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Mark Marsh
Group MembersAnnegret Pelchen-Matthews, Kristina Theusner, Kurt Vermeive, Tom Kershaw, Jane Turner, Sarah Main, Stephen Morris
Cell Biology of Virus Entry and AssemblyOur research is currently focused on two areas: * Understanding the mechanisms of assembly of HIV and how this might be used to understand and combat pathogenesis. * Understanding the molecular and cellular mechanisms that control the cell surface expression of the receptors for HIV and related viruses. Enveloped virus entry and egress is tightly linked to cellular membranes. Understanding the cellular and molecular mechanisms underlying these events informs us not only of the viruses themselves, but also of some basic properties of these membrane systems. HIV assemblyPrimate lentiviruses, which include the human immunodeficiency viruses (HIV-1 and -2) and the simian immunodeficiency viruses (SIV), infect and replicate in monocytic cells (primarily macrophages and dendritic cells) and CD4 +ve T lymphocytes. Within infected cells, the key proteins that make up virus particles must be bought together to ensure fully infectious viruses are made. We aim to understand the mechanisms that operate in infected cells that enable HIV to assemble effectively, and how these mechanisms are exploited to optimise cell-to-cell transfer of viruses from infected to uninfected cells. In many infected or transfected model cell lines and T cells, HIV particles have been seen to assemble at the plasma membrane. However, in HIV infected macrophages in tissue culture, and probably in dendritic cells as well, virus particles have been found to assemble primarily on intracellular membranes. These membranes are sub-domains of the plasma membrane that are sequestered within the cell, but are linked to the cell surface by closely apposed membrane sheets (Figure 1). These domains are present in uninfected cells, are morphologically complex (they comprise internal vesicular and 'sponge-like membrane arrays), and contain a number of plasma membrane markers including the tetraspanins CD9, CD53, CD81 and CD82, the lipid PI(4,5)P2 and AP-2 positive clathrin coated pits. But not markers of endocytic organelles such as CD63. Following infection, HIV particles can be seen to assemble on this restricted region of the plasma membrane and mature virus particles accumulate within the organelles. Following interaction with T cells, virions appear in synapse structures between infected macrophages and T cell targets, suggesting that HIV may be delivered from the assembly compartment to the synapse to mediate efficient cell-to-cell transfer to target cells. We aim to understand how virus components target this specific plasma membrane domain, the function of this 'organelle' in uninfected cells, and its regulation during cell-to-cell transmission of HIV. Intrinsic molecular signals must be present to target the viral envelope protein (Env) and structural proteins encoded in the Gag polyprotein to the assembly compartment. Several signals have been identified and studied in some detail. One in the cytoplasmic domain of Env is highly conserved in HIV and SIVs. In collaboration with Jim Hoxie (University of Pennsylvannia, Philadelphia), we have shown that this signal is required for pathogenesis in a SIV model and that deletion of this signal renders virus infectious but non-pathogenic. We aim to understand the cellular mechanisms through which this signal and others facilitate the production of SIV/HIV particles and contribute to disease. Virus entryHIV and other enveloped viruses infect cells by membrane fusion, either at the plasma membrane or with the limiting membrane of intracellular organelles following endocytosis. These events usually follow the engagement of the virus with receptors on the target cell. For HIV, two different surface glycoproteins combine to form the functional virus receptor. One, CD4, binds Env and triggers an initial conformational change that reveals a binding site for the second receptor component. This second component is a member of the chemokine receptor family of G protein-coupled receptors (GPCRs). Interaction with these molecules drives further conformational changes in Env that initiate membrane fusion. Two chemokine receptors have been linked to HIV infection in vivo. The CC chemokine receptor 5 (CCR5) is implicated in the transfer of virus to uninfected individuals and the establishment of infection in the majority of cases. Use of a second chemokine receptor, CXC chemokine receptor 4 (CXCR4), can emerge late in infection in some individuals. Normally these chemokine receptors have important roles in the development and function of the immune system, and other tissues, where they initiate events associated with the binding of small peptide ligands termed chemokines. For HIV to infect a cell, the receptor molecules must be present on the plasma membrane. Significantly, the chemokine ligands for CCR5 and CXCR4 can protect cells from infection by inducing endocytosis of the chemokine receptors. We are analyzing the molecular mechanisms involved in the recruitment of chemokine receptors into endocytic organelles and the subsequent fate of internalised receptors. Although focused on chemokine receptors, these studies have relevance to understanding the down modulation, desensitisation and resensitisation of 7TM GPCRs in general. Binding of chemokine agonists to CCR5 induces rapid internalisation of receptor molecules through endocytic clathrin coated pits and vesicles. Following internalisation, CCR5 molecules enter early sorting endosomes but are rapidly transferred to recycling endosomes. From this site the receptors can recycle to the cell surface. Under normal circumstances the chemokine ligand is removed from the receptor during transit through the endosome system and a resensitised receptor is returned to the cell surface. Ligand removal is not, however, pre-requisite for recycling but occupied receptors that return to the cell surface re-engage the endocytic machinery and are reinternalised to recycling endosomes. Thus resensitisation may be an iterative process involving multiple cycles through the endocytic pathway. We aim to understand the trafficking signals in activated CCR5 molecules and sorting mechanisms that target internalised receptor molecules to recycling endosomes and hence regulate CCR5 cell surface expression.
Morphology of the HIV assembly compartment in infected human monocyte-derived macrophages.Primary human macrophages infected with HIV-1 BaL for 14 days were fixed and embedded in Epon resin for transmission EM. Virus particles, identified by their characteristic morphology, were seen in complex virus-containing compartments (VCC) in the juxta-nuclear region. Note the clathrin-coated pits associated with these compartments (red arrows) and an immature virion (blue arrow). The presence of viral budding figures and immature viruses suggests that HIV assembles directly in this compartment. L, lipid droplets, M, mitochondrion. Scale Bar = 1 µm. See also Deneka et al. (2007).
In CHO cells, b arrestin 2-GFP traffics with agonist-activated CCR5 to a perinuclear, transferrin receptor-containing compartmentUpon activation by chemokine binding, CCR5 (red) is internalised and accumulates in recycling endosomes, showing strong overlap with the transferrin receptor (blue), which predominantly localises to this compartment. Interestingly, b -arrestins (over expressed b arr2-GFP shown in green), proteins that function as adaptors at the cell surface to couple CCR5 to the clathrin-mediated endocytic machinery, remain bound to CCR5 as it traffics to recycling endosomes. We are currently investigating a further role for these multifunctional adaptor proteins in the post-endocytic trafficking of CCR5. Scale Bar = 5 µm. PublicationsRecent Publications list is maintained and updated dynamically by UCL.
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