Suppression of Human T-cell Leukemia Virus I Gene Expression by Pokeweed Antiviral Protein

Human T-cell leukemia virus I (HTLV-I) is a deltaretrovirus that is the causative agent of adult T-cell leukemia and the neurological disorder HTLV-I-associated myelopathy/tropical spastic paraparesis. Currently, no effective antiretroviral treatment options are available to restrict the development of diseases associated with the virus. In this work, we investigated the activity of pokeweed antiviral protein (PAP) on HTLV-I, when expressed from a proviral clone in 293T cells or in an HTLV-I immortalized cell line. PAP is a plant-derived N-glycosidase that exhibits antiviral activity against a number of viruses; however, its mode of action has not been clearly defined. Here, we describe the mechanism by which PAP inhibited production of HTLV-I. We show that PAP depurinated nucleotides within the gag open reading frame and suppressed the synthesis of viral proteins in part by decreasing the translational efficiency of HTLV-I gag/pol mRNA. Observed reduction in levels of viral mRNAs were not due to enhanced degradation; rather, decreased amounts of viral transactivator protein, Tax, led to feed-back inhibition of transcription from the viral promoter. Therefore, PAP efficiently suppressed HTLV-I gene expression at both translational and transcriptional levels, resulting in substantially diminished virus production. Significantly, no changes in viability or rates of cellular transcription or translation were observed in cells expressing PAP, indicating that this protein was not toxic. Antiviral activity, together with the absence of cytotoxicity, supports further investigation of this enzyme as a novel therapeutic agent against the progression of HTLV-I infection. Human T-cell leukemia virus I (HTLV-I) is a deltaretrovirus that is the causative agent of adult T-cell leukemia and the neurological disorder HTLV-I-associated myelopathy/tropical spastic paraparesis. Currently, no effective antiretroviral treatment options are available to restrict the development of diseases associated with the virus. In this work, we investigated the activity of pokeweed antiviral protein (PAP) on HTLV-I, when expressed from a proviral clone in 293T cells or in an HTLV-I immortalized cell line. PAP is a plant-derived N-glycosidase that exhibits antiviral activity against a number of viruses; however, its mode of action has not been clearly defined. Here, we describe the mechanism by which PAP inhibited production of HTLV-I. We show that PAP depurinated nucleotides within the gag open reading frame and suppressed the synthesis of viral proteins in part by decreasing the translational efficiency of HTLV-I gag/pol mRNA. Observed reduction in levels of viral mRNAs were not due to enhanced degradation; rather, decreased amounts of viral transactivator protein, Tax, led to feed-back inhibition of transcription from the viral promoter. Therefore, PAP efficiently suppressed HTLV-I gene expression at both translational and transcriptional levels, resulting in substantially diminished virus production. Significantly, no changes in viability or rates of cellular transcription or translation were observed in cells expressing PAP, indicating that this protein was not toxic. Antiviral activity, together with the absence of cytotoxicity, supports further investigation of this enzyme as a novel therapeutic agent against the progression of HTLV-I infection. IntroductionHuman T-cell leukemia virus I (HTLV-I) 2The abbreviations used are: HTLV-Ihuman T-cell leukemia virus IPAPpokeweed antiviral proteinELISAenzyme-linked immunosorbent assayMTT3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromidePIPES1,4-piperazinediethanesulfonic acidLTRlong terminal repeatGAPDHglyceraldehyde-3-phosphate dehydrogenaseGFPgreen fluorescent protein. is a human deltaretrovirus that causes adult T-cell leukemia/lymphoma (1Uchiyama T. Yodoi J. Sagawa K. Takatsuki K. Uchino H. Blood. 