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Antimicrobial Agents and Chemotherapy, July 2007, p. 2600-2604, Vol. 51, No. 7
0066-4804/07/$08.00+0     doi:10.1128/AAC.00212-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Selective Inhibition of Porcine Endogenous Retrovirus Replication in Human Cells by Acyclic Nucleoside Phosphonates{triangledown}

Minyi Shi,1,2 Xin Wang,2 Erik De Clercq,3 Sonshin Takao,1 and Masanori Baba2*

Frontier Science Research Center, Kagoshima University, Kagoshima 890-8544, Japan,1 Division of Antiviral Chemotherapy, Center for Chronic Viral Diseases, Graduate School of Medical and Dental Sciences, Kagoshima University, Kagoshima 890-8544, Japan,2 Rega Institute for Medical Research, Katholieke Universiteit Leuven, B-3000 Leuven, Belgium3

Received 13 February 2007/ Returned for modification 29 March 2007/ Accepted 24 April 2007


   ABSTRACT
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Several anti-human immunodeficiency virus type 1 reverse transcriptase inhibitors were evaluated for their antiviral activities against porcine endogenous retrovirus in human cells. Among the test compounds, zidovudine was found to be the most active. The order of potency was zidovudine > phosphonylmethoxyethoxydiaminopyrimidine = phosphonylmethoxypropyldiaminopurine > tenofovir ≥ adefovir > stavudine.


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Xenotransplantation, the grafting of cells, tissues, or organs into different species, may be one of the solutions to overcome the extreme shortage of human allografts for transplantation (3). Pigs are considered to be the most suitable donors because of the resemblance of their organ sizes and functions to those of humans. However, there are two major obstacles to successful xenotransplantation, namely, immunological rejection and a risk of zoonosis. Recently, porcine endogenous retrovirus (PERV) has attracted much attention due to its omnipresent nature in pigs and vertical transmission in the host DNA. PERV is a type C retrovirus that is permanently integrated into the host genomic DNA as a provirus. Multiple copies of PERV proviral DNA exist in all of the breeds examined to date. PERV comprises three subtypes, PERV-A, -B, and -C, based on the divergence of their envelope genes, whereas their protease and reverse transcriptase (RT) sequences are highly conserved. PERV particles released from a variety of porcine cells have been shown to infect a certain range of human cell lines in vitro (14, 18, 27). Fortunately, PERV infection in vivo has not been demonstrated in retrospective surveys of the patients who received living porcine tissues, such as liver, kidney, and islet cells (8, 16, 17). However, Paradis and colleagues reported that long-lived microchimerism was found in some patients treated by extracorporeal splenic perfusion, which might increase a potential risk of PERV infection through its activation (16). Thus, it seems premature to exclude the possibility of PERV transmission from donor organs to recipients upon xenotransplantation. Furthermore, it is also possible that recombination between PERV and human endogenous retrovirus generates novel retroviruses pathogenic to humans (23).

Were PERV infection in humans to occur and subsequently develop into disease, antiviral chemotherapy would be the first option for the prophylaxis and treatment of the infection. Among anti-human immunodeficiency virus type 1 (HIV-1) drugs, nucleoside/nucleotide RT inhibitors (NRTIs) are the most likely to block the replication of retroviruses other than HIV-1. In fact, murine leukemia virus replication was examined in human cells and found to be susceptible to some antiviral drugs licensed for the treatment of HIV-1 infection (20). Similar studies with NRTIs were also conducted for PERV replication, and zidovudine (AZT) and didanosine proved to be potent and selective inhibitors (21, 22). Considering the fact that the acyclic nucleoside phosphonate tenofovir (PMPA) is highly effective in the treatment of HIV-1 infection and is active against a variety of retroviruses in vitro and in vivo (2, 4, 24), the inhibitory effects of certain acyclic nucleoside phosphonates on PERV replication in human cells are of particular interest in terms of xenotransplantation.

