Abstract
Our recent studies have demonstrated that extracellular, recombinant human immunodeficiency virus type I (HIV-1) Vpr protein is highly neurotoxic in the microenvironment of differentiated mature human neurons and undifferentiated neuronal precursors. Although most of the direct neurotoxic effects of HIV-1 have been attributed previously to the envelope gene product, gp120, and the Tat regulatory protein, it was demonstrated that Vpr protein caused apoptosis comparable to that induced by gp120 protein in a dose-dependent manner in the neuronal system. Having observed the neurocytopathic effects of extracellular Vpr protein previously, the effects of virally expressed Vpr on nondividing, terminally differentiated human neurons were investigated. An HIV-1-based three-plasmid expression vector system was utilized to study the effects of intracellularly expressed Vpr. These virion preparations were then used to transduce neurons generated from the human neuronal precursor NT2 cell-line. Intracellularly expressed Vpr induced apoptosis within terminally differentiated neurons, as demonstrated by TUNEL assays. Additionally, virions lacking Vpr expression did not significantly induce apoptosis within these neurons. These results suggest that HIV-1 Vpr may also be leading directly to selective neurotoxicity through intracellular expression. Furthermore, human apoptosis gene microarray comparisons exhibited an up-regulation of Bcl-2-related mRNA, as well as other apoptosis genes involved in the mitochondrial apoptotic pathway, for the Vpr-transduced neuronal cells, when compared to Vpr-negative controls. Thus, Vpr delivered intracellularly, as well as extra-cellularly, is involved in the induction of significant neuronal apoptosis and may be one of the molecular mechanisms in HIV-1-induced encephalopathy.
Similar content being viewed by others
References
Adie-Biassette H, Levy Y, Colombel M, Poron F, Natcher S, Keohane C, Gray F (1995). Neuronal apoptosis in HIV infection in adults. Neuropathol Appl Neurobiol 21: 218–227.
Ayyavoo V, Mahboubi A, Mahalingam S, Ramalingam R, Kudchodkar S, Williams VW, Green DR, Weiner DB (1997). HIV-1 Vpr suppresses immune activation and apoptosis through regulation of nuclear factor κB. Nature Med 3: 1117–1123.
Bagasra O, Lavi E, Khalili K, Pestaner JP, Tawadros R, Pomerantz RJ (1996). Cellular reservoirs of HIV-1 in the central nervous system of infected individuals: Identification by the combination of in situ polymerase chain reaction and immunohistochemistry. AIDS 10: 573–585.
Blomer U, Naldini L, Kafri T, Trono D, Verma IM, Gage FH (1997). Highly efficient and sustained gene transfer in adult neurons with a lentivirus vector. J Virol 71: 6641–6649.
Boldin MP, Mett IL, Varfolomeev EE, Chumakov I, Shemer-Avni Y, Camonis JH, Wallach D (1995). Self-association of the ‘death domains” of the p55 tumor necrosis factor (TNF) receptor and Fas/APO1 prompts signaling for TNF and Fas/APOl effects. J Biol Chem 270: 387–391.
Budihardjo I, Oliver H, Lutter M, Wang X (1999). Biochemical pathways of caspase activation during apoptosis. Annu Rev Cell Develop Biol 15: 269–290.
Canto-Nogues C, Sanchez-Ramon S, Munoz-Fernandez M (2001). HTV-1-infected cortical neurons in brain autopsy from children with HIV-1 related encephalopathy. 263. 1st IAS conference on HIV pathogenesis and treatment, Buenos Aires, Argentina, July 2001.
Cohen EA, Dehni G, Sodoroski JG, Haseltine WA (1990). Human immunodeficiency virus vpr product is a virion-associated regulatory protein. J Virol 64: 3097–3099.
DeNoronha C, Sherman M (2001). HIV-1 Vpr disrupts nuclear lamin structure leading to nuclear blebbing and cell cycle arrest. Abstract #141. 8th National conference on retroviruses and opportunistic infections, Chicago, Illinois, February 4 to 8.
Desagher S, Osen-Sand A, Nichols A, Eskes R, Montessuit S, Lauper S, Maundrell K, Antonsson B, Martinou J-C (1999). Bid-induced conformational change of Bax is responsible for mitochondrial cytochrome c release during apoptosis. J Cell Biol 144: 891–901.
Ensoli F, Cafaro A, Fiorelli V, Vannelli B, Ensoli B, Thiele C (1995). HIV-1 infection of primary human neuroblasts. Virology 210: 221–225.
Ferri KF, Jacotot E, Blanco J, Este JA, Zamzami N, Susin SA, Xie Z, Brothers G, Reed JC, Penninger JM, Kroemer G (2000). Apoptosis control in syncytic induced by the HIV type 1-envelope glycoprotein complex: Role of mitochondrial and caspases. J Exp Med 192: 1081–1092.
