Date Available

1-29-2018

Year of Publication

2016

Degree Name

Doctor of Philosophy (PhD)

Document Type

Doctoral Dissertation

College

Medicine

Department/School/Program

Molecular and Cellular Biochemistry

First Advisor

Dr. Rebecca E. Dutch

Abstract

Human metapneumovirus (HMPV) is a leading cause of respiratory tract infections worldwide across all age groups, and is particularly devastating in the pediatric, elderly and immunocompromised populations. Despite its high prevalence and burden on human health, there are currently no treatments or vaccines against HMPV infections. HMPV is an enveloped virus that belongs to the paramyxovirus family. Paramyxoviruses in general form by assembly of virus components at the plasma membrane followed by budding and release of virus particles into the extracellular matrix to spread infection. The process of forming new virus particles requires complex interactions between viral and cellular components and the requirements for particle production differ substantially among paramyxoviruses. Several key aspects of the life cycle of HMPV remain unknown. The work presented here provides significant advances in understanding the mechanisms underlying assembly and spread of HMPV in human bronchial airway epithelial cells. We provide evidence that HMPV induces reorganization of the actin cytoskeleton and microtubules at late stages of infection leading to the formation of complex networks of branched filaments and intercellular extensions, structures that have not been previously reported for paramyxoviruses. Our results indicate a novel mode of HMPV spread directly from cell-to-cell across intercellular extensions. We identified an important role of actin and the Rho GTPases Rac1 and Cdc42 in direct cell-to-cell spread of HMPV. While roles for paramyxovirus matrix and fusion proteins in membrane deformation have been previously demonstrated for several paramyxoviruses, we show that the HMPV phosphoprotein (P) associates with actin and induces formation of membrane extensions, suggesting a role for the P protein in HMPV exit from the cell. Additionally, infection of differentiated, polarized human airway tissues showed that release of HMPV particles at the apical side is inefficient and revealed that spread of HMPV in these tissues can occur in the presence of neutralizing antibodies. HMPV infection also resulted in reorganization of the actin cytoskeleton in these tissues mainly at the apical side. Collectively, the data provided in this dissertation reveal a novel mechanism by which HMPV uses the cytoskeleton for cell-to-cell transmission and provide critical insights into spread of respiratory viruses within the airway epithelium.

Digital Object Identifier (DOI)

http://dx.doi.org/10.13023/ETD.2016.009

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