Endocytosis-mediated HIV-1 entry and its significance in the elusive behavior of the virus in astrocytes

Abstract

Astrocytes protect neurons but also evoke a proinflammatory response to injury and viral infections including HIV. We investigated the mechanism of HIV-1 infection in primary astrocytes, which showed minimal but productive viral infection independent of CXCR4. As with ectopic-CD4-expressing astrocytes, lysosomotropic agents led to increased HIV-1 infection in wild-type but not Rab 5, 7, and 11-ablated astrocytes. Instead, HIV-1 infection was decreased in Rab-depleted astrocytes, corroborating viral entry by endocytosis. HIV-1 produced persistent infection in astrocytes (160 days); no evidence of latent infection was seen. Notably, one caveat is that endosomal modifiers enhanced wild-type HIV-1 infection (M- and T-tropic) in astrocytes, suggesting endocytic entry of the virus. Impeding endocytosis by inhibition of Rab5, 7 or 11 will inhibit HIV infection in astrocytes. Although the contribution of such low-level infection in astrocytes to neurological complications is unclear, it may serve as an elusive viral reservoir in the central nervous system.

HIV-1 differentially modulates autophagy in neurons and astrocytes

“…Briefly, 17 μg of each of the respective HIV-1 DNA vectors was transfected in 100 mm culture dishes (BD Falcon) using Lipofectamine 2000 (Invitrogen) as described earlier (Vijaykumar et al, 2008;Chauhan et al, 2014). Similarly, HIV-1 NLENY1 was pseudotyped with VSV-G envelope using 4.0 μg of VSV-G expression vector.…”

“…Similarly, HIV-1 NLENY1 was pseudotyped with VSV-G envelope using 4.0 μg of VSV-G expression vector. Lentiviral vector pLVX expressing GFP was pseudotyped with HIV-1 HXB2 envelope (X4) or VSV-G envelope, using cotransfection of 4.0 μg of HXB2 envelope vector or pcDNA VSV-G in combination with 12.0 μg of pLVX, 10.0 μg pGag-Pol, 3.0 μg each of pcDNA-Rev, and Vpr plasmids (Chauhan, 2014;Chauhan et al, 2014). Supernatants containing viral particles were harvested 72 h after transfection and centrifuged at 2000 rpm for 10 min to remove cellular debris, then treated with 5 IU/mL RNase-free DNase for 30 min at 25°C.…”

“…Given that HIV-1-infected monocytes or lymphocytes enter the brain (Price, 1996) and that a part from R5-tropic HIV-1, a subset of primary X4 viruses can infect macrophages and microglia (Ghorpade et al, 1998;Simmons et al, 1998;Verani et al, 1998;Ohagen et al, 1999), we tested both X4-and R5-HIV-1 virus particles. To investigate autophagy and HIV-1-induced direct toxicity, X4-or R5-tropic HIV-1 viruses were assembled in 293T cells by transfection of HIV-1 full-length infectious DNA (Vijaykumar et al, 2008;Chauhan et al, 2014). Assembled viruses harvested at 72 h after transfection were titrated for p24 capsid protein and used in subsequent experiments.…”

“…To determine the effects of HIV-1 particles and supernatants from HIV-1-infected macrophages, cultures of HFA were established as demonstrated by GFAP staining (Fig. 4A) (Mehla et al, 2012;Chauhan et al, 2014). HFA were doseresponsively treated with HIV-1 (10-500 ng/mL p24) for 48 h, using X4-and R5-tropic HIV-1 particles assembled in HEK 293T cells.…”

“…VSV-pseudotyped HIV-1 infection is a fusion-mediated viral entry process (endocytosis) without CXCR4 and CD4. Astrocytes are minimally infected by natural HIV-1 infection, but VSV-pseudotyped HIV-1 initiates a rapid and potent viral infection (Vijaykumar et al, 2008;Chauhan et al, 2014). VSVpseudotyped lentiviral vector carrying the GFP gene under the CMV-promoter was packaged in HEK 293T cells and used as a control.…”