Finkel E (2001). The mitochondrion: Is it central to apoptosis? Science 292: 624–626.
Gasmi M, Glynn J, Jin M-J, Jolly D-J, Yee J-K, Chen S-T (1999). Requirements for efficient production and transduction of human immunodeficiency virus type 1 based vectors. J Virol 73: 1828–1834.
Gelbard HA, James HJ, Sharer LR, Perry SW, Saito Y, Kazee AM, Blumberg BM, Epstein LM (1995). Apoptotic neurons in brains from pediatric patients with HIV-1 encephalitis and progressive encephalopathy. Neuropathol Appl Neurobiol 21: 208–217.
Glabinski AR, Ransohoff RM (1999). Chemokines and chemokine receptors in CNS pathology. J NeuroVirol 5: 3–12.
Gummuluru S, Emerman M (1999). Cell cycle and Vprmediated regulation of human immunodeficiency virus type 1 expression in primary and transformed T cell lines. J Virol 73: 5422–5430.
He J, Choe S, Walker R, DiMarzio P, Morgan DO, Landau NR (1995). Human immunodeficiency virus type 1 viral protein R (Vpr) arrests cells in the G2 phase of the cell cycle by inhibiting p34 cdc2 activity. J Virol 69: 6705–6711.
Heinzinger NK, Bukrinsky MI, Haggerty SA, Ragland AM, Kewalramani V, Lee MA, Gendelman HE, Ratner L, Stevenson M, Emerman M (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 USA 91: 7311–7315.
Hesselgesser J, Horuk R (1999). Chemokines and chemokine receptor expression in the central nervous system. J NeuroVirol 5: 3–12.
Hirka G, Prakash K, Kawashima H, Plotkin SA, Andrews PW, Gonczol E (1991). Differentation of human embryonal carcinoma cells induces human immunodeficiency virus permissiveness which is stimulated by human cytomegalovirus coinfection. J Virol 65: 2732–2735.
Hrimech M, Yao X-J, Bachand F, Rogeau N, Cohen EA (1999). Human immunodeficiency virus type 1 (HIV-1) Vpr functions as an immediate-early protein during HIV-1 infection. J Virol 73: 4101–4109.
Huang MB, Weeks O, Zhao L-J, Saltarelli M, Bond VC (2000). Effects of extracellular human immunodeficiency virus type 1 vpr protein in primary rate cortical cell cultures. J NeuroVirol 6: 202–220.
Jacotot E, Ferri KF, Hamel CE, Brenner C, Druillennec S, Hoebeke J, Rustin P, Metivier D, Lenoir C, Geuskens M, Vieira HLA, Loeffler M, Belzacq A-S, Briand J-P, Zamzami N, Edelman L, Xie Z-H, Reed JC, Roques BP, Kroemer G (2001). Control of mitochondrial membrane permeabilization by adenine nucleotide translocator interacting with HIV-1 viral protein rR and Bcl-2. J Exp Med 193: 509–519.
Jowett JBM, Planelles V, Poon B, Shah NP, Chen M-L, Chen ISY (1995). The human immunodeficiency virus type 1 vpr gene arrests infected T cells in the G2 + M phase of the cell cycle. J Virol 69: 6304–6313.
Kohleisen B, Hutzler P, Shumay E, Ovod V, Erfle V (2001). HIV-1 Nef co-localizes with the astrocyte-specific cytoskeleton protein GFAP in persistently nef-expressing human astrocytes. J NeuroVirol 7: 52–55.
Kroemer G, Reed JC (2000). Mitochondria control of cell death. Nature Med 6: 513–519.
Kulkosky J, Laptev A, Shetty S, Srinivasan A, BouHamdan M, Prockop DJ, Pomerantz RJ (1999). Human immunodeficiency virus type 1 Vpr alters bone marrow cell function. Blood 93: 1906–1915.
Lane JH, Tarantal AF, Pauley D, Marthas M, Miller CJ, Lackner AA (1996). Localization of simian immunodeficiency virus nucleic acid and antigen in brains of fetal macaques inoculated in utero. Am J Pathol 149: 1097–1104.
Lavi E, Strizki M, Ulrich AM, Zhang W, Fu L, Wang O, O’Connor M, Hoxie JA, Gonzalez-Scarano F (1997). CXCR-4 (Fusin), a co-receptor for the type 1 human immunodeficiency virus (HIV-1), is expressed in the human brain in a variety of cell-types, including microglia and neurons. Am J Path 151: 1035–1042.
