, respectively, when aligned to the sequences of Fig. 1), pro/pol gene fragments of AF435966 differed from all PERV 1 sequences on both nucleotide and amino acid sequences due to multiple frame-shift mutations. In the separate comparative analysis of both sequences, AF435967 was found to be 1A throughout the whole sequence and AF435966 was classified to the recombined clones AF038601, AJ133817 and AY099323 with the 5' end of the intermediate 1A fragment located in the gag gene. Due to its high polymorphism, the exact 5' end of the recombination was not investigated further (data not shown). The env genes of AF435966 and AF435967 did not show increased sequence polymorphism and, therefore, were included in the subsequent study (see below).
Recombination patterns in PERV 1 env sequences
As the env gene is crucial for retrovirus host tropism and, therefore, determines which PERV are capable of infecting human cells, we focused on recombination events of this gene. In total, we screened 82 complete PERV 1 env genes (Table 1), which have been submitted to GenBank by Bosch et al. (2000) (n=9), Herring et al. (2001) (n=6), Lee et al. (2002) (n=31), Oldmixon et al. (2002) (n=11) and additional groups (n=25). The env genes of the 18 PERV 1 full-length sequences described above were included. A total of 58 fragments harboured an ORF. Designation of the env sequences to PERV 1A, -B and -C was carried out by comparison to AF417223, Y12239 and AF038600, respectively (Akiyoshi et al., 1998; Le Tissier et al., 1997; Oldmixon et al., 2002), which showed maximal sequence diversity in the nucleotide polymorphism patterns. Of these sequences, 38 env sequences (46·3 %) were classified completely to one of the three original subfamilies (Fig. 2), whereas 44 (53·7 %) were hybrid sequences (Table 1). The hybrid sequences were classified to 15 distinct recombination patterns (Fig. 2). The recombined clones included Y12238, which has been assigned previously to 1A.
|
1 subfamilies (Table 2). The first 837 nt of AF296168 showed low identity to PERV 1A, -B and -C, whereas the 3' end was classified to 1A. Four additional sequences described to be 1B (Bosch et al., 2000) harboured regions differing from the known PERV 1 subfamilies. These env sequences were suggested to be derived from recombinations with retroviral genomes not yet known.
|
1 sequences derived from in vitro studies with PK15 cells (Fig. 1) as well as in the genome of different breeds (Fig. 2). In addition to the recombined env sequences, hybrid sequences in genomic pig DNA have been observed recently for pro/pol (Klymiuk et al., 2002). Misincorporation of nucleotides during reverse transcription of the retroviral genome has been shown as the cause as well as the result of recombination processes (Mikkelsen & Pedersen, 2000). However, we found no evidence for the accumulation of polymorphic nucleotide positions around the recombination sites of the five recombinant full-length PERV 1 clones (data not shown).
The PERV 1 sequences analysed in this study have been derived from genomic pig DNA of cell lines and different breeds as well as from retroviruses after infection experiments. Due to the particular conditions of the in vitro experiments and the putative preference of detecting individual sequences by the different techniques used, the data may not represent exactly the real genomic PERV 1 load in the pigs. Compared to the proposed number of 50 PERV 1 loci in the pig genome, the high number of env genes examined in this study indicated breed-specific and/or individual sequence polymorphisms of the genomic PERV 1 load. Concise examination of additional pig breeds may lead to the detection of further PERV recombination patterns. The appearance of a low number of PERV 1C sequences is in accordance with previous reports (Akiyoshi et al., 1998; Bosch et al., 2000; Klymiuk et al., 2002; Le Tissier et al., 1997; Mang et al., 2001).
In the env genes, we observed a high number of different recombination patterns. Compared to the C-region of the surface subunit and to the transmembrane subunit, we found a smaller number of recombination events in the receptor-binding domain (RBD). This may be caused by increased sequence polymorphisms in the RBD between the subfamilies, as low sequence similarity reduces the rate of recombination (Negroni & Buc, 2001). On the other side, only a minor part of the clones harbouring recombinant RBD may give rise to infectious viruses and/or to developmental advantages under invariant environmental conditions. Three retroviruses with hybrid sequences in the RBD (AF417227, AF417228 and AF417229) have been shown to be human tropic and replication competent (Oldmixon et al., 2002); however, it is not clear if the recombination has influenced host tropism. Additional data on host tropism are also not available for AF296168 and AJ288587.
Having carried out the polymorphism pattern comparison, we assigned here the proviral nucleotide sequences to the different host tropism that has been described previously for the PERV 1 proviruses. In addition, the PERV 1 env sequences of the three subfamilies A, B and C were defined showing maximal sequence diversity in the polymorphism patterns. These results will contribute to subsequent approaches to protect the recipient from infections with PERV in xenotransplantation. Chimeric env sequences containing fragments with low identity to PERV 1A, -B and -C indicated the potential of retroviral genomes not yet known to get involved in recombination events with unknown consequences for host tropism and pathogenicity of recombinant PERV 1 proviruses. Recombinational patch repair resulting in new retroviral genomes has been described previously in defective retroviral genomes (Mikkelsen & Pedersen, 2000; Negroni & Buc, 2001). Although this has not been determined yet for mutant PERV 1 sequences, the potential infectious risk cannot be ruled out for defective PERV 1 loci.
