Characterization of germline porcine endogenous retroviruses from Large White pig

Linda Scobie1, Samantha Taylor1, Nicola A. Logan1, Sharon Meikle1, David Onions1, Clive Patience2 and Gary Quinn2

1 Department of Veterinary Pathology, Institute of Comparitive Medicine, University of Glasgow, Switchback Road, Bearsden, Glasgow G61 1QH, UK
2 Immerge BioTherapeutics Incorporation, 300 Technology Square, Cambridge, MA 02139, USA

Correspondence
Linda Scobie
l.scobie{at}vet.gla.ac.uk


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Porcine endogenous retroviruses (PERV) are of concern when the microbiological safety aspects of xenotransplantation are considered. Four unique isolates of PERV B have been identified previously from a lambda library constructed from genomic DNA from a Large White pig. This study shows that none of these isolates are replication competent when transfected into permissive human or pig cells in vitro, and the removal of flanking genomic sequences does not confer a human tropic replication competent (HTRC) phenotype on these PERV proviruses. Analysis of the envelope sequences revealed that PERV B demonstrated high similarity to the envelope sequences derived from replication-competent PERV, indicating that lack of replication competence does not appear to be attributable to this region of the provirus. These data complement recent findings that HTRC PERV are recombinants between the PERV A and PERV C subgroups, and that these recombinants are not present in the germline of miniature swine. Together, these results indicate that these individual PERV B proviruses are unlikely to give rise to HTRC PERV.


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The use of pig organs in human transplant recipients has raised questions as to the zoonotic potential of pig micro-organisms present in the donor organ or tissue (Deng et al., 2000; Krach et al., 2001; Stoye et al., 1998; Takeuchi & Weiss, 2000). The majority of porcine pathogens can be removed by keeping animals in specific-pathogen-free conditions. However, these methods do not eliminate those viruses transmitted within the genome, in particular, porcine endogenous retrovirus (PERV) which has been reproducibly shown to infect human cells in vitro (Czauderna et al., 2000; Krach et al., 2001; Le Tissier et al., 1997; Niebert et al., 2002; Patience et al., 1997; Specke et al., 2001a; Wilson et al., 1998, 2000) and, under certain circumstances, in vivo (Deng et al., 2000; van der Laan et al., 2000). However, Specke et al. (2001b) demonstrated that, despite productive infection of cell lines derived from several species with PERV in vitro, none of the corresponding animal models tested showed evidence of PERV infection, regardless of the method of infection or the source of virus used. Nevertheless, the threat of zoonotic potential in vivo has raised concerns about the presence of endogenous retrovirus sequences in the pig genome. Currently, three major subfamilies of infectious PERV (A, B and C) have been identified. Of these, PERV A and B have been shown to productively infect certain human cells in vitro (Takeuchi et al., 1998).

To date, the majority of infectious PERVs have been derived from either the tissue culture-adapted porcine kidney cell line PK-15 or PK-15-infected 293 cells or have been identified as recombinant viruses (Bartosch et al., 2002; Czauderna et al., 2000; Krach et al., 2001; Le Tissier et al., 1997; Niebert et al., 2002; Oldmixon et al., 2002; Wilson et al., 1998). Human tropic replication competent (HTRC) PERV isolated from human cells infected by co-cultivation with miniature swine (MS) peripheral blood mononuclear cells (PBMC) is a recombinant between the PERV A and PERV C subtypes (Oldmixon et al., 2002). Recent data have shown that these recombinant PERVs are not present in the germline of MS (Scobie et al., 2004) and our data suggest that these are possibly exogenous PERVs (Wood et al., 2004).

We have previously isolated seven full-length proviral PERV B clones from a Large White pig, of which four were present at unique insertion sites (Herring et al., 2001). In the present study, we demonstrate that these full-length PERV B isolates are not replication competent in either human 293 or porcine ST-IOWA cells, both ordinarily permissive to PERV B infection. In certain cells, some of these PERV B isolates also exhibit limited long terminal repeat (LTR) transcriptional activity, possibly because of the reduced number of 39 bp enhancer repeats in comparison with replication-competent PERV clones. LTR sequences from all four isolates were highly similar (>97 %).

