Author ORCID Identifier

https://orcid.org/0000-0001-7207-0639

Date Available

9-12-2020

Year of Publication

2020

Degree Name

Doctor of Philosophy (PhD)

Document Type

Doctoral Dissertation

College

Medicine

Department/School/Program

Molecular and Cellular Biochemistry

First Advisor

Dr. Rebecca Dutch

Abstract

Pneumoviruses including human metapneumovirus (HMPV) and respiratory syncytial virus (RSV) are significant causes of respiratory tract infections globally. Children, elderly, and immunocompromised patients are at the greatest risk for developing severe infections, which can have devastating outcomes. Although these viruses are ubiquitous with significant impacts on human health, there are no antivirals or vaccines available. The only FDA approved therapy is a monoclonal antibody for RSV, given prophylactically during the infectious season, and this treatment is only available for high risk infants. The work presented in this thesis aims to increase our understanding of how these viruses enter, replicate, and spread to better characterize the basic molecular mechanisms used, opening avenues for potential antiviral therapies. We first analyzed the fusion protein of HMPV and how low pH is important for entry of some viral strains. We analyzed previously uncharacterized strains and found that residues initially hypothesized to be critical for low pH fusion are not always required, suggesting a more complex regulation of fusion. We then explored the role of the proteolytic cleavage event which is required for HMPV F as well as many other important respiratory pathogens, including influenza. We found that many proteases involved in activating influenza HA are also important for activating HMPV F, which has not previously been reported. We then used our understanding of cleavage to employ a treatment strategy targeting host proteases involved in this activation to prevent entry and spread. We next conducted a side-by-side comparison of infection, spread, and inhibition using a physiologically relevant 3-D human airway epithelial model system. We found that RSV and HMPV demonstrate significantly different infection and spread kinetics as well as phenotypes during infection, highlighting an interesting dichotomy between two closely related viruses. We further analyzed therapeutic potential for several monoclonal antibodies, finding that prophylactic interventions prevent entry and spread, but treatment after entry suggests that both HMPV and RSV can be inhibited during entry. However, RSV likely spreads through cellular release and re-entry whereas HMPV utilizes a mechanism that is antibody independent after establishing the initial infection. Lastly, we examined the concept of viral co-infections, as co-infections with RSV and HMPV have been reported to cause more severe disease in patients. We provide evidence that RSV and HMPV co-infected cells can occupy the same inclusion bodies, but further investigation suggests that HMPV and RSV replication synergy may be limited. Collectively, the data presented in this dissertation provide new understanding of pneumovirus infections and reveals important information about the molecular mechanisms of pneumovirus entry and spread.

Digital Object Identifier (DOI)

https://doi.org/10.13023/etd.2020.414

Funding Information

  • National Institutes of Health R01AI051517 (2015-2020)
  • National Institutes of Health 5R21AI117300 (2016-2018)
  • Medimmune 3048113265 (2016-2019)

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