RSV and IGF1R: More than just a scratch on the “cell” surface
Respiratory viruses have co-existed with humans for hundreds or even thousands of years and continue to persist and cause disease on a yearly basis. These viruses pose a significant threat to the most vulnerable, especially premature babies and the elderly. The 1918 influenza pandemic and currently, the coronavirus pandemic are two examples that show the deadly toll exerted by viruses on a large and acute scale. With modern medicine, we can intervene to prevent infections among the most vulnerable. An understanding of how virus enters the cell is critical for devising these interventions.
The culprit in this case is human respiratory syncytial virus (RSV). RSV is highly prevalent and by age of two, most children will have already experienced RSV infection. In most cases, RSV is non-threatening and causes cold-like symptoms. Unfortunately, in some newborns, immunocompromised or elderly patients, RSV infection is serious and requires hospitalization. In some infants, especially premature babies, their small airways can become obstructed from this infection. Sadly, around 60,000 infants succumb in hospitals to RSV yearly worldwide, yet RSV often flies under the radar and does not get much attention outside the clinic.
Scientists and physicians have been aware of RSV for over 50 years but curiously how RSV infects airway cells has only started to become clear in the last decade. RSV is an enveloped virus meaning its infectious contents are protected by plasma membrane, the same material that protects our cells. On the envelope (or virus surface), RSV expresses three proteins – F (fusion), G (glycoprotein), and SH (small hydrophobic). The RSV-F protein is critical for viral fusion with the cell, hence the name. A major limitation to the understanding of RSV cell entry was that a specific entry receptor used by RSV-F protein to bind stably to airway cells and mediate infection was unclear until now. In a Nature paper from the lab of Dr. David Marchant at the University of Alberta, Griffiths et al. show that RSV-F binds and manipulates a human protein called IGF1R (insulin-like growth factor 1 receptor) to gain entry in airway cells.
Before diving into the details, Dr. Marchant and others had previously determined that nucleolin, another host protein, was critical for RSV cell fusion. Nucleolin is typically not expressed on the cell surface though. As implied by the name, nucleolin is found in the nucleus of the cell. How would RSV then mediate an effective infection of airway cells through a protein found predominantly in the nucleus? This question led to a hypothesis that a surface molecule on airway cells must be involved in RSV infection.
In the paper, Griffiths et al. found that nucleolin would traffic from the nucleus to the cell surface in response to RSV infection. This could facilitate RSV host cell entry by enhancing the levels of nucleolin at the cell surface. One possibility could be that RSV triggers a surface receptor to cause the shuffling of nucleolin from the nucleus to the surface to promote infection. By targeting specific cell surface receptors with either small molecule inhibitors or antibodies, the authors honed on IGF1R, as cells treated with IGF1R inhibitors or antibodies became resistant to RSV infection. In mosquito cells, which cannot be infected by RSV, forced expression of IGF1R was sufficient to allow RSV infection. Remarkably, RSV-F protein and IGF1R interacted in a cell-free system with exquisite affinity and specificity through a technique known as microscale thermophoresis. Together, this proposes a means by which RSV mediates a successful infection through IGF1R-dependent triggering by RSV-F.
Griffiths et al. then demonstrated a role for an intracellular signaling pathway that relays IGF1R triggering by RSV to recruit nucleolin to the cell surface. This involves a kinase (a molecular switch), knowns as PKC-z (protein kinase C zeta). Inhibition of PKC-z was sufficient to prevent RSV infection and reflected the similar effects observed by blocking nucleolin or IGF1R. Moreover, kinase activity assays showed that PKC-zwas dependent on IGF1R triggering by RSV and this occurred very early (within 10 minutes) after infection. RSV primarily infects human ciliated bronchial epithelial cells (also called club cells) and indeed these cells showed expression of all three of the players (IGF1R, PKC-z, and nucleolin) supporting that this process happens in the airways during RSV infection.
Most of the experiments involved cell lines using traditional cell culture, so to validate a role for IGF1R and PKC-z in the airways, the authors moved to a system that more closely mimics lung physiology through organoids of bronchial epithelial cells in air-liquid interface (ALI) cultures. Microscopic examination of these ALI cultures before and after RSV infection showed a similar process of nucleolin trafficking to the surface after infection and this could be reduced significantly by IGF1R or PKC-z inhibition. Last, PKC-z inhibition could significantly reduce infection and pathology from RSV infection in a mouse model. Therefore, RSV host cell entry depends on triggering IGF1R signaling through PKC-z in vivo.
This impressively demonstrates IGF1R as RSV entry receptor, yet a few questions do remain unanswered. As I mentioned earlier, RSV also expresses two other proteins on the surface aside from the F protein which are the G and SH proteins. Whether these proteins play a role in vivo during the process of virus-mediated cell entry remains unclear. One possibility may be that these proteins tether the virus to the host cell surface while nucleolin traffics from the nucleus to the surface, a suggestion raised by the authors for the G protein. IGF1R also belongs to the family of proteins known as receptor tyrosine kinases which have complex biology, and perhaps RSV may also exploit IGF1R in other ways to mediate viral fusion. Additionally, whether IGF1R is the only cell entry receptor for RSV may be also contested and perhaps triggering of other receptors that engage PKC-z signaling may be relevant, although when asked about this Dr. Marchant says, “We found that IGF1R ticks all the boxes”. RSV infection also only causes severe disease in some patients and whether this is because of previous environmental exposures, features of the immune response, or variability of expression of IGF1R or other molecules requires future investigation.
In my opinion, this study will be seminal in the field of respiratory virus biology because it illustrates many sequential molecular processes that occur early during an infection. In the fight against respiratory viruses, each one of these events could be potentially targetable by pre-existing or novel therapeutics. In the case of RSV, an antibody that targets the F protein known as palivizumab has clinical use in high-risk infants as a preventative measure, although this only has limited efficacy. Perhaps targeted inhibition of IGF1R or PKC-z in the airways may have broader efficacy and help prevent hospitalizations from RSV. This paper also offers a framework that may be applicable for in-depth study of the biology of many other respiratory viruses, including the coronavirus responsible for the current pandemic.
By Carlos Castellanos
Link to the study
Schematic showing the binding of RSV to its receptor (IGF1R) and subsequent recruitment of its coreceptor (NCL) – from Griffiths et al. Nature 2020