Ten compounds, PMPA, adefovir (PMEA), (R)-9-(2-phosphonylmethoxypropyl)-2,6-diaminopurine (PMPDAP), [6-(2-phosphonylmethoxy)ethoxy]-2,4-diaminopyrimidine (PMEO-DAPy) (5), AZT, stavudine (d4T), 4'-ethynylstavudine (4'-Ed4T) (15), 4'-azidothymidine (4'-AZT) (12), lamivudine (3TC), and the nonnucleoside RT inhibitor (NNRTI) nevirapine (NVP), were used for anti-PERV assays. AZT 5'-triphosphate (AZT-TP) and d4T 5'-triphosphate (d4T-TP) were used for PERV RT inhibition assays. The porcine embryonic kidney cell line PK15, the human kidney cell line 293T, and primary embryonic kidney cells derived from Kagoshima minipigs (KK5 and DRK4) were used for experiments. PERV was obtained from PK15 cell culture supernatants. The culture supernatants were collected, filtered, and used immediately for the infection of 293T cells. The culture supernatants were never kept frozen to avoid decreasing PERV infectivity, and their RT activity was frequently monitored to ensure that 293T cells were always infected with an equal amount of PERV. 293T cells were seeded in a 24-well plate (1 x 105 cells/well) and incubated for 24 h at 37°C. The cells were then exposed to 2 ml of PK15 cell culture supernatants containing 8 µg/ml Polybrene and further incubated in the absence or presence of test compounds. After a 24-h incubation, genomic DNA was extracted from the cells and subjected to real-time PCR.

The antiviral activities of test compounds were determined by the inhibition of PERV proviral DNA synthesis in the infected 293T cells. The amount of proviral DNA was determined by real-time PCR using the sense primer 5'-AGCTCCGGGAGGCCTACTC-3', the antisense primer 5'-ACAGCCGTTGGTGTGGTCA-3', and the Taqman probe 5'-6-carboxyfluorescein-CCACCGTGCAGGAAACCTCGAGACT-6-carboxytetramethylrhodamine-3' (Applied Biosystems, Roche, Branchburg, NJ). The final concentrations of the primer pairs and probe were 200 and 100 nM, respectively. The primer pair amplifies a region of the pol gene of PERV. In each experiment, serial dilutions of DNA extracted from the infected cells in the absence of test compounds were used for drawing a standard curve. The detection limit of this assay was determined to be 10 DNA copies, according to a previous report (7). The cytotoxicities of test compounds were determined in parallel with their antiviral activities by a tetrazolium dye method, according to the manufacturer's instructions (Tetracolor One; Seikagaku Corporation, Tokyo, Japan) (28).

AZT-TP and d4T-TP were examined for their inhibitory effects on PERV RT activity using a commercial RT assay kit (Roche, Mannheim, Germany). The concentrated culture supernatants of PK15, KK5, and DRK4 cells were used as enzyme sources of PERV RT. The culture supernatants were filtered and ultracentrifuged at 35,000 x g for 2 h at 4°C. After centrifugation, the pellets were resuspended in lysis buffer supplied by the assay kit and subjected to a reverse transcription reaction for 60 min, according to the manufacturer's instructions, except that MgCl2 in the reaction mixture was replaced by MnCl2 (19). The activities of the compounds were evaluated by adding their serial dilutions to the reaction mixture. The compounds were also evaluated for their inhibitory effects on HIV-1 RT activity using recombinant HIV-1 RT supplied in the assay kit. For RT nucleotide sequence analysis, genomic DNA was extracted from PK15 and KK5 cells with a DNA extraction kit (Wako, Tokyo, Japan). The extracted DNA was amplified by PCR using the sense primer 5'-CTTGGGAGTGGGACGGGTAA-3' and the antisense primer 5'-GGGGCTGCTAAGGTCGCAAA-3', which cover the whole polymerase region of PERV RT. The purified PCR products were sequenced directly with a cycle-sequencing kit (BigDye Terminator v1.1; Applied Biosystems, Foster City, CA) by an automated DNA sequencer (model 310; Applied Biosystems).

To determine the inhibitory effects of test compounds on PERV replication in human cells, an antiviral assay system was developed. In comparison with other methods (21, 22), the measurement of proviral DNA synthesis by quantitative real-time PCR appeared to be rapid and sensitive enough for evaluation of RT inhibitors. As the target cells, a variety of cell lines were examined for their susceptibilities to PERV infection. Among the cell lines, 293T cells proved to be the most suitable for evaluation of compounds (data not shown). Furthermore, the amount of proviral DNA was determined at various time points after PERV infection, and it was found that a 24-h incubation period was the most appropriate for detection of proviral DNA by real-time PCR (data not shown).

