Abstract
Neurocognitive disorders continue to occur in HIV-infected individuals, despite successful antiretroviral therapy. HIV can persist in the brain for decades, where it infects mainly microglial cells and astrocytes. Brain tissues from HIV-infected individuals have been shown to harbor HIV proviruses and to express early viral products with neurotoxic properties, like Tat. Egress of HIV from astrocytes to the periphery in animals further supports a critical role of astrocytes as HIV reservoirs. In vitro studies show that astrocytes can harbor latent HIV proviruses that can be activated by various agents and initiate productive infection of immune cells. Cell culture studies of HIV-infection of astrocytes have depended heavily on rapidly dividing cells derived from tumors or from fetal tissue. However, in adult brains the majority of astrocytes are nondividing. Therefore, cell culture models are needed to investigate the unique properties of latent HIV proviruses in differentiated astrocytes and to compare these with the properties of other HIV reservoirs.
This protocol gives guidelines for the culture of the human neural stem cell line HNSC.100 and a stable subpopulation with latent HIV-1 provirus, HNSCLatGFP1.2. The HNSC.100 cell line provides a single cell model system for the study of HIV persistence in proliferating progenitor cells as well as fully differentiated, nondividing astrocytes. The HNSCLatGFP1.2 cell line contains a full-length HIV-1 provirus derived from NL4-3 with GFP-coding sequences in a defective Env reading frame, enabling handling under Biosafety level 2 conditions and convenient observation of provirus reactivation by monitoring GFP expression. The latent provirus can be reactivated by latency reversing agents which allows the analysis of novel latency reversing agents as well as inhibitors of reactivators of latency.
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References
Gonzalez-Scarano F, Martin-Garcia J (2005) The neuropathogenesis of AIDS. Nat Rev Immunol 5(1):69–81. https://doi.org/10.1038/nri1527
Valcour V, Chalermchai T, Sailasuta N, Marovich M, Lerdlum S, Suttichom D, Suwanwela NC, Jagodzinski L, Michael N, Spudich S, van Griensven F, de Souza M, Kim J, Ananworanich J, Group RSS (2012) Central nervous system viral invasion and inflammation during acute HIV infection. J Infect Dis 206(2):275–282. https://doi.org/10.1093/infdis/jis326
Jia P, Zhao Z, Hulgan T, Bush WS, Samuels DC, Bloss CS, Heaton RK, Ellis RJ, Schork N, Marra CM, Collier AC, Clifford DB, Gelman BB, Sacktor N, Morgello S, Simpson DM, McCutchan JA, Barnholtz-Sloan JS, Franklin DR, Rosario D, Letendre SL, Grant I, Kallianpur AR, Group CS (2017) Genome-wide association study of HIV-associated neurocognitive disorder (HAND): a CHARTER group study. Am J Med Genet B Neuropsychiatr Genet 174(4):413–426. https://doi.org/10.1002/ajmg.b.32530
Chen NC, Partridge AT, Sell C, Torres C, Martin-Garcia J (2017) Fate of microglia during HIV-1 infection: from activation to senescence? Glia 65(3):431–446. https://doi.org/10.1002/glia.23081
Brack-Werner R (1999) Astrocytes: HIV cellular reservoirs and important participants in neuropathogenesis. AIDS 13(1):1–22. https://doi.org/10.1097/00002030-199901140-00003
Azevedo FA, Carvalho LR, Grinberg LT, Farfel JM, Ferretti RE, Leite RE, Jacob Filho W, Lent R, Herculano-Houzel S (2009) Equal numbers of neuronal and nonneuronal cells make the human brain an isometrically scaled-up primate brain. J Comp Neurol 513(5):532–541. https://doi.org/10.1002/cne.21974
Sofroniew MV, Vinters HV (2010) Astrocytes: biology and pathology. Acta Neuropathol 119(1):7–35. https://doi.org/10.1007/s00401-009-0619-8
Churchill MJ, Wesselingh SL, Cowley D, Pardo CA, McArthur JC, Brew BJ, Gorry PR (2009) Extensive astrocyte infection is prominent in human immunodeficiency virus-associated dementia. Ann Neurol 66(2):253–258. https://doi.org/10.1002/ana.21697
King JE, Eugenin EA, Buckner CM, Berman JW (2006) HIV tat and neurotoxicity. Microbes Infect 8(5):1347–1357. https://doi.org/10.1016/j.micinf.2005.11.014