Analysis of the prevalence of these PERV B proviruses in both HTRC and non-HTRC MS and human decay-accelerating factor (hDAF) transgenic pigs, using flanking sequence-PCR described previously (Herring et al., 2001), demonstrated that the inheritance of these polymorphic loci did not correlate with the HTRC-transmitting phenotype (Oldmixon et al., 2002).


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Cell culture.
The following cell lines were used in this study: porcine testes, ST-IOWA; human embryonic kidney, 293; human umbilical vein endothelial cell, HuVEC; human cervical carcinoma, HeLa; human fibrosarcoma, HT1080. HuVEC (ATCC CRL1730) and HT1080 (ATCC CCL121) were obtained from the American Type Culture Collection. All cell lines were routinely maintained in DMEM (Invitrogen) supplemented with 10 % fetal bovine serum (FBS; Hyclone), 100 U penicillin ml–1 and 100 µg streptomycin ml–1 (Invitrogen), with the exception of HuVECs which were grown in human endothelial basal growth medium (Invitrogen) and 10 % FBS. The retroviral packaging cell line TELCeB was cultured as described previously (Cosset et al., 1995).

Analysis of replication competence of genomic PERV isolates.
PERV-containing lambda phage DNA was purified from clones previously isolated from a genomic library using the liquid lysate method described in Ausubel et al. (1997). Lambda clones 2441, 21321, 35121 and 310518 were used. Clone 2441 is an additional proviral insertion at the same locus as 310518; however, it possesses a different LTR nucleotide sequence and, therefore, was also tested. DNA from cosmid clone 78 was prepared using the Qiagen Plasmid Maxi kit according to the manufacturer's instructions (Qiagen). The PERV B and PERV A clones, PERV B-17 and PERV A-60, respectively (Bartosch et al., 2002), were provided by Y. Takeuchi (UCL, London, UK). Transfections of lambda phage DNA clones into mammalian cell lines were performed as described previously (Quinn et al., 2000). Following transfection, samples of supernatants were taken at 3 and 4 day intervals for up to 6 weeks and assayed for reverse transcriptase (RT) activity using the Cavidi HS-Mn2+ kit (Cavidi Tech AB) as described previously (Oldmixon et al., 2002).

For removal of flanking regions adjacent to the PERV B LTR sequences, both the cosmid 78 and lambda clone 310518 were each subcloned as two fragments using a unique NheI restriction site present in the PERV sequence. These were inserted into the promoterless cloning vector pCR4-Topo (Invitrogen) and then assayed for replication competence by transfection into 293 and ST-IOWA cells as described above.

PCR amplification and sequencing of PERV LTRs.
LTR sequences were amplified from each of the PERV provirus-containing lambda clones and also from the controls: PERV A-60 and PERV B-17. The primers used were based on the published PERV B sequence (GenBank accession no. AJ298074) and were as follows: LTR1 (5'-CCGGTACCTGAAAGGATGAAAATGCAACC-3') and LTR2 (5'-CCGCTAGCGCAGCCTGTGATCCTCCTA-3') and were each at a concentration of 10 pmol per µl. PCRs (50 µl) were performed using the PCR kit (Perkin Elmer) that contained 1·5 mM MgCl2 and 0·5 U Taq polymerase and a template of 200 ng lambda DNA was added. The reactions were cycled in an ABI 9700 thermocycler (Applied Biosystems) at 94 °C for 1 min 30 s followed by 30 cycles of 94 °C for 15 s, 66 °C for 1 min, 72 °C for 1 min, followed by 72 °C for 10 min to yield a product of approximately 800 bp. The amplified LTR products were cloned into the TA cloning vector pCR 2.1-Topo (Invitrogen), and subcloned into the luciferase reporter gene construct pGL3 basic (Promega) using the KpnI and NheI restriction enzyme sites. The constructs were designated pGL3-LTR followed by PERV or lambda clone identification. After subcloning, the sequence was determined on both strands using ABI Prism Dye terminator cycle sequencing on an ABI 373 automated sequencer (Applied Biosystems).