1977; 50: 481-492Crossref PubMed Google Scholar) and tropical spastic paraparesis, also called HTLV-I-associated myelopathy. The latter is a chronic and progressive disease of the nervous system, characterized by muscle weakness and sensory disturbance (2Gessain A. Barin F. Vernant J.C. Gout O. Maurs L. Calender A. de Thé G. Lancet. 1985; 2: 407-410Abstract PubMed Scopus (2399) Google Scholar, 3Osame M. Matsumoto M. Usuku K. Izumo S. Ijichi N. Amitani H. Tara M. Igata A. Ann. Neurol. 1987; 21: 117-122Crossref PubMed Scopus (564) Google Scholar). HTLV-I-infected individuals have a 2–3% estimated lifetime risk of developing adult T-cell leukemia, with a period of latency from 20 to 30 years (4Yamaguchi K. Takatsuki K. Clin. Haematol. 1993; 6: 899-915Scopus (41) Google Scholar, 5Tokudome S. Tokunaga O. Shimamoto Y. Miyamoto Y. Sumida I. Kikuchi M. Takeshita M. Ikeda T. Fujiwara K. Yoshihara M. Cancer Res. 1989; 49: 226-228PubMed Google Scholar). The majority of those infected with HTLV-I are therefore asymptomatic (6Tajima K. Int. J. Cancer. 1990; 45: 237-243Crossref PubMed Scopus (257) Google Scholar), because the virus maintains the expression of its genes at very low or undetectable levels. As a result, HTLV-I is not efficiently targeted by the immune system (7Yoshida M. Annu. Rev. Immunol. 2001; 19: 475-496Crossref PubMed Scopus (392) Google Scholar), and leukemia progresses after clonal expansion of T-cells infected with the virus (8Taylor G.P. Matsuoka M. Oncogene. 2005; 24: 6047-6057Crossref PubMed Scopus (181) Google Scholar, 9Franchini G. Nicot C. Johnson J.M. Adv. Cancer Res. 2003; 89: 69-132Crossref PubMed Scopus (68) Google Scholar). Due to the long period of latency prior to onset of leukemia, the disease appears mainly in individuals who have been infected with HTLV-I early in life (10Murphy E.L. Hanchard B. Figueroa J.P. Gibbs W.N. Lofters W.S. Campbell M. Goedert J.J. Blattner W.A. Int. J. Cancer. 1989; 43: 250-253Crossref PubMed Scopus (335) Google Scholar, 11Takatsuki K. Yamaguchi K. Kawano F. Hattori T. Nishimura H. Tsuda H. Sanada I. Nakada K. Itai Y. Cancer Res. 1985; 45: 4644s-4645sPubMed Google Scholar). The virus is transmitted through body fluids, including breast milk (12Höllsberg P. Hafler D.A. N. Engl. J. Med. 1993; 328: 1173-1182Crossref PubMed Scopus (292) Google Scholar, 13Moriuchi M. Inoue H. Moriuchi H. J. Virol. 2001; 75: 192-198Crossref PubMed Scopus (42) Google Scholar); therefore, mother-to-child transmission poses substantial risk for development of leukemia. Various forms of cytotoxic chemotherapy are currently used to treat adult T-cell leukemia; however, prognosis is poor, because the disease is aggressive, with a mean survival time of only 6 months. Few published accounts address the potential of antiretroviral therapy for limiting viral gene expression and/or inhibiting replication to reduce the viral load in individuals early after infection, which in turn would reduce the chances for progression of leukemia (14Gill P.S. Harrington Jr., W. Kaplan M.H. Ribeiro R.C. Bennett J.M. Liebman H.A. Bernstein-Singer M. Espina B.M. Cabral L. Allen S. N. Engl. J. Med. 1995; 332: 1744-1748Crossref PubMed Scopus (316) Google Scholar, 15Machuca A. Soriano V. J. Acquir. Immune Defic. Syndr. 2000; 24: 189-193Crossref PubMed Scopus (36) Google Scholar).In this report, we investigated the antiviral activity of a plant-derived protein against HTLV-I. Pokeweed antiviral protein (PAP) is a ribosome-inactivating protein synthesized by Phytolacca americana, the pokeweed plant (16Irvin J.D. Arch Biochem. Biophys. 1975; 169: 522-528Crossref PubMed Scopus (162) Google Scholar). Like all ribosome-inactivating proteins, PAP is a glycosidase that removes a specific adenine base from the large ribosomal RNA (17Endo Y. Tsurugi K. Lambert J.M. Biochem. Biophys. Res. Commun. 1988; 150: 1032-1036Crossref PubMed Scopus (177) Google Scholar). This depurination has traditionally been cited as the reason for antiviral activity, because resulting inhibition of host cell translation would limit virus proliferation (18Foà-Tomasi L. Campadelli-Fiume G. Barbieri L. Stirpe F. Arch. Virol. 