When six HIV-1 RT inhibitors were examined for their inhibitory effects on PERV replication in 293T cells, four compounds (AZT, PMEO-DAPy, d4T, and PMPDAP) displayed dose-dependent inhibition of proviral DNA synthesis (Fig. 1A to D). AZT and the other compounds did not reduce the viability of 293T cells at concentrations up to 10 and 100 µM, respectively, indicating that these compounds are selective inhibitors of PERV replication in human cells. In contrast, 3TC and NVP did not show any activities against PERV, even at a concentration of 100 µM (Fig. 1E and F). Among the active compounds, AZT proved to be the most potent inhibitor, followed by the acyclic nucleoside phosphonates PMEO-DAPy and PMPDAP. However, d4T was found to be a much weaker inhibitor of PERV than AZT (Fig. 1C). Table 1 shows the 50% effective concentrations (EC50s) and 50% cytotoxic concentrations (CC50s) of the compounds evaluated in this study. In addition to the six compounds described above, the acyclic nucleoside phosphonates licensed for treatment of HIV-1 and hepatitis B virus (HBV) infections, PMPA and PMEA, respectively, and the 4'-substituted thymidine analogs 4'-Ed4T and 4'-AZT were also examined. Again, AZT, PMEO-DAPy, and PMPDAP were highly potent inhibitors of PERV, with EC50s of 0.023, 0.18, and 0.28 µM, respectively. PMPA and PMEA also displayed significant inhibition of PERV replication, and their EC50s were 2.8 and 3.4 µM, respectively. Although d4T is as active as AZT against HIV-1 replication in vitro, it was approximately 300-fold less inhibitory to PERV replication (EC50, 7.8 µM) than AZT. More interestingly, 4'-Ed4T, 4'-AZT, and 3TC were totally inactive against PERV (Table 1).


Figure 1
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  		FIG. 1. Inhibitory effects of selected compounds on PERV replication in 293T cells. 293T cells were infected with PERV derived from PK15 cells and cultured in the presence of various concentrations of (A) AZT, (B) PMEO-DAPy, (C) d4T, (D) PMPDAP, (E) 3TC, and (F) NVP. After a 24-h incubation, the cells were collected and genomic DNA was extracted. Quantitative real-time PCR was performed to determine the amount of PERV proviral DNA in the infected cells using a primer pair and probe specific to the PERV pol gene (lines). The viable-cell number was determined by a tetrazolium dye cell proliferation assay (bars). The PCR and cell proliferation assays were performed in triplicate and in duplicate, respectively. The data represent means plus standard deviations. Representative results for two or three independent experiments are shown.

 

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  		TABLE 1. Inhibitory effects of selective compounds on PERV replication in 293T cellsa

 
The inhibitory effects of AZT-TP and d4T-TP on PERV RT derived from PK15, KK5, and DRK4 cells were examined to confirm that the reduction of proviral DNA synthesis by AZT and d4T in cell cultures was attributable to the inhibition of PERV RT activity. Since the RT activities of culture supernatants were different depending on their cell sources of origin (data not shown), concentrated cell culture supernatants that contained the same catalytic activity of PERV RT were used for the RT inhibition assay. Table 2 summarizes the 50% inhibitory concentrations (IC50s) of AZT-TP and d4T-TP for HIV-1 RT and PERV RT. Both AZT-TP and d4T-TP proved to be highly potent inhibitors of HIV-1 RT, with IC50s of 0.054 and 0.036 µM, respectively. Although AZT-TP displayed inhibition of PERV RT similar to or even more potent than that of HIV-1 RT, d4T-TP was less inhibitory to PERV RT than to HIV-1 RT. The PERV RT amino acid sequences in PK15 and KK5 cells are shown in Fig. 2. The amino acid homology of the RT region between the two sequences was 99%. Furthermore, the amino acid sequence of the catalytic site was found to be highly conserved as YVDD.


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  		TABLE 2. Inhibitory effects of AZT-TP and d4T-TP on HIV-1 and PERV RT activities

 

Figure 2
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  		FIG. 2. Amino acid sequences of the polymerase domain of PERV RT derived from PK15 and KK5 cells. The catalytic site is indicated by boldface letters.

 
Up to now, two groups have reported the efficacy of some NRTIs against PERV replication in cell cultures (21, 22). These studies demonstrated that, although significant anti-PERV activity was observed for AZT, other NRTIs licensed for the treatment of HIV-1 infection were not effective. The acyclic nucleoside phosphonates represent a new dimension to the therapy of viral infections (6). In this study, we have demonstrated for the first time that the acyclic nucleoside phosphonates PMEO-DAPy and PMPDAP are highly potent and selective inhibitors of PERV replication in human cells (Fig. 1 and Table 1). The clinically licensed phosphonates PMPA and PMEA were also active against PERV, although a higher concentration was required for sufficient inhibition of PERV replication than for PMEO-DAPy and PMPDAP. It is well known that PMPA and PMEA have a broad spectrum of antiviral activity against a variety of retroviruses, including hepatitis B virus (9, 29). In addition, these compounds were reported to be highly effective against murine leukemia virus and feline leukemia virus infections in vivo, both of which are type C retroviruses closely related to PERV (10, 24). Thus, further studies should be conducted to clarify that PMEO-DAPy or PMPDAP is necessary for the prevention of PERV infection instead of PMPA or PMEA.

