Investigation into sub-cellular CD4 distribution in human embryonic stem cell derived macrophages and its role in HIV-1 infection

Human macrophages are one of the main targets for HIV-1 infection, despite their moderately low surface expression levels of the main HIV-1 receptor, CD4. The site of HIV-1 fusion can occur at the surface or following uptake through an endosomal pathway and it might be anticipated that the site would affect the progress of HIV-1 through the cell to the nucleus. Previous pharmacological studies provide one line of evidence for an endosomal entry route which is dependent on Detergent Resistant Membranes (DRMs). However, these findings need confirmation using a genetic approach, as small molecules may have multiple non-specific effects. For this study, a novel genetic approach was developed to manipulate sub-cellular CD4 distribution and investigate whether it determines the HIV-1 entry pathway in macrophages. This was achieved by transducing human embryonic stem cells (hESC) with lentiviral vectors and differentiating these cells into homogeneous genetically modified macrophages. This cellular system by-passes the challenges posed by the refractoriness to direct genetic manipulation of heterogeneous primary macrophages. Firstly, as proof of principle, a short hairpin RNA targeting CD4 was expressed in hESC-macrophages, resulting in knockdown of CD4 and, as anticipated, strong inhibition of HIV-1 infection. Secondly, expression of LCK in hESC-macrophages effectively tethered CD4 at the cell surface, and sequestered HIV-1 into an unproductive pathway, presumably through surface fusion, rather than progressing successfully to the nucleus. Thirdly, endogenous CD4 was substituted with CD4 mutants designed to be excluded from DRMs, which resulted in reduced successful HIV-1 entry versus substituted control CD4. The results support the model in which the productive entry pathway of HIV-1 in macrophages occurs via fusion after a raft-dependent endocytic uptake pathway, and requires CD4 localization to lipid rafts.This thesis is not currently available in ORA