Kramer-Hammerle S, Rothenaigner I, Wolff H, Bell JE, Brack-Werner R (2005) Cells of the central nervous system as targets and reservoirs of the human immunodeficiency virus. Virus Res 111(2):194–213. https://doi.org/10.1016/j.virusres.2005.04.009
Spector C, Mele AR, Wigdahl B, Nonnemacher MR (2019) Genetic variation and function of the HIV-1 tat protein. Med Microbiol Immunol 208(2):131–169. https://doi.org/10.1007/s00430-019-00583-z
Seth P, Major EO (2005) Human brain derived cell culture models of HIV-1 infection. Neurotox Res 8(1–2):83–89. https://doi.org/10.1007/BF03033821
Gorry PR, Ong C, Thorpe J, Bannwarth S, Thompson KA, Gatignol A, Vesselingh SL, Purcell DF (2003) Astrocyte infection by HIV-1: mechanisms of restricted virus replication, and role in the pathogenesis of HIV-1-associated dementia. Curr HIV Res 1(4):463–473. https://doi.org/10.2174/1570162033485122
Vincendeau M, Kramer S, Hadian K, Rothenaigner I, Bell J, Hauck SM, Bickel C, Nagel D, Kremmer E, Werner T, Leib-Mosch C, Brack-Werner R (2010) Control of HIV replication in astrocytes by a family of highly conserved host proteins with a common rev-interacting domain (Risp). AIDS 24(16):2433–2442. https://doi.org/10.1097/QAD.0b013e32833e8758
Narasipura SD, Kim S, Al-Harthi L (2014) Epigenetic regulation of HIV-1 latency in astrocytes. J Virol 88(5):3031–3038. https://doi.org/10.1128/JVI.03333-13
Lutgen V, Narasipura SD, Barbian HJ, Richards M, Wallace J, Razmpour R, Buzhdygan T, Ramirez SH, Prevedel L, Eugenin EA, Al-Harthi L (2020) HIV infects astrocytes in vivo and egresses from the brain to the periphery. PLoS Pathog 16(6):e1008381. https://doi.org/10.1371/journal.ppat.1008381
Schneider M, Tigges B, Meggendorfer M, Helfer M, Ziegenhain C, Brack-Werner R (2015) A new model for post-integration latency in macroglial cells to study HIV-1 reservoirs of the brain. AIDS 29(10):1147–1159. https://doi.org/10.1097/QAD.0000000000000691
Villa A, Snyder EY, Vescovi A, Martinez-Serrano A (2000) Establishment and properties of a growth factor-dependent, perpetual neural stem cell line from the human CNS. Exp Neurol 161(1):67–84. https://doi.org/10.1006/exnr.1999.7237
Rubio FJ, Bueno C, Villa A, Navarro B, Martinez-Serrano A (2000) Genetically perpetuated human neural stem cells engraft and differentiate into the adult mammalian brain. Mol Cell Neurosci 16(1):1–13. https://doi.org/10.1006/mcne.2000.0854
Kunze C, Borner K, Kienle E, Orschmann T, Rusha E, Schneider M, Radivojkov-Blagojevic M, Drukker M, Desbordes S, Grimm D, Brack-Werner R (2018) Synthetic AAV/CRISPR vectors for blocking HIV-1 expression in persistently infected astrocytes. Glia 66(2):413–427. https://doi.org/10.1002/glia.23254
Rothenaigner I, Kramer S, Meggendorfer M, Rethwilm A, Brack-Werner R (2009) Transduction of human neural progenitor cells with foamy virus vectors for differentiation-dependent gene expression. Gene Ther 16(3):349–358. https://doi.org/10.1038/gt.2008.173
Rothenaigner I, Kramer S, Ziegler M, Wolff H, Kleinschmidt A, Brack-Werner R (2007) Long-term HIV-1 infection of neural progenitor populations. AIDS 21(17):2271–2281. https://doi.org/10.1097/QAD.0b013e3282f12f27
Zhang H, Zhou Y, Alcock C, Kiefer T, Monie D, Siliciano J, Li Q, Pham P, Cofrancesco J, Persaud D, Siliciano RF (2004) Novel single-cell-level phenotypic assay for residual drug susceptibility and reduced replication capacity of drug-resistant human immunodeficiency virus type 1. J Virol 78(4):1718–1729. https://doi.org/10.1128/jvi.78.4.1718-1729.2004
Acknowledgments
We thank Johanna Götz for excellent technical assistance. Furthermore, we thank Martha Schneider for generation of the HNSCLatGFP1.2 cell line. We are grateful to Ulrike Protzer, Helmholtz Zentrum München, Institute of Virology, for continuous support and encouragement.
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Bauer, A., Brack-Werner, R. (2022). Modeling HIV Latency in Astrocytes with the Human Neural Progenitor Cell Line HNSC.100. In: Poli, G., Vicenzi, E., Romerio, F. (eds) HIV Reservoirs. Methods in Molecular Biology, vol 2407. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1871-4_10
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DOI: https://doi.org/10.1007/978-1-0716-1871-4_10
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