Abstract
Background
Human immunodeficiency virus type 1 (HIV-1) is a lentivirus and shares with other members of this retroviral subfamily the ability to replicate in nondividing cells, in particular, cells of the monocyte/macrophage lineage. This feature relies on the presence of a specific nuclear localization signal (NLS) within the viral matrix protein (MA p17), which to some degree can be complemented by the activity of the viral vpr gene product. The MA p17 NLS ensures efficient transportation of the viral preintegration complex into the nucleus of an infected macrophage and confers persistence of HIV-1 in quiescent T cells, and therefore presents an attractive target for therapeutic intervention.
Materials and Methods
Nuclear localization signals (NLS) in general and the HIV-1 MA p17 NLS in particular are characterized by a stretch of positively charged amino acids including one or more lysine residues. A series of compounds potentially capable of binding and reacting with lysine by forming Schiff base adducts was synthesized. Our special consideration was to make compounds that would preferentially bind to two closely contiguous amino functions, as opposed to isolated single lysine residues. We assumed that this approach might specifically target the compound to NLS while affecting other regions less, thus reducing nonspecific cytotoxicity. Antiviral activity was assessed in primary monocytes and in peripheral blood lymphocytes (PBL) infected with HIV-1ADA strain. Viral replication was monitored by reverse transcriptase (RT) activity in the supernatant. Efficiency of nuclear importation of the viral preintegration complex was estimated by the formation of 2-LTR circle forms of HIV-1 DNA and also by in situ PCR techniques.
Results
Arylene bis(methyl ketone) compounds with a nitrogenous third substituent, especially a pyrimidinic side-chain, inhibited HIV-1 replication in human monocytes at an IC50 as low as 1 nM. These compounds did not block HIV-1 replication in peripheral blood lymphocyte cultures. The inhibitory effect observed in monocyte cultures appeared in the context of markedly reduced nuclear importation of viral DNA in the presence of the drug. No cytotoxic effects of the compounds was observed in vitro at concentrations as high as 10 µM. An amidinohydrazone derivative of the most active compound was about 100 times less active than the parent, indicating that carbonyl groups were instrumental in the antiviral effect.
Conclusions
These early results suggest that retroviral replication in nondividing cells is susceptible to pharmaceutical intervention targeted against the NLS activity of HIV-1 proteins in the viral preintegration complex. The compounds described efficiently block translocation of viral DNA to the nuclei of infected primary monocytes, and inhibit viral replication. This inhibition is effective only in nondividing cells and is not seen in proliferating cultures, such as activated PBLs. Thus, drugs that target HIV-1 NLS may be useful to specifically block the macrophage arm of HIV infection and could thereby be of value in treating macrophage-specific manifestations of HIV disease, such as HIV-1 dementia. In combination with other drugs, potential therapeutics exploiting this target may also help to control the progression of HIV-1 infection and disease.
Similar content being viewed by others
References
Connor RI, Mohri H, Cao Y, Ho DD. (1993) Increased viral burden and cytopathicity correlate temporally with CD4+ T-lymphocyte decline and clinical progression in human immunodeficiency virus type 1-infected individuals. J. Virol. 67: 1772–1777.
Roos MTL, Lange JMA, de Goede REY, et al. (1992) Viral phenotype and immune response in primary human immunodeficiency virus type 1 infection. J. Infect. Dis. 165: 427–432.
Zhu T, Mo H, Wang N, et al. (1993) Genotypic and phenotypic characterization of HIV-1 patients with primary infection. Science 261: 1179–1181.
Schuitemaker H, Koot M, Koostra NA, et al. (1992) Biological phenotype of human immunodeficiency virus type 1 clones at different stages of infection: Progression of disease is associated with a shift from monocytotropic to T-cell-tropic populations. J. Virol. 66: 1354–1360.
Gendelman HE, Orenstein JM, Baca LM, et al. (1989) The macrophage in the persistence and pathogenesis of HIV infection [see comments]. AIDS 3: 475–495.
Koenig S, Gendelman HE, Orenstein JM, et al. (1986) Detection of AIDS virus in macrophages in brain tissue from AIDS patients with encephalopathy. Science 233: 1089–1093.
Gartner S, Markovits P, Markovits DM, Kaplan MH, Gallo RC, Popovic M. (1986) The role of mononuclear phagocytes in HTLV-III/LAV infection. Science 233: 215–219.
Popovic M, Gartner S. (1987) Isolation of HIV-1 from monocytes but not T lymphocytes. Lancet 2: 916.
Rosenberg Z, Fauci A. (1990) Immunopathogenic mechanisms of HIV infection: Cytokine induction of HIV expression. Immunol. Today 11: 176–180.
Rosenberg ZF, Fauci AS. (1993) Immunology of HIV infection. In: Paul WE (ed). Fundamental Immunology. Raven Press, New York, pp. 1375–1397.
Navia BA, Jordan BD, Price RW. (1986) The AIDS dementia complex. I. Clinical features. Ann. Neurol 19: 517–524.
