Extracellular Vesicles Carry HIV Env and Facilitate Hiv Infection of Human Lymphoid Tissue

Abstract

Cells productively infected with HIV-1 release virions along with extracellular vesicles (EVs) whose biogenesis, size, and physical properties resemble those of retroviruses. Here, we found that a significant number of EVs (exosomes) released by HIV-1 infected cells carry gp120 (Env), a viral protein that mediates virus attachment and fusion to target cells, and also facilitates HIV infection in various indirect ways. Depletion of viral preparations of EVs, in particular of those that carry gp120, decreases viral infection of human lymphoid tissue ex vivo. Thus, EVs that carry Env identified in our work seem to facilitate HIV infection and therefore may constitute a new therapeutic target for antiviral strategy.

Figure 1

Analysis of EVs and HIV-1 captured with CD81 MNPs. A preparation of HIV_SF162 (containing both viruses and EVs) was incubated with CD81 MNPs and the captured particles were stained for CD45 with anti-CD45-eFluor 450 and for gp120 with 2G12-Alexa Fluor 647 antibodies. (A) Particles (EVs and/or virions) captured with CD81 MNPs carrying gp120. (B) EVs (CD45+) captured with CD81 MNPs carrying gp120. (C) 2G12 specificity control: EVs released by uninfected PBMCs captured with CD81 MNPs negative for 2G12-Alexa Fuor 647. (D) Isotype control: Staining with mouse IgG1k-eFluor 450 and human IgG1k-Alexa Fluor 647 antibodies. Presented are data from one representative experiment out of five.

Figure 2

Analysis of EVs expressing gp120 in HIV_SF162 preparation. (A) A preparation of HIV_SF162 (containing both viruses and EVs) was incubated with CD81-MNPs and the captured particles were stained for AChE with anti-AChE-Alexa Fluor 647 and for gp120 with 2G12-Pacific Blue antibodies. (B) Isotype control: Staining with mouse IgG1k-Alexa Fluor 647 and human IgG1k-Pacific Blue. Presented are data from one representative experiment out of three.

Figure 3

Analysis of EVs captured by CD45 MNPs in HIV_SF162 preparation. (A) A preparation of HIV_SF162 (containing both viruses and EVs) was incubated with CD45-MNPs and the captured particles were stained for gp120 with 2G12-Alexa Fluor 647 antibodies. (B) Isotype control: Staining with human IgG1k-Alexa Fluor 647. Indicated are the numbers of acquired events from both preparations.

Figure 4

Analysis of EVs captured by PG16-MNPs. (A) A preparation of HIV_SF162 (containing both viruses and EVs) was incubated with PG16-MNPs and the captured particles were stained with anti-AChE-Alexa Fluor 647 and anti-CD45-eF450 antibodies. (B) Isotype control: Staining with mouse IgG1k-Alexa Fluor 647 and mouse IgG1k eFluor450. Indicated are the numbers of acquired events from both preparations. Presented are data from one of two experiments.

Figure 5

Analysis of EVs expressing gp120 in HIV_LAI.04 preparation. (A) A preparation of HIV_LAI.04 (containing both viruses and EVs) was incubated with CD81-MNPs and the captured particles were stained with anti- AChE-Alexa Fluor 647 and anti-gp120 2G12-Pacific Blue antibodies. (B) Isotype control: Staining with mouse IgG1k-Alexa Fluor 647 and human IgG1k-Pacific Blue. Presented are data from one representative experiment out of three.

Figure 6

Infection of human tonsillar tissue ex vivo with EV-depleted preparations. Donor-matched human tonsillar tissue blocks were inoculated with HIV_SF162 preparations that underwent one or two rounds of depletion using MNPs coupled to antibodies. Left two bars: Viral preparations were depleted with msIgG-MNPs (“mock-depleted”, control, open bar) or anti-CD45 antibody coupled MNPs (black bar). In the two-round depletion experiments the viral preparations were depleted with MNPs coupled to either 2G12 or to PG16 and next depleted with either MNPs coupled to msIgG (“mock-depleted”, open bars) or to anti-CD45 (black bars). Virus released in the medium over 16 days of infection from 27 tissue blocks for each donor was evaluated with p24 measurement and expressed as percent of control. Presented are means ± standard errors of the means (SEM) from experiments with tissues from four donors.