Marchant, D.; (2005) Characterisation of a post-entry restriction to HIV in human cells. Doctoral thesis , University of London.

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Abstract

HIV-2 infected monocyte derived macrophages (MDM) at much lower efficiency compared to HIV-1 or to infection of PBMC. After a brief initial burst of replication, HIV-2 viral production was terminated. HIV-1 however rebounded cyclically in culture over a period of 20 days. Early entry events of HIV-2 in MDM were accommodated efficiently and replication could be restimulated with LPS indicating that HIV-2 enters a latent phase in MDM. The amino acid charge of the HIV-1 V3 loop is negatively correlated with macrophage tropism (Zhong et al., 1995), and I demonstrate that the HIV-2 V3 loop charge is also negatively correlated with MDM tropism, albeit weakly. This led me to investigate other determinants of HIV cellular tropism. An HIV-2 primary isolate that is unable to replicate in MDM was molecularly cloned (MCR) and the restriction (termed Lv2) mapped to virus env and gag (Schmitz et al., 2004). I show that a variety of primary HIV-1 and HIV-2 viruses are susceptible to Lv2 restriction. Lv2 is a post-entry, post-reverse transcription restriction to HIV infection. To incorporate a role for Env, which acts at the cell surface, a model where the Env delivers a susceptible Gag into a restrictive cellular compartment was developed (Schmitz et al., 2004). The compartmentalisation model of Lv2 was tested using compounds that affect endocytic pathways (hypertonic sucrose) and lipid rafts (Methyl- p-cyclodextrin) in restrictive cells. With these methods I show that restricted virus can be rescued from Lv2 if a lipid raft-dependent endocytic pathway is inhibited. Fusion of restricted virus into HeLa/CD4 cells containing a tailless CD4 that located outside lipid rafts was fully permissive. The restrictive pathway was further defined using dominant negative mutants that specifically inhibit defined endocytic pathways clathrin, caveolae and non-clathrin non-caveolae mediated. A role for an Arf6 non-clathrin non-caveolae mediated endocytic pathway in Lv2 restriction was demonstrated. Lastly, env swapped viruses demonstrate that delivery to the restriction pathway is Env dependent. In keeping with the Lv2 model, the unrestricted virus was unaffected by any of these treatments. Thus the route of entry, determined by the viral Env, can influence cellular tropism by avoiding intracellular blocks to infection. The saturable nature of Lv2 restriction was investigated. HIV-2 Env pseudotypes of El and N-tropic MLV pseudotypes saturated Lv2. This observation led me to investigate if human tripartite motif protein (TRIM) 5a and members of the human TRIM family of proteins, shown to restrict MLV, mediated Lv2. The TRIM family of proteins have been implicated in retroviral restrictions and even so far as to have broad anti-viral activity (Nisole et al., 2005). By choosing TRIM proteins with SPRY(B30.2) domains, and an RNA interference (RNAi) screening approach, TRIMs 1,18 and 34 were identified as having Lv2 restriction activity. These proteins are expressed in restrictive HeLa/CD4 cells but not in permissive ones, thus acting as intracellular determinants of retroviral cell-tropism.

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