26/09/11 Virology journal club #1 - is nucleolin the RSV receptor?

This week's journal club (Monday 26/09/2011) - the first of the coming academic year - was on the recently published paper identifying the molecule nucleolin as a functional receptor for respiratory synycitial virus (RSV) - a virus who's receptor has proved elusive to scientists; it was taken by a post-doc in our lab: Olivier Touzelot. This Nature Medicine study (get the paper here) was released in mid-August 2011 by a group at the The Hospital for Sick Children, Toronto, Ontario, Canada.

Abstract:

Human respiratory syncytial virus (RSV) causes a large burden of disease worldwide¹. There is no effective vaccine or therapy, and the use of passive immunoprophylaxis with RSV-specific antibodies is limited to high-risk patients^{2, 3, 4, 5}. The cellular receptor (or receptors) required for viral entry and replication has yet to be described; its identification will improve understanding of the pathogenesis of infection and provide a target for the development of novel antiviral interventions. Here we show that RSV interacts with host-cell nucleolin via the viral fusion envelope glycoprotein and binds specifically to nucleolin at the apical cell surface in vitro. We observed decreased RSV infection in vitro in neutralization experiments using nucleolin-specific antibodies before viral inoculation, in competition experiments in which virus was incubated with soluble nucleolin before inoculation of cells, and upon RNA interference (RNAi) to silence cellular nucleolin expression. Transfection of nonpermissive Spodoptera frugiperda Sf9 insect cells with human nucleolin conferred susceptibility to RSV infection. RNAi-mediated knockdown of lung nucleolin was associated with a significant reduction in RSV infection in mice (P = 0.0004), confirming that nucleolin is a functional RSV receptor in vivo.

RSV virus and nucleolin (Ncl) co-localisation in airway epithelial cells. Notice a lot of RSV without Ncl - yet they are both found in the same general distribution? Is something else playing a part, possiblu mediating the interaction between the two?

In our discussion, a number of points were raised regarding this paper and it's results:

• Why exactly was nucleolin focused on? If you look at the supplementary figures, a lot of proteins could have bound to the virus - especially as they were previously narrowing their search down cell surface proteins - what was wrong with the other humans ones (which had higher hits than nucleolin). 
• Sometimes data shown can be too good. Comments were given that in some of the figures (the vopa ones especially), the blots looked very neat with no spurious bands. Apparently this techniques has been carried out a lot before without so near as much luck as this case. The paper didn't say how theirs worked while others did not. Maybe these guys are just better at it than others...
• Their co-localisation data wasn't very convincing - a lot of RSV was seen attached to the cells without nucleolin - although, this might reflect the nature of nucleolin movement within the cell. And, in the mouse tissue - a lot of nucleolin positive staining was seen without viral infection.
• When transfecting in the nucleolin gene inside non-permissive insect cells - not a lot of protein was produced, and it was had a very strange distribution being only found in one single small area of the cell. Yet, this allowed near 100% infection by the virus. Maybe this does reflect how the receptor behaves naturally however - which may explain why only a little is required (also may explain the strange co-localisation data).
• Finally, this paper highlights how difficult it is to work with a gene/protein that is so necessary for cell-viability - how can you easily knock-down a gene that is expressed very highly in every cell and retain viability?

 While we all agreed it was a pretty good paper - even if all this data was taken as correct, it would still not explain the entire story of RSV infection of a cell, despite being completely effective in allowing insect cells to be infected. As nucleolin is expressed in every cell, you would expect RSV to infect all tissues of a human, but it only infects the respiratory epithelium. However, this may be an effect of viral release, with virus particles only being released into the space of the lung. Clearly this work needs repeated in a more appropriate model system.