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
|---|
Bartosch, B., Weiss, R. A. & Takeuchi, Y. (2002). PCR-based cloning and immunocytological titration of infectious porcine endogenous retrovirus subgroup A and B. J Gen Virol 83, 2231–2240.
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 Verlag.
Boneva, R. S., Folks, T. M. & Chapman, L. E. (2001). Infectious disease issues in xenotransplantation. Clin Microbiol Rev 14, 1–14.
Bosch, S., Arnauld, C. & Jestin, A. (2000). Study of full-length porcine endogenous retrovirus genomes with envelope gene polymorphism in a specific-pathogen-free Large White swine herd. J Virol 74, 8575–8581.
Czauderna, F., Fischer, N., Boller, K., Kurth, R. & Tonjes, R. R. (2000). Establishment and characterization of molecular clones of porcine endogenous retroviruses replicating on human cells. J Virol 74, 4028–4038.
Deng, Y. M., Tuch, B. E. & Rawlinson, W. D. (2000). Transmission of porcine endogenous retroviruses in severe combined immunodeficient mice xenotransplanted with fetal porcine pancreatic cells. Transplantation 70, 1010–1016.[CrossRef][Medline]
Herring, C., Quinn, G., Bower, R. & 10 other authors (2001). Mapping full-length porcine endogenous retroviruses in a large white pig. J Virol 75, 12252–12265.
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., Müller, M., Brem, G. & Aigner, B. (2002). Characterization of porcine endogenous retrovirus pro-pol nucleotide sequences. J Virol 76, 11738–11743.
Krach, U., Fischer, N., Czauderna, F. & Tonjes, R. R. (2001). Comparison of replication-competent molecular clones of porcine endogenous retrovirus class A and class B derived from pig and human cells. J Virol 75, 5465–5472.
Lee, J. H., Webb, G. C., Allen, R. D. & Moran, C. (2002). Characterizing and mapping porcine endogenous retroviruses in Westran pigs. J Virol 76, 5548–5556.
Le Tissier, P., Stoye, J. P., Takeuchi, Y., Patience, C. & Weiss, R. A. (1997). Two sets of human-tropic pig retrovirus. Nature 389, 681–682.[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.
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]
Niebert, M., Rogel-Gaillard, C., Chardon, P. & Tonjes, R. R. (2002). Characterization of chromosomally assigned replication-competent gamma porcine endogenous retroviruses derived from a large white pig and expression in human cells. J Virol 76, 2714–2720.
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.
Takeuchi, Y., Patience, C., Magre, S., Weiss, R. A., Banerjee, P. T., Le Tissier, P. & Stoye, J. P. (1998). Host range and interference studies of three classes of pig endogenous retrovirus. J Virol 72, 9986–9991.
van der Laan, L. J., Lockey, C., Griffeth, B. C. & 8 other authors (2000). Infection by porcine endogenous retrovirus after islet xenotransplantation in SCID mice. Nature 407, 90–94.[CrossRef][Medline]
van Regenmortel, M. H. V., Fauquet, C. M., Bishop, D. H. L., Carstens, E. B., Estes, M. K., Lemon, S. M., Maniloff, J., Mayo, M. A., McGeoch, D. J., Pringle, C. R. & Wickner, R. B. (editors) (2000). Virus Taxonomy. Seventh Report of the International Committee on Taxonomy of Viruses. San Diego: Academic Press.
Weiss, R. A. (1998). Transgenic pigs and virus adaptation. Nature 391, 327–328.[CrossRef][Medline]
Wilson, C. A., Wong, S., VanBrocklin, M. & Federspiel, M. J. (2000). Extended analysis of the in vitro tropism of porcine endogenous retrovirus. J Virol 74, 49–56.
Received 11 April 2003;
accepted 19 May 2003.
This article has been cited by other articles:
|
|
 |
|
  T. Preuss, N. Fischer, K. Boller, and R. R. Tonjes Isolation and characterization of an infectious replication-competent molecular clone of ecotropic porcine endogenous retrovirus class C. J. Virol., October 1, 2006; 80(20): 10258 - 10261. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. Klymiuk, M. Muller, G. Brem, and B. Aigner Phylogeny, recombination and expression of porcine endogenous retrovirus {gamma}2 nucleotide sequences. J. Gen. Virol., April 1, 2006; 87(Pt 4): 977 - 986. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Niebert and R. R. Tonjes Evolutionary Spread and Recombination of Porcine Endogenous Retroviruses in the Suiformes J. Virol., January 1, 2005; 79(1): 649 - 654. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. Bartosch, D. Stefanidis, R. Myers, R. Weiss, C. Patience, and Y. Takeuchi Evidence and Consequence of Porcine Endogenous Retrovirus Recombination J. Virol., December 15, 2004; 78(24): 13880 - 13890. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| INT J SYST EVOL MICROBIOL | MICROBIOLOGY | J GEN VIROL |
| J MED MICROBIOL | ALL SGM JOURNALS | |
