Interactions between Respiratory Syncytial Virus and Cell Surface Nucleolin

• Author / Creator

  Griffiths, Cameron

• Respiratory syncytial virus (RSV) is a ubiquitous respiratory pathogen that infects almost everyone by the age of two. In high-risk populations, such as infants and elderly individuals, RSV can infect the lower respiratory tract and cause severe symptoms, such as bronchiolitis or pneumonia. As a result, RSV is a leading cause of infant hospitalization. Despite the prevalence of RSV and the severity of infection, many questions remain about the basic biology of the virus. In particular, the RSV entry process has not been completely elucidated. In this thesis, I explored the RSV entry mechanism and how RSV interacts with a critical cell surface receptor, nucleolin (NCL). In normal cells, NCL is primarily found in the nucleus and the levels of NCL on the cell surface are very low, which is uncharacteristic for an important viral receptor. While examining cell surface NCL, I created a series of NCL truncation mutants and used them to characterize the NCL domains that are required for surface expression. I found that some domains of NCL were needed for surface expression, while the presence of others restricted surface expression. Nuclear trafficking of NCL was dispensable for surface expression. I also identified a novel phenomenon of NCL transferring between the surface of cells in a contact-dependent manner. To understand how NCL was acting as an RSV receptor, I measured the levels of NCL on the cell surface and surrounding viral particles during RSV entry. Using cellular fractionations and flow cytometry, I found that NCL translocated from the nucleus to the cell surface during the RSV entry process. I then used live cell imaging and imaging flow cytometry to show that the recruitment of NCL to the cell surface was focused around RSV particles, as viral fusion with the host cell plasma membrane occurred. Using inhibitors of cellular kinases and cell surface receptors, I found that both insulin-like growth factor (IGF1R) signaling and protein kinase C zeta isoform (PKCζ) activity played an important role in RSV entry. I then used imaging flow cytometry to show that PKCζ activity was a critical mediator of the RSV-induced recruitment of NCL to the cell surface. Using an in vitro kinase assay, I found that IGF1R was an upstream regulator of PKCζ activity. Furthermore, I observed that stimulation of IGF1R caused an increase in cell surface NCL expression. In addition to initiating a signaling cascade during RSV entry, I characterized IGF1R as a novel RSV receptor that played a role in RSV binding to host cells. Taken together, my results identified a novel RSV entry mechanism, where RSV first binds to IGF1R. This activates IGF1R, which subsequently signals through PKCζ to induce cell surface trafficking of NCL. On the cell surface, NCL then accumulates around RSV particles, at which point viral fusion occurs.

• Subjects / Keywords

  • Infection
  • Signaling
  • Nucleolin
  • RSV
  • Receptor
  • Virus

• Graduation date

  Spring 2020

• Type of Item

  Thesis

• Degree

  Doctor of Philosophy

• DOI

  https://doi.org/10.7939/r3-57nv-ka49

• License

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