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.

The primary human immunodeficiency virus type 1 (HIV-1) - specific CD8+ T cell response plays an important role in control of early viral replication. The nature of this response may thus be among the factors determining the prognostically-important persisting viral load. Here, the primary HIV-specific CD8+ T cell response was characterised in a small cohort of patients, to gain insight into quantitative and qualitative features of the response and their relationship to the efficiency of control of virus replication. The majority of patients studied established intermediate or high persisting viral loads. In these individuals, no association was found between the persisting viral load established and the total magnitude of the early HIV- specific CD8+ T cell response, its epitope breadth or specificity. However the response was observed to be heavily biased towards the most immunodominant epitopes. Kinetic analyses of epitope-specific responses revealed that in most patients, responses expanded asynchronously, with rapidly-expanded responses being immunodominant in primary infection. Mechanisms that may account for the rapid initial expansion of selected responses in acute viral infection, including high avidity and pre-existence of cross-reactive memory cells, were explored using murine models. Notably, the most rapidly-expanded epitope-specific response(s) typically did not reach peak magnitude until several weeks after the peak in acute viral replication and the fastest response was observed in the one patient studied who established a low persisting viral load. Phenotypic studies suggested that HIV-specific CD8+ T cells exhibit an "immature" phenotype from primary infection onwards in all patients. Although these studies need to be extended to a larger number of patients, the results obtained suggest that the kinetics and synchronicity of expansion of epitope-specific CD8+ T cell responses in primary HIV-1 infection may be among the factors that impact on the efficiency of control of primary viraemia.

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