Byland, R.; (2006) Trafficking of primate lentiviral envelope proteins. Doctoral thesis , University of London.

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Abstract

The assembly of enveloped viruses requires the correct trafficking of the viral envelope or membrane proteins to the site of virus assembly. To understand the molecular and cellular mechanisms in human immunodeficiency virus envelope protein (Env) trafficking, I analysed the activities of putative endocytosis signals in the cytoplasmic domain of Env. Morphological and biochemical analysis of HxB2 Env as well as CD4-Env chimeras revealed two functional endocytosis motifs, the membrane proximal GY712XX0 motif and the C-terminal dileucine motif. Both of these motifs work with equivalent efficiency and show no obvious additive effect. RNAi knock down experiments show that endocytosis mediated by both signals is at least partly dependent on the clathrin pathway and the clathrin adaptor AP-2. Motifs of the Yxx0 type have been implicated in transport to the late endosome and HIV has been reported to assemble on membranes of this compartment. I studied the intracellular itineraries of HIV Env and found clear evidence of trafficking through early endosomes. However, no obvious colocalisation with CD63 or LAMP-1 was observed. Further analysis suggested that Env is transported to an endosomal compartment positive for the tetraspanin CD81, a compartment that has recently been recognised as the site of HIV assembly in macrophages. Env was targeted to this compartment independently of other viral components in HeLa cells as well as in macrophages. In order to identify other cellular components interacting with Env and the machinery responsible for Env incorporation into virions I performed yeasts- hybrid assays. I found that Vps37B, a component of ESCRT-I, which is required for HIV assembly, can interact with both HIV and SIV Env cytoplasmic tails, and that the ubiquitin E3-ligase WWP2 binds SIV Env. These findings may enable us to establish a molecular mechanism for the incorporation of Env into budding HIV particles.

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