The productive entry pathway of HIV-1 in macrophages is dependent on endocytosis through lipid rafts containing CD4.
Macrophages constitute an important reservoir of HIV-1 infection, yet HIV-1 entry into these cells is poorly understood due to the difficulty in genetically manipulating primary macrophages. We developed an effective genetic approach to manipulate the sub-cellular distribution of CD4 in macrophages, and investigated how this affects the HIV-1 entry pathway. Pluripotent Stem Cells (PSC) were transduced with lentiviral vectors designed to manipulate CD4 location and were then differentiated into genetically modified macrophages. HIV-1 infection of these cells was assessed by performing assays that measure critical steps of the HIV-1 lifecycle (fusion, reverse transcription, and expression from HIV-1 integrants). Expression of LCK (which tethers CD4 to the surface of T cells, but is not normally expressed in macrophages) in PSC-macrophages effectively tethered CD4 at the cell surface, reducing its normal endocytic recycling route, and increasing surface CD4 expression 3-fold. This led to a significant increase in HIV-1 fusion and reverse transcription, but productive HIV-1 infection efficiency (as determined by reporter expression from DNA integrants) was unaffected. This implies that surface-tethering of CD4 sequesters HIV-1 into a pathway that is unproductive in macrophages. Secondly, to investigate the importance of lipid rafts (as detergent resistant membranes - DRM) in HIV-1 infection, we generated genetically modified PSC-macrophages that express CD4 mutants known to be excluded from DRM. These macrophages were significantly less able to support HIV-1 fusion, reverse-transcription and integration than engineered controls. Overall, these results support a model in which productive infection by HIV-1 in macrophages occurs via a CD4-raft-dependent endocytic uptake pathway.
Sir William Dunn School of Pathology, University of Oxford, Oxford, United Kingdom.
Bonnie
B
van Wilgenburg
Sir William Dunn School of Pathology, University of Oxford, Oxford, United Kingdom.
Michael D
MD
Moore
Sir William Dunn School of Pathology, University of Oxford, Oxford, United Kingdom.
William S
WS
James
Sir William Dunn School of Pathology, University of Oxford, Oxford, United Kingdom.
Sally A
SA
Cowley
D000818:Animals; D015704:CD4 Antigens / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D004705:Endocytosis / Q000276:immunology*; D015870:Gene Expression; D055785:Gene Knockdown Techniques; D023061:Gene Order; D005822:Genetic Vectors; D015658:HIV Infections / Q000235:genetics / Q000276:immunology / Q000378:metabolism / Q000821:virology; D015497:HIV-1 / Q000502:physiology*; D006801:Humans; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck) / Q000235:genetics / Q000378:metabolism; D008264:Macrophages / Q000502:physiology* / Q000821:virology*; D021962:Membrane Microdomains; D051379:Mice; D021381:Protein Transport; D048348:Reverse Transcription; D053586:Virus Internalization; D014779:Virus Replication
D015704:CD4 Antigens; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck)