Annegret Pelchen-Matthews BSc ARCS PhD DIC

Morphological analysis of HIV assembly in macrophages

Following my PhD in Molecular Neurobiology, I joined Mark Marsh to work on the cell biology of virus infection, focusing particularly on the primate immunodeficiency viruses, HIV-1 and SIVmac. I have analysed the cellular trafficking of the HIV receptor CD4 and the co-receptors CXCR4 and CCR5, as well as the HIV and SIV envelope glycoproteins. Recently, I have used various electron microscopy methods, including immunolabelling of ultrathin cryosections, to analyse HIV assembly in its natural host cells, particularly macrophages. We have shown that in macrophages, HIV assembles in cell surface-connected intracellular membrane compartments; these sites may provide a reservoir of infectious virus. The ability of myeloid cells (macrophages and dendritic cells) to sequester virus particles in surface-connected compartments may be particularly important during virus entry at mucosal surfaces and may play a role in virus transmission from these cells to T cells, the main target cells of HIV.

I have been interested in HIV/AIDS since the virus was discovered in the early 1980s, and particularly in the impact of HIV/AIDS in Africa.

David Nkwe BSc

Analysis of HIV-1 budding in primary human macrophages

In macrophages, HIV-1 assembles into internal plasma membrane connected compartments, contrary to cell surface assembly observed in T-cells. Assembly and budding of HIV-1 at the plasma membrane is facilitated by the virus’ exploitation of various host cell proteins, such as the ESCRT machinery and accessory proteins. It is unclear how the internal plasma membrane used by the virus in macrophages differs from the cell surface plasma membrane, or indeed whether the internal plasma membrane is the only site for virus assembly in macrophages. To address this latter question, I am using different molecular biology approaches, targeting the virus-ESCRT machinery interactions, to arrest HIV-1 budding process and generate stable budding intermediates in order to examine the location and distribution of these sites in infected cells. Increased understanding of how HIV interacts with host cell components might offer novel targets for new therapeutic approaches.

Joe Grove BSC, PhD

Analysis of virus receptor interactions

Viruses are obligate parasites that rely on host cell machinery to replicate. To commandeer a target cell a virus particle must first cross the plasma membrane and deliver its genetic material to an appropriate intracellular compartment, a process termed virus entry. I am interested in the various mechanisms by which viruses achieve this and the cell biology that underpins these processes.

I am currently investigating the G-protein coupled chemokine receptor CCR5 and its role in the entry of HIV. Specifically, I am interested in the behavior of CCR5 at the plasma membrane and how it is internalized following treatment with specific ligands. By studying these aspects of CCR5 biology we hope to better understand protein trafficking to and from the plasma membrane and the implications this may have for understanding HIV entry. I am using live cell and electron microscopy, alongside basic molecular cell biology, to tackle these questions and hope to apply these techniques to other virus systems in the future.

Picorna virus entry and release

A critical part of the replication cycle of Picornaviruses, a group of non-enveloped, positive strand RNA viruses, is their interaction with the mucosal surface of the respiratory or gastrointestinal tract. In the case of human enteroviruses, which replicate in the gastrointestinal epithelial cells, this interaction is poorly understood. The limited data available suggest that the biology of enterovirus infections in polarized cells differs in several respects from that observed in non-polarised cells commonly used to study these viruses. Decay accelerating factor (DAF/CD55) binding enteroviruses, including echovirus 11-207 (EV11-207) are found in the gastrointestinal tract of infected patients. We are using confocal microscopy, electron microscopy and biochemical analysis to understand the entry and the exit stages of the enterovirus lifecycle, focusing on EV11-207 and the model of human polarised epithelial cell line Caco2.

In polarized Caco2 cells, EV11-207 particles are transported to perinuclear compartments upon binding to DAF at the apical membrane.

Polarized Caco-2 monolayers growing on filters were exposed to EV11-207 (100 PFU/cell) at 4°C for 30 min to allow virus attachment. The cells were then washed and warmed to 37°C for 30 min. Fixed and permeabilized cells were stained with anti-ZO-1 mAb (red), anti-EV11-207 pAb (green) and DAPI.

Sebastian Giese Boehringer Ingelheim Fonds Graduate Student

Analysis of cell-to-cell virus transfer

In macrophages, HIV undergoes assembly in intracellular compartments (IC) that are connected to the cell surface by closely apposed membrane sheets. Internal assembly may allow cell-to-cell transmission of HIV to be spatially and temporally co-ordinated through virological synapses (VS, see figure below). These are regions of intimate contact between infected and uninfected leukocytes and have been proposed to facilitate cell-to-cell spread of viruses.
The aim of my PhD project is to investigate whether the virus-filled IC of HIV-infected monocyte-derived macrophages (MDM) are involved in the formation of VS between MDM and CD4+ T cells. I test whether proteins characteristic of the IC are present in these VS and try to establish live-cell imaging assays that could provide direct evidence for the recruitment of pre-formed IC to VS.