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. 2015 Apr 24;29(7):755-66.
doi: 10.1097/QAD.0000000000000605.

Cell-to-cell Contact Facilitates HIV Transmission From Lymphocytes to Astrocytes via CXCR4

Free PMC article

Cell-to-cell Contact Facilitates HIV Transmission From Lymphocytes to Astrocytes via CXCR4

Guan-Han Li et al. AIDS. .
Free PMC article


Objectives: HIV reservoir in the brain represents a major barrier for curing HIV infection. As the most abundant, long-lived cell type, astrocytes play a critical role in maintaining the reservoir; however, the mechanism of infection remains unknown. Here, we determine how viral transmission occurs from HIV-infected lymphocytes to astrocytes by cell-to-cell contact.

Design and methods: Human astrocytes were exposed to HIV-infected lymphocytes and monitored by live-imaging, confocal microscopy, transmission and three-dimensional electron microscopy. A panel of receptor antagonists was used to determine the mechanism of viral entry.

Results: We found that cell-to-cell contact resulted in efficient transmission of X4 or X4R5-using viruses from T lymphocytes to astrocytes. In co-cultures of astrocytes with HIV-infected lymphocytes, the interaction occurred through a dynamic process of attachment and detachment of the two cell types. Infected lymphocytes invaginated into astrocytes or the contacts occurred via filopodial extensions from either cell type, leading to the formation of virological synapses. In the synapses, budding of immature or incomplete HIV particles from lymphocytes occurred directly onto the membranes of astrocytes. This cell-to-cell transmission could be almost completely blocked by anti-CXCR4 antibody and its antagonist, but only partially inhibited by anti-CD4, ICAM1 antibodies.

Conclusion: Cell-to-cell transmission was mediated by a unique mechanism by which immature viral particles initiated a fusion process in a CXCR4-dependent, CD4-independent manner. These observations have important implications for developing approaches to prevent formation of HIV reservoirs in the brain.

Conflict of interest statement

Conflicts of interest

The authors declare that there are no conflicts of interest.