Levy DN, Refaeli Y, Weiner DB (1995). Extracellular Vpr protein increases cellular permissiveness to human immunodeficiency virus replication and reactivates virus from latency. J Virol 69: 1243–1252.
Li X, Traganos F, Melamed MR, Darzynkiewicz Z (1995). Single step procedure for labeling DNA strand breaks with fluorescein- or BODIPY-conjugated deoxynucleotides. Detection of apoptosis and bromodeoxyuridine incorporation. Cytometry 20: 172–180.
Mahalingam S, Ayyavoo V, Patel M, Kieber-Emmons T, Weiner D (1997). Nuclear import, virion incorporation, and cell cycle arrest/differentiation are mediated by distinct functional domains of human immunodeficiency virus type 1 Vpr. J Virol 71: 6339–6347.
Mahalingam S, Khan SA, Jabbar MA, Monken CE, Collman RG, Srinivasan A (1995). Identification of residues in the N-terminal acidic domain of HIV-1 Vpr essential for virion incorporation. Virology 207: 297–302.
Mischiati C, Pironi F, Milani D, Giacca M, Mirandola P, Capitani S, Zauli G (1999). Extracellular HIV-1 Tat protein differentially activates the JNK and ERK/MAPK pathways in CD4 T cells. AIDS 13: 1637–1645.
Mizrachi Y, Rodriquez I, Sweetnam PM, Rubinstein A, Volsky DJ (1994). HIV type 1 infection of human cortical neuronal cells: Enhancement by select neuronal growth factors. AIDS Res Hum Retrovir 10: 1593–1596.
Mukhtar M, Duke H, BouHamdan M, Pomerantz RJ (2000). Anti-human immunodeficiency virus type 1 gene therapy in human central nervous system-based cells: An initial approach against a potential reservoir. Human Gene Ther 11: 347–359.
Mukhtar M, Pomerantz RJ (2000). Development of an in vitro blood-brain barrier model to study molecular neuropathogenesis and neurovirologic disorders induced by human immunodeficiency virus type 1 infection. J Human Virol 3: 324–334.
Naldini L, Blomer U, Gallay P, Ory D, Mulligan R, Gage FH, Verma IM, Trono D (1996). In vivo gene delivery and stable transduction of non-dividing cells by a lentiviral vector. Science 272: 263–267.
Nuovo GJ, Gallery F, MacConnell P, Braun A (1994). In situ detection of polymerase chain reaction-amplified HIV-1 nucleic acids tumor necrosis factor-alpha RNA in the central nervous system. Am J Pathol 144: 659–666.
Ohagen A, Ghosh S, He J, Huang K, Chen Y, Yuan M, Osathanondh R, Gartner S, Shi B, Shaw G, Gabuzda D (1999). Apoptosis induced by infection of primary brain cultures with diverse human immunodeficiency virus type 1 isolates: Evidence for a role of the envelope. J Virol 73: 897–906.
Patel CA, Mukhtar M, Pomerantz RJ (2000). Human immunodeficiency virus type 1 Vpr induces apoptosis in human neuronal cells. J Virol 74: 9717–9726.
Petito CK, Roberts B (1995). Evidence of apoptotic cell death in HIV encephalitis. Am J Pathol 146: 1121–1130.
Piller SC, Jans P, Gage PW, Jans DA (1998). Extracellular HIV-1 virus protein R causes a large inward current and cell death in cultured hippocampal neurons: Implications for AIDS pathology. Proc Natl Acad Sci USA 95: 4595–4600.
Pleasure SJ, Lee VM (1993). NTera 2 cells: A human cell line which displays characteristics expected of a human committed neuronal progenitor cell. J Neurosci Res 35: 585–602.
Pleasure SJ, Page C, Lee VM (1992). Pure, post mitotic, polarized human neurons derived from Ntera 2 cells provide a system for expressing exogenous proteins in terminally differentiated neurons. J Neurosci 12: 1802–1815.
Poon B, Grovit-Ferbas K, Stewart KA, Chen ISY (1998). Cell cycle arrest by Vpr in HIV-1 virions and insensitivity to antiretroviral agents. Science 281: 266–269.
Popov S, Rexach M, Zybarth G, Reiling N, Lee M-A, Ratner L, Lane CM, Moore MS, Blobel G, Bukrinsky M (1998). Viral protein R regulates nuclear import of the HIV-1 pre-integration complex. EMBO J 17: 909–917.
Sharpe AH, Hunter JJ, Chassier P, Jaenisch R (1984). Role of abortive retroviral infection of neurons in spongiform CNS degeneration. Nature 246: 181–183.
Sherman MP, DeNoronha C, Greene S, Heusch M, Greene WC (2001). Identification of HIV-1 Vpr as a shuttling protein. Abstract #139. 8th National conference on retro-viruses and opportunistic infections. Chicago, Illinois, February 4 to 8.
