Nkwe, DO; (2014) Analysis of HIV-1 Assembly on Intracellular Plasma Membrane-connected Compartments of Primary Human Macrophages. Doctoral thesis , UCL (University College London).

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Abstract

In primary human monocyte-derived macrophages (MDM), HIV assembles on complex intracellular plasma membrane-connected compartments (IPMCs). Currently, it is unclear whether, in addition to assembly at IPMCs, HIV uses the cell surface of MDM, as viruses that bud at the cell surface may dissociate and be lost from the samples. As the endosomal sorting complex required for transport (ESCRT) machinery is required for the final scission events that release assembled virus from the plasma membrane, this question was addressed by depleting the ESCRT components Tsg101 and ALIX, or generating mutant HIV-1 strains defective in recruiting the ESCRT machinery, to arrest HIV budding and visualise all budding events. Using siRNA, ALIX and Tsg101 were depleted efficiently in MDM. Although the effects on virus release were minimal, Tsg101 had a greater effect than ALIX for HIV release in MDM. In the second approach, I generated mutants (HIV-1 PTAP−, YP−, PTAP−YP−, Δp6) defective in recruiting the ESCRT machinery, by mutating the sequences that bind Tsg101 and ALIX. The mutants were characterised on HEK 293T cells, and the release of PTAP−, PTAP−YP− and Δp6 was inhibited. Analysis by electron microscopy (EM) showed that the mutants indeed produced arrested viruses. As the mutants are defective in release, I developed a method to rescue HIV-1 PTAP− and PTAP−YP− viruses for infecting MDM. The cells were infected with the rescued mutants, and analysed using immunofluorescence and EM. By confocal microscopy, 77% of the cells had viruses in the IPMCs. Using EM, immature viruses were found predominantly (97%) in the IPMCs. Estimates of membrane area revealed enrichment of HIV in the IPMCs. This study provides the first conclusive evidence that HIV is targeted to IPMCs in MDM. This may shield the virus from immune surveillance during virus assembly, with potential impact on cell-to-cell transmission and disease progression.

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