Luciferase reporter assays.
pGL3-LTR constructs for each lambda clone were transfected into 293, HuVEC, ST-IOWA and HT1080 cells using Lipofectamine Plus (Life Technologies) according to the manufacturer's guidelines. Each well of a 12-well dish (Costar) was seeded with 2x104 cells and transfected with 100 ng of the pGL3-LTR constructs. Cells were harvested and assayed for luciferase activity after 48 h. Transfected cells were washed with PBS and lysed with 200 µl lysis buffer (Promega) at 25 °C for 5 min. The lysate was harvested using a cell scraper and then centrifuged at 13 000 g for 1 min at room temperature to remove cell debris. Lysates (40 µl) were assayed for luciferase activity using a luminometer (Luminoscan Ascent), immediately after the addition of 40 µl luciferase substrate (Promega). Luciferase assays were carried out in triplicate for each vector, cell line and drug treatment. In all cases, cells were co-transfected with the green fluorescent protein (GFP) vector BSP2-GFP (kindly provided by M. Morrison, University of Glasgow, UK) to determine transfection efficiency. GFP plasmid (100 ng) was transfected into cell lines with Lipofectamine Plus as described above and transfection efficiency was determined by fluorescence microscopy (Leica Instruments).

Drug treatment regimens.
Cells were exposed to a particular drug for 24 h prior to transfection with the pGL3-LTR constructs. Plasmid DNA (100 ng) was transfected into 293 cells using Lipofectamine Plus and 1 µg DNA was transfected into HuVECs using Gene Juice (Novagen), according to each of the respective manufacturers' guidelines. Cells were incubated for 3 h following transfection, washed with PBS and incubated in normal media at 37 °C for 48 h. They were then harvested for luciferase assay as described above. Cell viability counts were carried out simultaneously on parallel wells with and without drug treatment to control for cell death in the culture. Cells were exposed to different drugs under the following conditions: 0·1 µg cyclosporin A (CsA) ml–1 (Sandoz), phorbol 12,13–didecanoate (PDD; Sigma) and phorbol 12-myristate 13-acetate (PMA; Sigma) at 100 nM and 10 nM, respectively, dexamethasone (dex) at 20 mM and 0·1 mM, prednisilone (Sigma) and 17-{beta} oestradiol (oest; Sigma), at 10 µM and 10 nM, respectively, TNF-{alpha} (AMS Biotechnology) at 10 ng ml–1 and 10 pg ml–1 and interferon-{gamma} (IFN-{gamma}; AMS Biotechnology) at 50 and 2·5 ng ml–1.

PERV B loci screening in MS and Large White pigs.
Using flanking sequence primers described previously in Herring et al. (2001), PCR for the PERV B proviruses 78, 35121, 310518 and 21321 and their adjacent flanking sequences was performed on the genomic DNA of a selection of animals. DNA was isolated from MS, which were designated either HTRC or non-HTRC PERV transmitters, as previously defined by co-culture of activated PBMC with 293 cells (Oldmixon et al., 2002). In addition, PCR was performed on DNA taken from 10 unrelated Large White pigs, which were transgenic for hDAF, in order to compare the prevalence of the PERV B proviruses between the breeds. PCR conditions were as described previously (Herring et al., 2001).