Shi B, De Girolami U, He J, Wang S, Lorenzo A, Busciglio J, Gabuzda D (1996). Apoptosis induced by HIV-1 infection of the central nervous system. J Clin Invest 98: 1979–1990.
Shi B, Raina J, Lorenzo A, Busciglio J, Gabuzda D (1998). Neuronal apoptosis induced by HIV-1 Tat protein and TNF-α: Potentiation of neuro-toxicity mediated by oxidative stress and implications for HIV-1 dementia. J NeuroVirol 4: 281–290.
Shimuzu S, Narita M, Tsujimoto Y (1999). Bcl-2 family proteins regulate the release of apoptogenic cytochrome c by the mitochondrial channel VDAC. Nature 399: 483–487.
Shostak LD, Ludlow J, Fisk J, Pursell S, Rimel BJ, Nguyen D, Rosenblatt JD, Planelles V (1999). Roles of p53 and cas-pases in the induction of cell cycle arrest and apoptosis by HIV-1 vpr. Exp Cell Res 251: 156–165.
Speth C, Stockl G, Mohhsenipour I, Wurzner R, Stoiber H, Lass-Florl C, Dierich M (2001). Human immunodeficiency virus type 1 induces expression of complement factors in human astrocytes. J Virol 75: 2604–2615.
Stewart SA, Poon B, Jowett JBM, Chen IS (1997). Human immunodeficiency virus type 1 Vpr induces apoptosis following cell cycle arrest. J Virol 71: 5579–5592.
Stewart SA, Poon B, Jowett JB, Xie Y, Chen IS (1999). Lentiviral delivery of HIV-1 vpr protein induces apoptosis in transformed cells. Proc Natl Acad Sci USA 96: 12039–12043.
Stewart SA, Poon B, Song JY, Chen IS (2000). Human immunodeficiency virus type 1 Vpr induces apoptosis through caspase activation. J Virol 74: 3105–3111.
Truckenmiller ME, Kulaga H, Coggiano M, Wyatt R, Snyder SH, Sweetnam PM (1993). Human cortical neuronal cell line: A model for HIV-1 infection in an immature neuronal system. AIDS Res Hum Retroviruses 9: 445–453.
Vodicka MA, McDonald D, Lucero G, Emerman M, Hope TJ (2001). HIV-1 Vpr is a CRM+ dependent nucleocytoplasmic shuttling protein. Abstract #140. 8th National conference on retroviruses and opportunistic infections. Chicago, Illinois, February 4 to 8.
Yao X-J, Mouland AJ, Subbramanian RA, Forget J, Rougeau N, Bergeron D, Cohen EA (1998). Vpr stimulates viral expression and induces cell killing in human immunodeficiency virus type 1-infected dividing Jurkat T cells. J Virol 72: 4686–4693.
Yao X-J, Subbramanian RA, Rogeau N, Boisvert F, Bergeron D, Cohen EA (1995). Mutagenic analysis of human immunodeficiency virus type 1 Vpr: Role of a predicted N-terminal alpha-helical structure in Vpr nuclear localization and virion incorporation. J Virol 69: 7032–7044.
Yasuda J, Miyao T (2001). T cell apoptosis causes peripheral T cell depletion in mice transgenic for the HIV-1 Vpr gene. Virology 285: 181–192.
Yuan X, Malsuda Z, Matsuda M, Essex M, Lee TH (1990). Human immunodeficiency virus vpr gene encodes a virion-associated protein. AIDS Res Hum Retroviruses 6: 1265–1271.
Zhao L-J, Lilin W, Mukherjee S, Narayan O (1994). Biochemical mechanism of HIV-1 Vpr function. Oligomerization mediated by the N-terminal domain. J Biol Chem 296: 32131–32137.
Zufferey R, Nagy D, Mandel RJ, Naldini L, Trono D (1997). Multiply attenuated lentiviral vector achieves efficient gene delivery in vivo. Nature Biotech 15: 871–875.
Author information
Authors and Affiliations
Corresponding author
Additional information
Research was supported in part by NIH N.R.S.A.; training Grant T32-A107533; and USPHS grants MH63009, NS27405, and MH58526 to RJP.
Rights and permissions
About this article
Cite this article
Patel, C.A., Mukhtar, M., Harley, S. et al. Lentiviral expression of HIV-1 Vpr induces apoptosis in human neurons. Journal of NeuroVirology 8, 86–99 (2002). https://doi.org/10.1080/13550280290049552
Received:
Revised:
Accepted:
Issue Date:
DOI: https://doi.org/10.1080/13550280290049552