Wiley CA, Schrier RD, Nelson JA, Lampert PW, Oldstone MBA. (1986) Cellular localization of human immunodeficiency virus infection within the brains of acquired immune deficiency syndrome patients. Proc. Natl. Acad. Sci. U.S.A. 83: 7089–7093.
Wiley CA, Nelson JA. (1990) Human immunodeficiency virus: Infection of the nervous system. Curr. Top. Microbiol. Immunol. 160: 157–172.
Salahuddin SZ, Rose RM, Groopman JE, Markham PD, Gallo RC. (1986) Human T lymphotropic virus type III infection of human alveolar macrophages. Blood 68: 281–287.
Pearce TE, Nowakowski M, Eden E, et al. (1993) Uniform detection of HIV-1 in alveolar macrophages of pediatric but not adult AIDS patients. J. Leukoc. Biol. 53: 722–726.
Meitzer MS, Kornbluth RS, Hansen B, Dhawan S, Gendelman HE. (1993) HIV infection of the lung. Role of virus-infected macrophages in the pathophysiology of pulmonary disease. Chest 103: 103S–108S.
Schuitemaker H, Meyaard L, Kootstra NA, et al. (1993) Lack of T-cell dysfunction and programmed cell death in human immunodeficiency type-1 infected chimpanzees correlates with absence of monocytotropic variants. J. Infect. Dis. 168: 1140–1147.
Bukrinsky MI, Sharova N, Dempsey MP, et al. (1992) Active nuclear import of human immunodeficiency virus type 1 preintegration complex. Proc. Natl. Acad. Sci. U.S.A. 89: 6580–6584.
Stevenson M, Bukrinsky M, Haggerty S. (1992) HIV-1 replication and potential targets for intervention. AIDS Res. Hum. Retrovir. 8: 107–117.
Lewis P, Emerman M. (1994) Passage through mitosis is required for oncoretroviruses but not for the human immunodeficiency virus. J. Virol. 68: 510–516.
Bukrinsky MI, Sharova N, McDonald TL, Pushkarskaya T, Tarpley WG, Stevenson M. (1993) Association of integrase, matrix, and reverse transcriptase antigens of human immunodeficiency virus type 1 with viral nucleic acids following acute infection. Proc. Natl. Acad. Sci. U.S.A. 90: 6125–6129.
Heinzinger N, Bukrinsky M, Haggerty S, et al. (1994) The Vpr protein of human immunodeficiency virus type 1 influences nuclear localization of viral nucleic acids in non-dividing host cells. Proc. Natl. Acad. Sci. U.S.A. 91: 7311–7315.
Bukrinsky MI, Haggerty S, Dempsey MP, et al. (1993) A nuclear localization signal within HIV-1 matrix protein that governs infection of non-dividing cells. Nature 365: 666–669.
Chelsky D, Ralph R, Jonak G. (1989) Sequence requirements for synthetic peptidemediated translocation to the nucleus. Mol. Cell. Biol. 9: 2487–2492.
von Schwedler U, Kornbluth RS, Trono D. (1994) The nuclear localization signal of the matrix protein of human immunodeficiency virus type 1 allows the establishment of infection in macrophages and quiescent T lymphocytes. Proc. Natl. Acad. Sci. U.S.A. 91: 6992–6996.
Gulizia J, Dempsey MP, Sharova N, et al. (1994) Reduced nuclear import of human immunodeficiency virus type 1 preintegration complexes in the presence of a prototypic nuclear targeting signal. J. Virol. 68: 2021–2025.
Ulrich P, Cerami A. (1984) Trypanocidal 1,3-arylene diketone bis(guanylhydrazone)s. Structure-activity relationships among substituted and heterocyclic analogues. J. Med. Chem. 27: 35–40.
Gendelman HE, Orenstein JM, Martin MA, et al. (1988) Efficient isolation and propagation of human immunodeficiency virus on recombinant colony-stimulating factor 1-treated monocytes. J. Exp. Med. 167: 1428–1441.
Bukrinsky MI, Stanwick TL, Dempsey MP, Stevenson M. (1991) Quiescent T lymphocytes as an inducible virus reservoir in HIV-1 infection. Science 254: 423–427.
Nuovo GJ, Margiotta M, MacConnell P, Becker J. (1992) Rapid in situ detection of PCR-amplified HIV-1 DNA. Diagn. Mol. Pathol. 1: 98–102.
Nuovo GJ, Gallery F, MacConnell P, Becker J, Bloch W. (1991) An improved technique for the detection of DNA by in situ hybridization after PCR-amplification. Am. J. Pathol. 139: 1239–1244.
Nuovo GJ. (1992) PCR in Situ Hybridization: Protocols and Applications. Raven Press, New York.
Nuovo GJ, Gallery F, MacConnell P, Braun A. (1994) In situ detection of polymerase chain reaction-amplified HIV-1 nucleic acids and tumor necrosis factor-α RNA in the central nervous system. Am. J. Pathol. 144: 659–666.