Figure 1
Figure 1. Infection of astrocytes following the co-cultures with HIV-infected lymphocytes or PBMCs
HIV infection of astrocytes was observed after 3 days when co-cultured with NLENG1-infected (A) JKT cells, (B) MT4 cells, and (C) PBMCs. GFAP-positive astrocytes: red; NLENG1-infected cells: green. Magnification: 200x.
Figure 2
Figure 2. Interactions of astrocytes with NLENG1-infected JKT cells
(A) A NLENG1-infected JKT cell (arrow) is shown partially invaginated into an astrocyte resulting in infection with the virus as shown by acquisition of green fluorescence. (B) An astrocyte (arrow head) wrapped around a NLENG1-infected JKT cell (arrow) and a Velcro-like interdigitated interface was formed by processes extending from both cells as shown by an outline. (C) An NLENG1-infected JKT cell made contact with astrocytes by extending long processes (arrow). (D) NLENG1-infected MT4 cells adhered to astrocytes along their entire cell surface (arrow). Magnification: 630x in A and B; 200x in C and D.
Figure 3
Figure 3. Interactions between astrocytes and HIV-infected lymphocytes as viewed by correlative 3D electron microscopy
(A) Fluorescent image with phase exposure was taken prior to processing the sample for EM and 3D EM. HIV-infected JKT cells show green fluorescence. Magnification: 200x. (B) Low-resolution 3D image was re-constructed using SBF-SEM and 3D software. “A” = astrocyte, “T” = T cell. (C) An overlay image of A and B. (D) High-resolution 3D images show that an NLENG1-infected JKT cell, T1 (brown), was partially wrapped by A1 (purple). The villus-like processes from both cells extended and reached the opposite cell, some of which were interdigitated. Inserts above show the views from different angles. (E) Branched processes from A1 (purple) stretched out in a claw-like structure onto T2 (brown). Viral particles were attached to these processes. Inserts at the bottom show the views from different angles. (F) T3 (brown) sited by A2 (purple) which produced processes and wrapped T3. A large filopodium from T3 protruded into A2. Viral particles spread on the astrocyte. Inserts at the bottom show the views of the filopodium from T3 at different angles. (G) A section of SBF-SEM showed multiple areas of the interdigitated contact between the processes from A2 and T3. (H) Serial, discontinuous sections from the boxed region as shown in Fig. 3G were aligned. Multiple tight contacts between the two cells were observed (block arrow) and the viruses from the infected JKT cell were seen budding into intercellular spaces or directly onto the membrane of the astrocyte (thin arrow). (I) Diagram shows that virological synapses are formed and the viral particles are budding between the areas of tight contacts. The immature, budding viral particles make contact with an astrocyte.
Figure 4
Figure 4. Filopodial extensions and HIV budding onto the membrane of astrocytes
(A) Bridge-like structures were observed between an astrocyte and HIV-infected JKT cells. Magnification: 2,500x. (B) Tight connections were seen at the contact sites of the bridge-like processes between the astrocyte and the bottom lymphocyte in (A). Viral particles were seen attached to the cell membrane and processes of the astrocyte. Magnification: 20,000x. (C) A bridge was formed between the processes from the astrocyte and the upper-left lymphocyte in A. Magnification: 2,500x. (D) A budding virus interacted with an astrocyte process (block arrow) from C. Magnification: 50,000x. The insert shows the site of contact of the budding virus with the astrocyte. Magnification: 80,000x. (E) A bridge in D was further magnified (50,000x). A budding virus was seen inside the virological synapse and made a tight contact with the astrocyte membrane (block arrow). Another mature viral particle was also docked on the astrocyte membrane inside the synapse (thin arrow). (F) The diagram is a composite of the above photomicrographs, depicting the areas of tight contacts between the two types of cells with immature viral particles that directly bud onto the astrocyte membrane.
Figure 5
Figure 5. Chemokine receptor, CXCR4, mediates cell-to-cell transmission of HIV from lymphocytes to astrocytes
(A) HIV cell-to-cell transmission was significantly blocked by both anti-CXCR4 antibody (20 μg/ml) and CXCR4 antagonist AMD3100 (40–100 μM), and partially inhibited by anti-CD4 antibody (20 μg/ml) and fusion inhibitor T20 (100–500 nM) as well as anti-ICAM1 and anti-LFA1 antibodies (20 μg/ml). There was no inhibition with antibodies to DC-SIGN (20 μg/ml) and α4β7 integrin (30ug/ml). The results were the average of 3–5 experiments, shown as mean ± SEM. * p<0.05, ** p<0.01, **** p<0.0001. (B) The blocking of HIV infection of astrocytes by anti-CXCR4 could be partially reversed after the antibody was removed from the co-culture. (C) Astrocytes were pretreated with antibodies to CD4 or CXCR4 (20 μg/ml each) or control media for 1 hr; antibodies were maintained in the co-cultures with NLENG1-infected JKT cells for 24 hr before fixing and processing for correlative IA-SEM. Membrane protrusions from astrocytes toward JKT membranes were absent in the co-cultures with anti-CXCR4 antibody, but present in the controls and the ones with anti-CD4 antibody. Two images for each treatment were from two independent experiments. Colors in images: astrocyte: blue; HIV-infected JKT cell: gold; and HIV virion: red. (D) A hypothesis is proposed based on the ultrastructural observations and the inhibition of CXCR4 on the transmission of HIV from lymphocytes to astrocytes. The CXCR4-binding sites on the surface of the envelope of budding or immature HIV is in an “open” state which allows the virus to directly bind to CXCR4 on the membrane of astrocytes, and hidden following a conformational change that is triggered during HIV maturation. However, the pre-bound virus would trigger the fusion process of HIV envelope with the astrocyte membrane while the maturation is completed, leading to HIV transmission from lymphocytes to astrocytes. This process cannot occur with cell-free mature HIV in astrocytes that lack CD4 expression since the CXCR4-binding sites are hidden in the envelope of mature HIV particles.

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