1982; 71: 323-332Crossref PubMed Scopus (47) Google Scholar). However, mutants of the protein have been expressed that maintain antiviral activity in the absence of rRNA depurination, thereby separating the effect on viruses from host cell translation (19Picard D. Kao C.C. Hudak K.A. J. Biol. Chem. 2005; 280: 20069-20075Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 20Tumer N.E. Hwang D.J. Bonness M. Proc. Natl. Acad. Sci. U.S.A. 1997; 94: 3866-3871Crossref PubMed Scopus (129) Google Scholar). Pokeweed antiviral protein has been shown to depurinate some viral RNAs, indicating that antiviral activity may be due to broader substrate specificity that is not limited to rRNA depurination (21Hudak K.A. Wang P. Tumer N.E. RNA. 2000; 6: 369-380Crossref PubMed Scopus (91) Google Scholar). Incubation of purified PAP with lymphocytes prior to their infection with HIV-I reduced the number of virus particles released into the medium (22Zarling J.M. Moran P.A. Haffar O. Sias J. Richman D.D. Spina C.A. Myers D.E. Kuebelbeck V. Ledbetter J.A. Uckun F.M. Nature. 1990; 347: 92-95Crossref PubMed Scopus (193) Google Scholar). In addition, incubation of HIV-I genomic RNA with purified PAP resulted in release of purines, suggesting that PAP depurinated the RNA in this assay (23Rajamohan F. Venkatachalam T.K. Irvin J.D. Uckun F.M. Biochem. Biophys. Res. Commun. 1999; 260: 453-458Crossref PubMed Scopus (84) Google Scholar); however, the effect of PAP on retroviral gene expression in cells has not been described. Accordingly, our goal was to examine whether PAP is able to target and inhibit HTLV-I and to determine the mechanism of such activity.We show here that PAP efficiently inhibited HTLV-I gene expression at both translational and transcriptional levels in the absence of host cell toxicity. PAP depurinated nucleotides within the gag open reading frame of genomic RNA and reduced its translational efficiency in vitro. Decreased amounts of the viral transactivator protein, Tax, led to feed-back inhibition of HTLV-I gene expression, diminishing gag/pol, env, and tax/rex mRNA levels. Due to this combined effect, PAP significantly reduced virus production.DISCUSSIONPAP exhibits antiviral activity against a number of different viruses; however, the mechanistic details of this activity have not been described (19Picard D. Kao C.C. Hudak K.A. J. Biol. Chem. 2005; 280: 20069-20075Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 22Zarling J.M. Moran P.A. Haffar O. Sias J. Richman D.D. Spina C.A. Myers D.E. Kuebelbeck V. Ledbetter J.A. Uckun F.M. Nature. 1990; 347: 92-95Crossref PubMed Scopus (193) Google Scholar, 36Tomlinson J.A. Walker V.M. Flewett T.H. Barclay G.R. J. Gen. Virol. 1974; 22: 225-232Crossref PubMed Scopus (87) Google Scholar). As a ribosome-inactivating protein, PAP also depurinates a conserved adenine within the sarcin/ricin loop of the large rRNA, which has been reported to inhibit the binding of elongation factor 2 and slow the rate of elongation during protein translation (37Stirpe F. Bailey S. Miller S.P. Bodley J.W. Nucleic Acids Res. 1988; 16: 1349-1357Crossref PubMed Scopus (124) Google Scholar, 38Gessner S.L. Irvin J.D. J. Biol. Chem. 1980; 255: 3251-3253Abstract Full Text PDF PubMed Google Scholar, 39Nilsson L. Nygård O. Eur. J. Biochem. 1986; 161: 111-117Crossref PubMed Scopus (43) Google Scholar). This inhibition of translation has traditionally been cited to explain antiviral activity, because a positive correlation has been shown between ribosome depurination and decline of virus infection (18Foà-Tomasi L. Campadelli-Fiume G. Barbieri L. Stirpe F. Arch. Virol. 1982; 71: 323-332Crossref PubMed Scopus (47) Google Scholar, 40Taylor S. Massiah A. Lomonossoff G. Roberts L.M. Lord J.M. Hartley M. Plant J. 1994; 5: 827-835Crossref PubMed Scopus (105) Google Scholar). However, very few ribosome-inactivating proteins are antiviral, suggesting that merely inhibiting ribosomes does not impart antiviral qualities. Moreover, our results show that PAP inhibited HTLV-I gene expression in the absence of cytotoxicity. In addition to their potential to inhibit translation, many ribosome-inactivating proteins induce a ribotoxic stress response that results in cell death (41Narayanan S. Surolia A. Karande A.A. Biochem. J. 2004; 377: 233-240Crossref PubMed Scopus (75) Google Scholar, 42Ouyang D.Y. Wang Y.Y. Zheng Y.T. Cell Mol. Immunol. 