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
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ABSTRACT |
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INTRODUCTION |
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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
).
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METHODS |
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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) ml1 (Sandoz), phorbol 12,13didecanoate (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- oestradiol (oest; Sigma), at 10 µM and 10 nM, respectively, TNF-
(AMS Biotechnology) at 10 ng ml1 and 10 pg ml1 and interferon-
(IFN-
; AMS Biotechnology) at 50 and 2·5 ng ml1.
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 ml1 (Sigma). Infectious titres were measured by X-Gal (5-bromo-4-chloro-3-indoyl
-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 4872 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
).
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RESULTS |
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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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DISCUSSION |
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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.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 12 January 2004;
accepted 6 April 2004.