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.

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Published in PLOS Pathogens, v. 12, no. 9, e1005922, p. 1-30.

This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.

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RED was supported by NIH grants R01AI051517 and 2P20 RR020171 from the National Center for Research Resources. UJB was supported by the Intramural Program of the NIH, National Institute of Allergy and Infectious Diseases. The University of Kentucky Proteomics Core and the Department Imaging Core were partially supported by a COBRE grant from the NIH/NIGMS (P20GM103486).

journal.ppat.1005922.s001.DOCX (92 kB)
S1 Table. Proteomic Analysis of Purified HMPV Particles.

journal.ppat.1005922.s002.DOCX (28 kB)
S2 Table. Proteomic Analysis of Exosomes Purified from Mock Infected Cells.