Assay for PERV B envelope function.
To assess envelope functions, both transient and stable transfections were carried out. For the transient assay, lambda DNA of PERV B clones 78, 35121, 310518 and 21321 was transfected into TELCeB cells (MLV-gag pol expressers) as described previously (Cosset et al., 1995) using Lipofectamine Plus in accordance with the manufacturer's recommendations. HT1080 and ST-IOWA cells were exposed to supernatants harvested 72 h post-transfection, filtered using a 0·45 µm-pore filter (Sartorius) and adjusted to a final concentration of 8 µg polybrene ml–1 (Sigma). Infectious titres were measured by X-Gal (5-bromo-4-chloro-3-indoyl {beta}-D-galactopyranoside) staining after 48 h as described previously (Cosset et al., 1995) and counting of lacZ-positive colonies. Infectious titres were determined in comparison to TELCeB PERV B pseudotype particles. The envelope regions from the same PERV B lambda clones were amplified in 50 µl reactions that contained 50 mM KCl, 10 mM Tris/HCl (pH 8·3), 1·5 mM MgCl2 150 nM each primer, 200 nM each dNTP and 2·5 U AmpliTaq (PE Biosystems) using 200 ng lambda DNA. For the env open reading frame PCR, the primers used were: env F (5'-GGATCCTAATACGACTCACTATAGGAACAGACCACCATGCATCCCACGTTAAGCCG-3') and env R (5'-CGCTCTAGACTAAGCGTAGTCTGGGACGTCGTATGGGTAGAACTGGGAAGGGTAGAGGTCAGT-3') and then cycled as follows: 95 °C for 3 min followed by 30 cycles of 94 °C for 1 min, 62 °C for 1 min, 72 °C for 2 min 10 s, followed by 10 min at 72 °C. PCR products were then cloned into the pCR2.1-Topo cloning vector (Invitrogen) according to the manufacturer's instructions. The PERV B envelope sequences were then excised from the pCR2.1-Topo vector by digestion with SpeI and XbaI and subcloned into the expression vector pCR3.1 (Invitrogen) at the XbaI site. The correct orientation of the env sequences was confirmed by digestion of the constructs with XhoI. Stable envelope functions were assessed by transfection of TELCeB cells with the various PERV B envelope clones followed by continuous selection with G418 (Gibco Life Technologies) until a bulk population of G418-resistant cells was obtained. Supernatants from confluent monolayers were then collected, filtered as described above and used to infect 293, ST-IOWA, HT1080, Mv-1-Lu and HeLa cells in the presence of polybrene using standard methodologies. Pseudotype infectivity was determined after 48–72 h by X-Gal staining and counting of lacZ-positive colonies, and was performed in comparison to the TELCeB PERV B env clones that have been reported previously (Takeuchi et al., 1998).

Nucleotide accession numbers.
Complete nucleotide sequences of PERV B-17 (AY099324) and PERV A-60 (AY099323) are available at GenBank (Bartosch et al., 2002). The sequence used for LTR sequence comparison was 293-PERV B-43 (AJ298074; Czauderna et al., 2000; Scheef et al., 2001). All PERV B LTR sequences have been deposited in GenBank. Accession numbers for LTR 21321, 2441, 310518, 78 and 35121 are AY056033, AY056032, AY056029, AY056035 and AY056034, respectively, and for PERV B env sequences isolated from Large White pigs are AY056024, AY056025, AY056027 and AY056028 for LTR 21321, 35121, 78 and 2441, respectively (Herring et al., 2001).


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Full-length PERV B isolates are not replication competent in vitro
Four intact and unique PERV B proviral sequences (78, 35121, 21321 and 310518) were isolated from a genomic DNA lambda phage library derived from the Large White pig (Herring et al., 2001). To assess whether these PERV B clones were capable of resulting in productive infection, each lambda clone was transfected into the human 293 and porcine ST-IOWA cell lines. As a control for transfection efficiency, all transfections were performed in the presence of an expression plasmid that encodes the marker GFP, and fluorescence microscopy was used to estimate transfection efficiency. Transfected cells were then analysed for the production of virus particles by the measurement of RT activity using a sensitive ELISA technique and, typically, cells were assayed twice a week for up to 60 days post-transfection. RT activity was negligible in all of the transfected cells, indicating that none of the lambda clones encoded replication-competent virus. Representative PERV B proviral clones 78 and 310518 are shown in Fig. 1. To test whether flanking genomic DNA sequences were suppressing provirus expression, clones were isolated without the flanking genomic DNA sequences and re-analysed as described above. However, removal of flanking genomic DNA did not have any effect on replication competence in comparison with control constructs (data not shown).



