Human metapneumovirus Induces Reorganization of the Actin Cytoskeleton for Direct Cell-to-Cell Spread

Abstract

Paramyxovirus spread generally involves assembly of individual viral particles which then infect target cells. We show that infection of human bronchial airway cells with human metapneumovirus (HMPV), a recently identified paramyxovirus which causes significant respiratory disease, results in formation of intercellular extensions and extensive networks of branched cell-associated filaments. Formation of these structures is dependent on actin, but not microtubule, polymerization. Interestingly, using a co-culture assay we show that conditions which block regular infection by HMPV particles, including addition of neutralizing antibodies or removal of cell surface heparan sulfate, did not prevent viral spread from infected to new target cells. In contrast, inhibition of actin polymerization or alterations to Rho GTPase signaling pathways significantly decreased cell-to-cell spread. Furthermore, viral proteins and viral RNA were detected in intercellular extensions, suggesting direct transfer of viral genetic material to new target cells. While roles for paramyxovirus matrix and fusion proteins in membrane deformation have been previously demonstrated, we show that the HMPV phosphoprotein extensively co-localized with actin and induced formation of cellular extensions when transiently expressed, supporting a new model in which a paramyxovirus phosphoprotein is a key player in assembly and spread. Our results reveal a novel mechanism for HMPV direct cell-to-cell spread and provide insights into dissemination of respiratory viruses.

Author Summary

Human metapneumovirus (HMPV) is an important human respiratory pathogen that affects all age groups worldwide. There are currently no vaccines or treatments available for HMPV, and key aspects of its life cycle remain unknown. We examined the late events of the HMPV infection cycle in human bronchial epithelial cells. Our data demonstrate that HMPV infection leads to formation of unique structures, including intercellular extensions connecting cells, and large networks of branched cell-associated filaments. Viral modulation of the cellular cytoskeleton and cellular signaling pathways are important for formation of these structures. Our results are consistent with the intercellular extensions playing a role in direct spread of virus from cell-to-cell, potentially by transfer of virus genetic material without particle formation. We also show that the HMPV phosphoprotein localizes with actin and can promote membrane deformations, suggesting a novel role in viral assembly or spread for paramyxovirus phosphoproteins.