The inhibitory effects of compounds were determined by the reduction of proviral DNA synthesis in human cells after 24 h of PERV infection using quantitative real-time PCR. AZT, a nucleoside active against a variety of retroviruses, including murine leukemia virus and feline leukemia virus (13, 20, 25), was also found to be the most potent inhibitor of PERV. It is well documented that the resistance of HIV-1 to RT inhibitors is associated with specific amino acid mutations of the enzyme. For instance, the single amino acid mutation M184V of HIV-1 RT confers complete resistance to 3TC and emitricitabine (26). The M184 amino acid composes the catalytic site of HIV-1 RT, together with the amino acids Y183, D185, and D186 (YMDD), while the catalytic site of PERV RT consists of YVDD (Fig. 2). This may be a reason that 3TC was totally inactive against PERV replication. However, the possibility that other factors may exist that are involved in the different drug susceptibility patterns between HIV-1 and PERV cannot be excluded, because the two 4'-substituted nucleoside analogs 4'-AZT and 4'-Ed4T did not inhibit PERV replication even at a concentration of 100 µM (Table 1). 4'-Ed4T is a novel NRTI that is highly active against various HIV-1 strains with RT mutations that engender resistance to some NRTIs and NNRTIs (15). The M184V mutation confers only partial resistance to this compound. Thus, the YVDD motif of PERV RT might not be able to explain such high-level resistance to 4'-Ed4T. It is possible that the conformation of PERV RT differs significantly from that of HIV-1 RT due to their modest amino acid homology. As expected, NVP was totally inactive against PERV (Fig. 1 and Table 1), because PERV RT may not have a hydrophobic pocket adjacent to the catalytic site, which is known as the NNRTI-binding site.

The Kagoshima strain of minipigs is considered to be an ideal organ donor in xenotransplantation and medical research due to size and other favorable profiles. Although PERV proviral DNA was integrated into the genomic DNA and was functionally active even in this strain, the production of PERV viral particles from the Kagoshima minipig kidney cells (KK5 and DRK4) was found to be approximately seven- to eightfold lower than that from PK15 cells (data not shown). Therefore, the antiviral assay of compounds for PERV derived from the Kagoshima strain was not successful because of its low infectivity for human cells (data not shown). Equal amounts (catalytic activities) of the enzymes were used for the cell-free RT inhibition assay of AZT-TP and d4T-TP; nevertheless, the enzymes obtained from KK5 and DRK4 cells showed slightly higher susceptibility to the compounds than that obtained from PK15 (Table 2). These results suggest that the acyclic nucleoside phosphonates also show potent and selective inhibition of PERV derived from Kagoshima minipigs in cell cultures.

The difference in anti-PERV activity between AZT and d4T in the 293T cells was found to be much greater than the difference in anti-RT activity between AZT-TP and d4T-TP in an enzyme assay (Tables 1 and 2), which is in agreement with a previous report (22). One explanation for this finding is the different intercellular metabolisms of AZT and d4T compared to AZT-TP and d4T-TP, respectively, as previously demonstrated by Balzarini and colleagues (1). Furthermore, profound drug-drug interactions may occur between NRTIs and immunosuppressants, although they are considered to be less likely than interactions between HIV-1 protease inhibitors and immunosuppressants (11).

In conclusion, in addition to AZT, the acyclic nucleoside phosphonates examined in this study may be promising candidates for the prevention of PERV transmission from porcine organs to human recipients in xenotransplantation, and they should be further examined for their antiviral potential in vivo.


   ACKNOWLEDGMENTS
 
We thank M. Yoshida and K. Miyoshi for kindly providing us the primary kidney cells obtained from Kagoshima minipigs.

This work was supported in part by a Grant-in-Aid for Scientific Research (S) (grant no. 17100007) from the Ministry of Education, Science, Sports, Culture and Technology of Japan.


   FOOTNOTES
 
* Corresponding author. Mailing address: Division of Antiviral Chemotherapy, Center for Chronic Viral Diseases, Faculty of Medicine, Kagoshima University, 8-35-1, Sakuragaoka, Kagoshima 890-8544, Japan. Phone: 81 99-275-5931. Fax: 81 99-275-5932. E-mail: m-baba{at}vanilla.ocn.ne.jp Back

{triangledown} Published ahead of print on 30 April 2007. Back


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Antimicrobial Agents and Chemotherapy, July 2007, p. 2600-2604, Vol. 51, No. 7
0066-4804/07/$08.00+0     doi:10.1128/AAC.00212-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.




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