2005; 2: 419-425PubMed Google Scholar). This response is characterized by damage to the rRNA, which triggers activation of a stress-induced kinase that may culminate in programmed cell death (43Iordanov M.S. Pribnow D. Magun J.L. Dinh T.H. Pearson J.A. Chen S.L. Magun B.E. Mol. Cell. Biol. 1997; 17: 3373-3381Crossref PubMed Google Scholar). In contrast, we have demonstrated previously that PAP expression in 293T cells resulted in cell survival rather than apoptosis (28Chan Tung K.W. Mansouri S. Hudak K.A. Int. J. Biochem. Cell Biol. 2008; 40: 2452-2461Crossref PubMed Scopus (9) Google Scholar). In the current study, the amount of PAP expression that caused 100-fold decline in HTLV-I protein levels was not sufficient to be toxic to cells, although 16% of the 28 S rRNA was depurinated. This level of rRNA depurination did not affect the rate of cellular translation, indicating that reduction in the level of viral proteins was not due to general defects in the cellular translation machinery as a result of rRNA depurination. Together with our findings indicating minimal PAP effect on cellular transcription rates, we conclude that antiviral activity was not a consequence of cytotoxicity.In addition to demonstrating lack of harm to host cells, examining the potential of PAP as a therapeutic option to limit HTLV-I progression would require understanding the steps in the virus life cycle affected by depurination. Here, we describe the mechanism through which PAP inhibited virus production (Fig. 8). Specifically, transient expression of PAP in 293T cells caused a substantial decline in the level of intracellular Gag and virus-associated p19 matrix protein and intracellular Tax protein. We show that the 5′ region of the gag/pol mRNA was depurinated by PAP and that this mRNA was less efficiently translated in a cell-free system, suggesting that depurination contributed to diminished levels of viral proteins. We have shown previously that depurination of Brome mosaic virus RNA3 caused ribosomes to stall at the missing base during elongation (32Gandhi R. Manzoor M. Hudak K.A. J. Biol. Chem. 2008; 283: 32218-32228Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). Expression of PAP also reduced HTLV-I gag/pol, env, and tax/rex mRNA levels in a dose-dependent manner. Decreased tax/rex mRNA levels may have also resulted in diminished Rex synthesis, which in turn would have reduced the amount of cytoplasmic gag/pol mRNA for translation. Therefore, post-transcriptional effects of PAP contributed to the decline in viral protein synthesis. Diminished levels of viral mRNAs were not due to enhanced degradation but rather were probably caused by limited synthesis, based on the results of our reporter assay. HTLV-I Tax protein is encoded from the HTLV-I doubly spliced tax/rex mRNA and is the transactivator of the viral promoter located within the 5′-LTR (35Sodroski J. Rosen C. Goh W.C. Haseltine W. Science. 1985; 228: 1430-1434Crossref PubMed Scopus (231) Google Scholar). Since Tax is required for enhanced transcription from the viral promoter, we expected that undetectable Tax would result in decreased levels of all HTLV-I-encoded mRNAs. Therefore, feedback inhibition of viral gene expression manifested as an efficient decline in the level of viral mRNAs and proteins, hence the significant reduction in virus production.Apart from pokeweed antiviral protein, other enzymes exist that modify the genomes of some viruses. For example, APOBEC3G is a family member of cytidine deaminases that acts as a host defense factor against some retroviruses (reviewed in Refs. 44Bieniasz P.D. Nat. Immunol. 