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Fig. 1. Replication competence of cloned PERV B proviruses isolated from a Large White pig genomic DNA library. Lambda phage DNA encoding the full-length PERV B proviruses was transfected into 293 cells. Replication competence of each virus was measured by assaying RT activity in the culture supernatant over 5 weeks. PERV A and PERV B are the replication-competent virus controls PERV A-60 and PERV B-17.

 
Next, we analysed the function of the Env protein of these four PERV B clones in both stable and transient MLV-based pseudotype assays. Clones were assayed alongside an active PK15-derived PERV B MLV pseudotype (Takeuchi et al., 1998). Under identical infection conditions, the infectious titre of the PK15 PERV B pseudotype was approximately 103 to 104 IU ml–1. In comparison, the infectious titre of the Large White-derived PERV B clone was undetectable, i.e. <4 IU ml–1 (data not shown). Complete sequence analysis of Env from these PERV B clones did not reveal any gross differences in comparison to the published PERV B envelope sequence Y12239; i.e. only one or two single amino acid changes were noted over the entire sequence. Interestingly and in contrast, the PERV B isolates and Y12239 sequence have regions that are significantly altered in comparison with germline PERV AF435966 and AF435967 sequences that were isolated from Large White pigs and have been shown to be replication competent (Fig. 2) (Herring et al., 2001). The mechanism(s) that results in the generation of these differences remains to be determined.



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Fig. 2. Alignment of amino acid region of Env that illustrates the most significant sequence variation between the PERV B isolates 78, 2441, 35121 and 21321, replication-competent PERV B isolate Y12239 and the replication-competent germline isolates AF435966 and AF435967 from Large White pigs (Niebert et al., 2002).

 
Analysis of LTR activity in PERV B clones
LTR regions comprising U3, R and U5 were amplified from PERV B lambda clones 2441, 21321, 35121, 310518 and cosmid 78 and subcloned into a luciferase reporter gene vector. Sequencing of the LTR regions revealed ~97–99 % similarity with both the PERV B published sequence (AJ298074) and the control PERV A and B LTRs [pGL3-LTR A60 and pGL3-LTR B17 (Fig. 3)].



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Fig. 3. Partial sequence alignment of the LTR enhancer region for genomic PERV B clones 78, 21321, 310518, 2441 and 35121. Dashes indicate missing bases. Bases in bold indicate changes in nucleotides between sequences. PERV LTRB (AJ298074) was used as the consensus sequence. PERV A-60 and PERV B-17 are the LTRs subcloned from the infectious PERV A and PERV B kindly provided by Y. Takeuchi.

 
Differences within the U3 region and the PERV B clones were found with respect to the 39 bp enhancer repeats. In contrast to three and four repeats for the PERV A and B controls, respectively, each of the genomic PERV B LTR clones contained two enhancer repeats (Fig. 3). Scheef et al. (2001) recently demonstrated that the number of enhancer repeats, thought to multimerize following serial passage in human cell lines, influence transcription of PERV RNA. However, in transient reporter gene analyses all PERV B clones demonstrated detectable LTR activity in comparison with PERV A and PERV B controls (Fig. 4). In ST-IOWA and HT1080 cells, clone 78 LTR (pGL3-LTR 78) exhibited levels of luciferase expression comparable to that of the PERV A and B control LTRs (Fig. 4b, d) and clones 21321 and 35121 exhibited equivalent levels to pGL3-LTR B17 in HuVEC cells (Fig. 4a). Luciferase expression in HT1080 was considerably lower for pGL3-LTR 21321, pGL3-LTR 35121 and pGL3-LTR 2441 when compared with controls (Fig. 4c). Thus, although not capable of productive infection in either pig or human cells, all the PERV B clones do appear to retain a functional LTR.



