CCR5-, DC-SIGN-dependent endocytosis and delayed reverse transcription after human immunodeficiency virus type 1 infection in human astrocytes

We sought to determine the pathway of HIV-1 entry into human astrocytes and the fate of HIV-1 by detecting viral DNA and GFP-tagged HIV-1 in HIV-1-infected primary astrocytes. Immunochemistry and FACS analysis were used to assess the expression of DC-SIGN in human purified cultures of astrocytes. HIV-1 LTR was detected by PCR in infected cultures of human embryonic astrocytes at their third passage. GFP-Vpr-labeled R5 tropic HIV-1 was used to infect astrocytes, and was followed by confocal microscopy. Forty percent of astrocytes expressed DC-SIGN at the membrane level. Viral DNA was detected 5 days after infection in human astrocytes, but not in the presence of anti-CCR5 and anti-DC-SIGN mAbs. T20, NH4Cl, and bafilomycin had no effect on viral DNA detection. We found that 67% of the fluorescent GFP-Vpr-labeled R5 tropic HIV-1 viruses were present in the endosomes of astrocytes at 24 h, but not in the presence of anti-CCR5 or DC-SIGN mAbs. Bafilomycin and NH4Cl each increased the amount of fluorescent HIV-1 detected outside endosomes. Titers of p24 remained low from day 1 to day 5 postinfection, in the presence or absence of NH4Cl. Astrocytes express DC-SIGN and HIV-1 penetrates into these cells through CCR5- and/or DC-SIGN-mediated endocytosis, via a pH-dependent pathway, with a delayed reverse transcription after infection without productive infection.