2004; 5: 1109-1115Crossref PubMed Scopus (359) Google Scholar and 45Cullen B.R. J. Virol. 2006; 80: 1067-1076Crossref PubMed Scopus (214) Google Scholar). These enzymes convert cytosines of negative-strand viral cDNA to uracils during reverse resulting in to in the B. P. G. M. L. D. Nature. 2003; PubMed Scopus Google Scholar, H. B. C. L. Nature. 2003; PubMed Scopus Google Scholar, M.H. M.S. Nat. Immunol. 2003; PubMed Scopus Google Scholar). The cDNA may be targeted for or of in proviral may result in synthesis of or viral proteins. HTLV-I appears to be to the antiviral effects of and of to are observed T. A. J. Virol. PubMed Scopus Google Scholar). a in the of HTLV-I protein has been shown to inhibit into thereby limiting its antiviral effect D. G. P. G. Proc. Natl. Acad. Sci. U.S.A. PubMed Scopus Google Scholar). Therefore, HTLV-I appears to this cellular factor that causes of its We that of virus to pokeweed antiviral protein may be that the of the PAP effect described here does not on the protein into viral particles and that no for of depurinated RNA have been described. The decline in protein synthesis from the viral RNA and its effect on transcriptional and post-transcriptional that PAP may have an as part of an antiviral in the IntroductionHuman T-cell leukemia virus I (HTLV-I) 2The abbreviations used are: HTLV-Ihuman T-cell leukemia virus IPAPpokeweed antiviral proteinELISAenzyme-linked immunosorbent assayMTT3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromidePIPES1,4-piperazinediethanesulfonic acidLTRlong terminal repeatGAPDHglyceraldehyde-3-phosphate dehydrogenaseGFPgreen fluorescent protein. is a human deltaretrovirus that causes adult T-cell leukemia/lymphoma (1Uchiyama T. Yodoi J. Sagawa K. Takatsuki K. Uchino H. Blood. 1977; 50: 481-492Crossref PubMed Google Scholar) and tropical spastic paraparesis, also called HTLV-I-associated myelopathy. The latter is a chronic and progressive disease of the nervous system, characterized by muscle weakness and sensory disturbance (2Gessain A. Barin F. Vernant J.C. Gout O. Maurs L. Calender A. de Thé G. Lancet. 1985; 2: 407-410Abstract PubMed Scopus (2399) Google Scholar, 3Osame M. Matsumoto M. Usuku K. Izumo S. Ijichi N. Amitani H. Tara M. Igata A. Ann. Neurol. 1987; 21: 117-122Crossref PubMed Scopus (564) Google Scholar). HTLV-I-infected individuals have a 2–3% estimated lifetime risk of developing adult T-cell leukemia, with a period of latency from 20 to 30 years (4Yamaguchi K. Takatsuki K. Clin. Haematol. 1993; 6: 899-915Scopus (41) Google Scholar, 5Tokudome S. Tokunaga O. Shimamoto Y. Miyamoto Y. Sumida I. Kikuchi M. Takeshita M. Ikeda T. Fujiwara K. Yoshihara M. Cancer Res. 1989; 49: 226-228PubMed Google Scholar). The majority of those infected with HTLV-I are therefore asymptomatic (6Tajima K. Int. J. Cancer. 1990; 45: 237-243Crossref PubMed Scopus (257) Google Scholar), because the virus maintains the expression of its genes at very low or undetectable levels. As a result, HTLV-I is not efficiently targeted by the immune system (7Yoshida M. Annu. Rev. Immunol. 2001; 19: 475-496Crossref PubMed Scopus (392) Google Scholar), and leukemia progresses after clonal expansion of T-cells infected with the virus (8Taylor G.P. Matsuoka M. Oncogene. 2005; 24: 6047-6057Crossref PubMed Scopus (181) Google Scholar, 9Franchini G. Nicot C. Johnson J.M. Adv. Cancer Res. 2003; 89: 69-132Crossref PubMed Scopus (68) Google Scholar). Due to the long period of latency prior to onset of leukemia, the disease appears mainly in individuals who have been infected with HTLV-I early in life (10Murphy E.L. Hanchard B. Figueroa J.P. Gibbs W.N. Lofters W.S. Campbell M. Goedert J.J. Blattner W.A. Int. J. Cancer. 1989; 43: 250-253Crossref PubMed Scopus (335) Google Scholar, 11Takatsuki K. Yamaguchi K. Kawano F. Hattori T. Nishimura H. Tsuda H. Sanada I. Nakada K. Itai Y. Cancer Res. 1985; 45: 4644s-4645sPubMed Google Scholar). The virus is transmitted through body fluids, including breast milk (12Höllsberg P. Hafler D.A. N. Engl. J. Med. 1993; 328: 1173-1182Crossref PubMed Scopus (292) Google Scholar, 13Moriuchi M. Inoue H. Moriuchi H. J. Virol. 2001; 75: 192-198Crossref PubMed Scopus (42) Google Scholar); therefore, mother-to-child transmission poses substantial risk for development of leukemia. Various forms of cytotoxic chemotherapy are currently used to treat adult T-cell leukemia; however, prognosis is poor, because the disease is aggressive, with a mean survival time of only 6 months. Few published accounts address the potential of antiretroviral therapy for limiting viral gene expression and/or inhibiting replication to reduce the viral load in individuals early after infection, which in turn would reduce the chances for progression of leukemia (14Gill P.S. Harrington Jr., W. Kaplan M.H. Ribeiro R.C. Bennett J.M. Liebman H.A. Bernstein-Singer M. Espina B.M. Cabral L. Allen S. N. Engl. J. Med. 1995; 332: 1744-1748Crossref PubMed Scopus (316) Google Scholar, 15Machuca A. Soriano V. J. Acquir. Immune Defic. Syndr. 2000; 24: 189-193Crossref PubMed Scopus (36) Google Scholar).In this report, we investigated the antiviral activity of a plant-derived protein against HTLV-I. Pokeweed antiviral protein (PAP) is a ribosome-inactivating protein synthesized by Phytolacca americana, the pokeweed plant (16Irvin J.D. Arch Biochem. Biophys. 1975; 169: 522-528Crossref PubMed Scopus (162) Google Scholar). Like all ribosome-inactivating proteins, PAP is a glycosidase that removes a specific adenine base from the large ribosomal RNA (17Endo Y. Tsurugi K. Lambert J.M. Biochem. Biophys. Res. Commun. 1988; 150: 1032-1036Crossref PubMed Scopus (177) Google Scholar). This depurination has traditionally been cited as the reason for antiviral activity, because resulting inhibition of host cell translation would limit virus proliferation (18Foà-Tomasi L. Campadelli-Fiume G. Barbieri L. Stirpe F. Arch. Virol. 1982; 71: 323-332Crossref PubMed Scopus (47) Google Scholar). However, mutants of the protein have been expressed that maintain antiviral activity in the absence of rRNA depurination, thereby separating the effect on viruses from host cell translation (19Picard D. Kao C.C. Hudak K.A. J. Biol. Chem. 2005; 280: 20069-20075Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 20Tumer N.E. Hwang D.J. Bonness M. Proc. Natl. Acad. Sci. U.S.A. 1997; 94: 3866-3871Crossref PubMed Scopus (129) Google Scholar). Pokeweed antiviral protein has been shown to depurinate some viral RNAs, indicating that antiviral activity may be due to broader substrate specificity that is not limited to rRNA depurination (21Hudak K.A. Wang P. Tumer N.E. RNA. 2000; 6: 369-380Crossref PubMed Scopus (91) Google Scholar). Incubation of purified PAP with lymphocytes prior to their infection with HIV-I reduced the number of virus particles released into the medium (22Zarling J.M. Moran P.A. Haffar O. Sias J. Richman D.D. Spina C.A. Myers D.E. Kuebelbeck V. Ledbetter J.A. Uckun F.M. Nature. 1990; 347: 92-95Crossref PubMed Scopus (193) Google Scholar). In addition, incubation of HIV-I genomic RNA with purified PAP resulted in release of purines, suggesting that PAP depurinated the RNA in this assay (23Rajamohan F. Venkatachalam T.K. Irvin J.D. Uckun F.M. Biochem. Biophys. Res. Commun. 1999; 260: 453-458Crossref PubMed Scopus (84) Google Scholar); however, the effect of PAP on retroviral gene expression in cells has not been described. Accordingly, our goal was to examine whether PAP is able to target and inhibit HTLV-I and to determine the mechanism of such activity.We show here that PAP efficiently inhibited HTLV-I gene expression at both translational and transcriptional levels in the absence of host cell toxicity. PAP depurinated nucleotides within the gag open reading frame of genomic RNA and reduced its translational efficiency in vitro. Decreased amounts of the viral transactivator protein, Tax, led to feed-back inhibition of HTLV-I gene expression, diminishing gag/pol, env, and tax/rex mRNA levels. Due to this combined effect, PAP significantly reduced virus production.

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