Abstract

Astrocytes are a significant CNS reservoir of proviral HIV-1 DNA and contribute to the pathogenesis of HIV-associated dementia

M. Churchill^1,2, P.R. Gorry^1,2,3, D. Cowley¹, L. Gray¹, C. Pardo⁴, J. McArthur⁴, B.J. Brew⁵, S.L. Wesselingh<sup>1,2

1</sup>Burnet Institute, Centre for Virology, Melbourne, Australia, ²Monash University, Department of Medicine, Melbourne, Australia, ³University of Melbourne, Department of Microbiology and Immunology, Melbourne, Australia, ⁴Johns Hopkins University School of Medicine, Department of Neurology, Baltimore, United States, ⁵St. Vincent's Hospital, Department of Neurology and St. Vincent's Centre for Applied Medical Research, Darlinghurst, Australia

Background: HIV-1 penetrates the central nervous system (CNS) frequently causing encephalitis (HIVE), HIV-associated dementia (HAD) or less severe neurocognitive impairment. The CNS is also a significant HIV-1 reservoir. Astrocytes are latently-infected resulting in astrocyte dysfunction. Because astrocyte infection is considered rare, estimated at 1-3% by early studies^[1-3], astrocytes are thought to be a minor HIV-1 reservoir with a secondary role in the pathogenesis of HAD. In this study, we re-evaluated the magnitude of astrocyte infection in vivo and potential role for HIV-1-infected astrocytes HAD pathogenesis.
Methods: Autopsied brains of 12 HIV-1+ cases who died with- (n=10) or without HAD (n=2) were studied. HAD cases consisted of subjects without HIVE (n=2), with mild (n=1), moderate (n=4) or severe HIVE (n=3). GFAP+ astrocytes and CD68+ macrophage-lineage cells were identified by immunohistochemistry and nuclei isolated using Laser-capture microdissection. Integrated HIV-1 DNA was detected by nested Alu-PCR. Astrocyte infection frequency was determined by multiplexed PCR on single nuclei to amplify the HIV-1 V3 Env region and cellular GAPDH.
Results: The results of Alu-PCR amplifications confirmed that astrocytes of 5 cases with severe or moderate HIVE harbored integrated HIV-1 DNA. Single-cell astrocyte amplifications from all 12 cases showed that astrocyte infection frequency is extensive, occurring in up to 19% of GFAP+ cells. Astrocyte infection frequency correlated with severity of HIVE and proximity to macrophages in vivo.
Conclusions: Astrocytes are extensively infected with HIV-1 in subjects with HAD and harbor integrated proviral DNA. Since astrocytes are terminally differentiated and essential for brain function, they are an extensive and permanent reservoir of HIV-1 DNA that complicates virus eradication strategies. In addition, our results suggest HIV-1-infected astrocytes contribute to the pathogenesis of HAD.
1. Bagasra et al., AIDS 10:573-585, 1996.
2. Takahashi et al., Ann. Neurol. 39:705-711, 1996.
3. Brack-Werner., AIDS 13:1-22, 1999.