Unravelling the movement of viral genomes through PD

A long-standing research interest in the lab has been in the movement of viral genomes through PD. Most encapsidated plant viruses are too large to pass through the PD pore, necessitating the cell-cell movement of the viral genome as some form of linear ribonucleoprotein complex. Currently we are investigating the replication and movement of two model plant viruses, tobacco mosaic virus (TMV) and potato virus X (PVX). We are focussing our studies on the early stages of replication and movement, as the events that occur in newly infected cells are poorly understood. For TMV, we have adapted methods for tagging RNA developed by Ilan Davis University of Oxford (http://www.bioch.ox.ac.uk/aspsite/research/brochure/Davis/) In this approach the vRNA is tagged in vitro with UTP-CY3 which is incorporated into the viral RNA (vRNA). Following injection of CY3-vRNA, we were able to track the movement of infectious vRNA as it enters a living cell. Significantly, we showed that the vRNA anchors immediately to the cortical actin/ER network prior to the formation of a viral replication complex (for details see Christensen et al., 2009).

Injection of a mixture of GFP and Cy3-labelled viral RNA into a single leaf trichome. Note that the viral RNA forms granules in the cytoplasm. The virus also encoded cytosolic DsRed, and cell-cell movement of the viral genome is evident from the diffuse red labelling of the subtending epidermal cell. With time (insets) the DsRed-expressing virus invaded the leaf lamina. In comparison, note that GFP does not exit the injected cell (arrowed). Chlorophyll autofluorescence is shown in blue (see Christensen et al., 2009).

Although viral MPs have been tagged extensively with fluorescent protein reporters, the localisation of the vRNA during infection has remained obscure due to an inability to detect vRNA in vivo. We have developed a protein-based tag for vRNA derived from the RNA-binding protein, Pumilio. Pumilio binds to RNA in a sequence-specific manner and contains two discrete RNA binding domains known as Pumilio homology (PUMH) domains. These PUMH domains contain 8 repeats that bind to specific nucleotides on the target RNA. We engineered two PUMH domains to recognise specific sequences in the genomic RNA of TMV and PVX, and were able to detect the vRNA of these viruses in living cells (For details see Tilsner et al., 2008). In recent work, we are examining the cell-cell movement strategy of PVX using a combination of mutagenesis, vRNA imaging and viral protein tagging.

Viral RNA of tobacco mosaic virus (TMV) localised with the RNA-binding protein Pumilio using bimolecular fluorescence complementation (BiFC). The vRNA is concentrated in the viral replication complex (VRC; arrow) and also in punctae at the cell surface
 

Viral RNA of potato virus X (PVX) revealed by Pumilio tagging. Note that the vRNA is localised in discrete ‘whorls’ within the VRC