Kramer, Beatrice; (2004) HIV-1 assembly in primary human macrophages. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Human immunodeficiency virus (HIV) is thought to assemble and bud at the plasma membrane of infected cells. However, published electron micrographs have indicated that virus can also be found in intracellular compartments in some infected cells. Intracellular assembly is particular prominent in macrophages, although the nature of the virus-containing compartment and the relevance of intracellular virus in HIV infection are unclear. This thesis shows that in human monocyte derived macrophages (MDM) infectious HIV-1 is assembled on intracellular membranes of multivesicular late endosomes. The nature of the compartment into which virus particles bud was identified by immunolabelling techniques with organelle-specific antibodies. This showed a strong co-localisation of viral antigen with late endosomal markers such as CD63. Immuno-electron microscopy detected CD63 on the viral membrane and internal vesicles of multivesicular bodies, suggesting that cellular proteins resident in late endosomes are incorporated in intracellular virus. To determine whether intracellular virus is released and is infectious, virus derived from MDM supernatants was precipitated with organelle-specific antibodies. This revealed the presence of late endosomal proteins, such as CD63 and LAMP-1, on the viral membrane of infectious particles. Plasma membrane proteins, in contrast, were not incorporated, suggesting that infectious virus released from MDM had assembled intracellularly. The detection of infectivity in MDM-homogenates further supports the finding that intracellularly assembled virus is infectious. Virus release seems to be an active process, as indicated by temperature sensitivity, and probably occurs through fusion of internal compartments with the plasma membrane. Immunoprecipitation of T-cell derived HIV-1 indicated that T-cells may also assemble virus intracellularly, alongside plasma membrane assembly. This was suggested by the detection of late endosomal and plasma membrane proteins on the viral membrane and supported by preliminary morphological analysis. These findings may have broad implications for our understanding of the biology of HIV infection and its cell-cell transmission.

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