Richter JD, Standiford D. (1992) Structure and regulation of nuclear localization signals. In: Feldherr CM (ed). Nuclear Trafficking. Academic Press, San Diego, pp. 90–121.
Mitsuya H, Yarchoan R, Kageyama S, Broder S. (1991) Targeted therapy of human immunodeficiency virus-related disease. F.A.S.E. B. J. 5: 2369–2381.
Yarchoan R, Pluda JM, Perno CF, Mitsuya H, Broder S. (1991) Anti-retroviral therapy of HIV infection: Current strategies and challenges for the future. Blood 78: 859–884.
Mitsuya H, Yarchoan R. (1994) Development of antiretroviral therapy for AIDS and related disorders. In: Broder S, Merigan TC, Bolognesi D (eds). Textbook of AIDS Medicine. Williams & Wilkins, Baltimore, pp. 721–742.
Roe TY, Reynolds TC, Yu G, Brown PO. (1993) Integration of murine leukemia virus DNA depends on mitosis. E.M.B.O. J. 12: 2099–2108.
Li G, Simm M, Potash MJ, Volsky DJ. (1993) Human immunodeficiency virus type 1 DNA synthesis, integration, and efficient replication in growth-arrested T cells. J. Virol. 67: 3969–3977.
Weinberg JB, Matthews TJ, Cullen BR, Malim MH. (1991) Productive human immunodeficiency virus type 1 (HIV-1) infection of nonproliferating human monocytes. J. Exp. Med. 174: 1477–1482.
Layne SP, Merges MJ, Dembo M, et al. (1992) Factors underlying spontaneous inactivation and susceptibility to neutralization of human immunodeficiency virus. Virology 189: 695–714.
Schuitemaker H, Kootstra NA, Koppelman MH, et al. (1992) Proliferation-dependent HIV-1 infection of monocytes occurs during differentiation into macrophages. J. Clin. Invest. 89: 1154–1160.
Yamasaki L, Lanford RE. (1992) Nuclear transport receptors: Specificity amid diversity. In: Feldherr CM (ed). Nuclear Trafficking. Academic Press, San Diego, pp. 122–174.
van Furth R, Diesselhoff-den Dulk MMC, Raeburn JA, Zwet TL, Crofton RW, van Oud Albas AB. (1980) Characteristics, origin and kinetics of human and murine mononuclear phagocytes. In: van Furth R (ed). Mononuclear Phagocytes. Functional Aspects. Martinus Nijhoff, Hague, pp. 279–300.
Koyanagi Y, O’Brien WA, Zhao JQ, Golde DW, Gasson JC, Chen IS. (1988) Cytokines alter production of HIV-1 from primary mononuclear phagocytes. Science 241: 1673–1675.
Watkins BA, Dorn HH, Kelly WB, et al. (1990) Specific tropism of HIV-1 for microglial cells in primary human brain cultures. Science 249: 549–553.
Stevenson M, Haggerty S, Lamonica C, Mann AM, Meier C, Wasiak A. (1990) HIV-1 replication is controlled at the level of T cell activation and proviral integration. E.M.B.O. J. 9: 1551–1560.
Zack JA, Arrigo SJ, Weitsman SR, Go AS, Haislip A, Chen IS. (1990) HIV-1 entry into quiescent primary lymphocytes: Molecular analysis reveals a labile, latent viral structure. Cell 61: 213–222.
Spina CA, Kwoh TJ, Chowers MY, Guatelli JC, Richman DD. (1994) The importance of nef in the induction of human immunodeficiency virus typa 1 replication from primary quiescent CD4 lymphocytes. J. Exp. Med. 179: 115–123.
Miller MD, Warmerdam MT, Gaston I, Greene WC, Feinberg MB. (1994) The human immunodeficiency virus-1 nef gene product: A positive factor for viral infection and replication in primary lymphocytes and macrophages. J. Exp. Med. 179: 101–113.
Pantaleo G, Graziosi C, Demarest JF, et al. (1993) HIV infection is active and progressive in lymphoid tissue during the clinically latent stage of disease. Nature 362: 355–358.
Ho WZ, Cherukuri R, Douglas SD. (1994) The macrophage and HIV-1. Immunol. Ser. 60: 569–587.
Folks TM, Powell D, Lightfoote M, et al. (1986) Biological and biochemical characterization of a cloned Leu-3− cell surviving infection with the acquired immune deficiency syndrome retrovirus. J. Exp. Med. 164: 280–290.
Acknowledgements
This work was supported in part by Grant AI 33776 from National Institutes of Health and 02059-15-RGR from AmFAR to MB.
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Dubrovsky, L., Ulrich, P., Nuovo, G.J. et al. Nuclear Localization Signal of HIV-1 as a Novel Target for Therapeutic Intervention. Mol Med 1, 217–230 (1995). https://doi.org/10.1007/BF03401569
Published:
Issue Date:
DOI: https://doi.org/10.1007/BF03401569