The Intracellular Virus-Containing Compartments in Primary Human Macrophages Are Largely Inaccessible to Antibodies and Small Molecules

Abstract

HIV-1 assembly and release occurs at the plasma membrane of human T lymphocytes and model epithelial cell lines, whereas in macrophages intracellular sites of virus assembly or accumulation predominate. The origin of the intracellular virus-containing compartment (VCC) has been controversial. This compartment is enriched in markers of the multivesicular body, and has been described as a modified endosomal compartment. Several studies of this compartment have revealed the presence of small channels connecting to the plasma membrane, suggesting that instead of an endosomal origin the compartment is a modified plasma membrane compartment. If the compartment is accessible to the external environment, this would have important implications for antiviral immune responses and antiviral therapy. We performed a series of experiments designed to determine if the VCC in macrophages was open to the external environment and accessible to antibodies and small molecules. The majority of VCCs were found to be inaccessible to exogenously-applied antibodies to tetraspanins in the absence of membrane permeabilization, while tetraspanin staining was readily observed following membrane permeabilization. Cationized ferritin was utilized to stain the plasma membrane, and revealed that the majority of virus-containing compartments were inaccessible to ferritin. Low molecular weight dextrans could access only a very small percentage of VCCs, and these tended to be more peripheral compartments. We conclude that the VCCs in monocyte-derived human macrophages are heterogeneous, but the majority of VCCs are closed to the external environment.

High-Multiplicity HIV-1 Infection and Neutralizing Antibody Evasion Mediated by the Macrophage-T Cell Virological Synapse

“…Our data relating to macrophage VS-mediated transfer show that MPER-directed NAb suffer the most loss of activity, and results with 10E8 Fab and T20 suggest this is at least partially mediated by steric hindrance (28). This would be consistent with limited access of IgG to virus within the VCC (7,8), to the rapid transfer of infectious HIV-1 from the VCC to contacting T cells ( Fig. 1) (4), and with electron microscopic imaging which shows tight apposition of MDM and T cell membranes at the synaptic junction (4).…”

“…Long-lived HIV-1-infected macrophages resist viral cytopathic effects and shelter replicationcompetent virions in surface-accessible (3)(4)(5)(6), but antibody-occluding (7,8), virus-containing compartments (VCCs) (6) for several weeks (9), which strongly implicates macrophages in HIV-1 persistence (10,11). Moreover, by efficiently spreading HIV-1 to uninfected CD4 ϩ T cells through direct cell-to-cell contact (4,12), macrophages may actively contribute to viral pathogenesis (10).…”

Molecular Determinants Directing HIV-1 Gag Assembly to Virus-Containing Compartments in Primary Macrophages

“…Notably, mature particles stored in macrophages remain infec-tious for weeks in the presence of a protease inhibitor (20). Moreover, viruses in VCCs are inaccessible to neutralizing antibodies (14,17) yet can be transferred to CD4 ϩ T cells that come into contact with the infected macrophages (21,22). These findings collectively support the hypothesis that VCC-associated viruses are likely to play important roles in HIV-1 pathogenesis.…”

“…In contrast, early ultrastructural studies of HIV-1-infected primary macrophages showed that both mature and immature HIV-1 particles and budding structures accumulate in intracellular compartments (10,11) which share some similarity with endosomes (5,11,12). Subsequent EM studies over the past decade revealed that at least some of these intracellular compartments, now termed virus-containing compartments (VCCs), are deep and convoluted invaginations of the PM and appear to form connections to the cell surface and extracellular space via tubular structures in a dynamic and transient manner (5,(13)(14)(15)(16)(17)(18)(19). Notably, mature particles stored in macrophages remain infec-tious for weeks in the presence of a protease inhibitor (20).…”

Cellular Pharmacology and Potency of HIV-1 Nucleoside Analogs in Primary Human Macrophages

“…Although the antiviral profile of antiretroviral therapy (ART) is partially defined in macrophages (2,(9)(10)(11), a direct link between potency and cellular pharmacology had not been reported. The relationship between the cellular pharmacology of ART and macrophages can impact viral loads, emergence of resistant HIV-1, and the long-term survival of infected individuals (8,12). For these reasons, understanding the dynamics of ART pharmacology in macrophages and subsequent targeted elimination of both the cells and the virus that they harbor are critical to eliminating HIV-1 infection.…”