SummaryHIV-1 disseminates to diverse tissues and establishes long-lived viral reservoirs. These reservoirs include the CNS, in which macrophage-lineage cells, and as suggested by many studies, astrocytes, may be infected. Here, we have investigated astrocyte infection by HIV-1. We confirm that astrocytes trap and internalize HIV-1 particles for subsequent release but find no evidence that these particles infect the cell. Astrocyte infection was not observed by cell-free or cell-to-cell routes using diverse approaches, including luciferase and GFP reporter viruses, fixed and live-cell fusion assays, multispectral flow cytometry, and super-resolution imaging. By contrast, we observed intimate interactions between HIV-1-infected macrophages and astrocytes leading to signals that might be mistaken for astrocyte infection using less stringent approaches. These results have implications for HIV-1 infection of the CNS, viral reservoir formation, and antiretroviral therapy.

Our findings lead to new insights into unique aspects of HIV transmission in the brain and at T cell-T cell synapses, which are thought to be a predominant mode of rapid HIV transmission early in the infection process.

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 3-demensional electron microscopy. A panel of receptor antagonists was used to determine 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 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 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.