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Fig. 4. PERV B pGL3-LTR constructs transfected into HuVECs (a), HT1080 (b), 293 (c) and ST-IOWA (d) cells. Results shown are the means of three separate experiments plus the standard deviation. pGL3-basic is the backbone vector-only control. The PERV B pGL3 constructs transfected are indicated on the x-axis.

 
Effect of immunosuppressive drugs and other chemical stimuli on LTR activity
It is probable that for clinical xenotransplantation the use of immunosuppressive drugs to overcome graft rejection will be required. We wished to address the potential circumstance where the PERV B proviral sequences might be transcribed weakly under normal conditions but may become more active in the presence of certain chemical agents, particularly immunosuppressive drugs. This possibility was examined by the addition of CsA, prednisilone, PDD, PMA, dex, oest, TNF-{alpha} and IFN-{gamma} to cells prior to transfection with the PERV pGL3-LTR constructs. PDD and PMA increased the expression of all the constructs on average by 14-fold at the lower concentration of PMA and 9-fold at the lower concentration of PDD (Fig. 5a, b). Neither CsA, prednisilone, TNF-{alpha}, IFN-{gamma}, oest nor dex appeared to have any significant effect on luciferase expression in any of the constructs (data not shown). Cells were additionally treated with CsA and prednisilone both individually and in combination with no significant increase or decrease in expression observed for any of the pGL3-LTR constructs (data not shown). For HuVEC cells, in contrast to 293 cells, none of the these drugs significantly affected luciferase expression from the pGL3-LTR constructs as seen for the 293 cells. All constructs were tested in HuVECs with all the drugs described in Methods (data not shown).



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Fig. 5. Response of all PERV B pGL3-LTR constructs to PMA (a) and PDD (b) in 293 cells. Negative controls are the pGL3-basic vector backbone and no DNA. Transfections were performed in triplicate and the mean values of three experiments expressed as RLU of luminescence after normalization against the pGL3-basic vector backbone plus standard deviations.

 
Flanking data analysis
To determine the prevalence of these PERV B proviruses, PCR was carried out on DNA isolated from both HTRC- and non-HTRC-transmitting MS and unrelated Large White pigs transgenic for the human complement regulator gene hDAF. By conducting PCR using primers that extended from the PERV B LTR region into the flanking genomic sequences (Herring et al., 2001), the presence or absence of each provirus in the genome was determined. All 10 of the hDAF pigs examined were non-HTRC-transmitting and the prevalence of the PERV B proviruses in the hDAF pigs was comparable to that observed previously in Herring et al. (2001), with proviruses 35121, 21321 and 310518 being present in 8 of 10 pigs, whereas provirus 78 is present in only 2 of 10 pigs (data not shown).

Proviruses 78, 21321 and 35121 were found to be present in 3 of 15, 5 of 15 and 6 of 15 MS analysed, respectively. However, provirus 310518 was absent in all pigs (Table 1). Prevalence of these PERV B proviral sequences in MS was also found not to be associated with either the HTRC-transmitting or non-HTRC-transmitting phenotypes, suggesting that there is no correlation between PERV B and infectious PERV present in these pigs (Table 1).


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Table 1. Summary of MS tested for the prevalence of the PERV B proviruses and their transmitting or non-transmitting status

 

   DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
In this study, we examined the functional characteristics of four unique full-length PERV B proviruses that were previously identified as the only full-length PERV moieties present in the genome of a Large White pig (Herring et al., 2001). We found that, even in the presence of intact gag, pol and env genes, these full-length PERV B proviruses did not display any RT activity up to 60 days post-transfection in permissive cell lines. The finding that none of the PERV B clones encode functional envelope sequences, as measured in a pseudotype assay, provides the most reasonable explanation for the lack of infectivity associated with these proviruses. Moreover, no major differences in either the envelope sequences or LTR enhancer region were identified in comparison with prototypical PERV B sequences.

Functional analysis of the LTR regions of these clones demonstrated detectable LTR activity indicating that the LTR would likely be functional in vivo. However, one notable difference between the PERV B proviral LTRs and those from replication-competent PERV LTRs is the reduced number of 39 bp repeats. It has been shown that the PERV LTR contains putative hormone receptor-binding sites (Quinn & Langford, 2001), and at least one of the commonly used immunosuppressive drugs, prednisilone, is a hormone derivative although no significant response to prednisilone was observed for any of the PERV B LTR constructs. Analysis of PERV isolated in vitro from pig cell lines has demonstrated that an increased ability to replicate can arise through duplication of the LTR enhancer sequences during continuous passage in human cells in culture (Scheef et al., 2001). However, this does not appear to affect the promoter activity of the same PERV B LTRs studied and their response to immunosuppressive drugs (Scheef et al., 2002). More recent data has also demonstrated little variation in the transcriptional activity of the PERV subtype A, B and C LTRs in various cell lines (Wilson et al., 2003). Scheef et al. (2002) also found no effect of the immunosuppressive drugs CsA and prednisilone on PERV A or PERV B LTR activity and our data with genomic PERV B LTRs support this observation. The response in HuVEC cells was also examined as these cells are the first point of contact between the recipient's tissue and PERV arising from a xenotransplant. No increased response in LTR activity was observed for any of the constructs in these cells. Wood et al. (2004) have identified MS that do not appear to carry PERV that infects either pig or human cells, referred to as ‘PERV null’ animals. Stimulation of PBMC, from these PERV null MS, with agents known to increase PERV expression was unable to induce PERV production (Wood et al., 2004). These data, and the lack of response in primary HuVEC cells, suggest that the stimulated PERV LTR response observed for all constructs might be specific to the 293 cell. In combination, these studies would indicate that treatment with these specific drugs might constitute no increased risk of inducing infectious PERV in other human cell types in vitro and possibly in vivo.

Our previous work has shown that isolates of HTRC PERV are recombinants between PERV A and PERV C in the post-VRA region of the envelope and, subsequently, MS were identified that are known to generate these recombinant HTRC PERV (Oldmixon et al., 2002). We have recently demonstrated that these recombinants are absent from the genome of both HTRC and non-HTRC MS (Scobie et al., 2004). Furthermore, it was also found from in vivo analysis that PERV A/C-recombinants from HTRC MS might indeed be exogenous PERV generated by an alternative mechanism possibly driven by increased PERV C expression (Wood et al., 2004). These data suggest that PERV A and PERV C play a pivotal role in the generation of HTRC recombinant PERV isolated from MS and that PERV B may not contribute to this phenotype. Our investigations into the prevalence of the PERV B proviral loci that were first identified in the Large White pig (Herring et al., 2001), showed no correlation between the HTRC phenotype of MS and the presence of these PERV B proviral sequences. Therefore, within these MS, PERV B does not appear to be involved in this recombination process.

In summary, following extensive analysis of the full-length PERV B proviral clones, we found no evidence to suggest that PERV B proviruses isolated from a Large White pig were transcriptionally active in human cells or that their presence in the genome of pigs correlated with the capacity to infect human cells. In addition, we found no evidence that drug families likely to be used during a xenotransplantion situation activate transcription of these viruses. Consequently, and in light of the evidence that recombination is a requirement for the HTRC phenotype (Oldmixon et al., 2002; Scobie et al., 2004; Wood et al., 2004), germline PERV B probably poses a low risk for xenozoonosis.


   ACKNOWLEDGEMENTS
 
This work was supported by the Scottish Hospitals Endowment Research Trust grant number RG6/02 and in part by Immerge BT. The authors would like to thank Dr Y. Takeuchi for kindly donating the PERV A-60 and PERV B-17 isolates.


   REFERENCES
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A. & Struhl, K. (1997). Current Protocols in Molecular Biology. New York: Wiley.

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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.[Abstract/Free Full Text]

Patience, C., Takeuchi, Y. & Weiss, R. A. (1997). Infection of human cells by an endogenous retrovirus of pigs. Nat Med 3, 282–286.[Medline]

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Received 12 January 2004; accepted 6 April 2004.