Hemagglutinin and fusion proteins precede measles virus ribonucleoproteins and marshal cell-to-cell spread in airway epithelium

ABSTRACT Measles virus (MeV) is the most contagious human respiratory pathogen. To understand how it spreads in the airways, we infected well-differentiated primary cultures of human airway epithelial cells (HAE). We discovered that the MeV attachment protein (hemagglutinin, H), the fusion (F) protein, and the assembly organizer protein (matrix, M) reach newly infected cells ahead of, and independently from, the replicative complex proteins. To characterize the mechanisms of MeV spread, we generated a replication-deficient adenoviral vector expressing the MeV M, F, and H proteins in a doxycycline-inducible manner. This viral vector formed infectious centers that preserved epithelial function like those from MeV infection. To further dissect this process, we generated Ad vectors expressing individual MeV proteins, termed Ad-M, Ad-F, and Ad-H. Transduction of HAE with single, double, and triple Ad vector combinations revealed that Ad-F and Ad-H are necessary and sufficient to form MeV-like infectious centers. MeV enters and spreads within airway epithelia via H protein binding to adherens junction protein, nectin-4. To disrupt spread, we introduced the point mutation H Y543A , a mutation previously described to inhibit nectin-4 binding, in the Ad-H vector. Co-transduction of Ad-F and Ad-H Y543A did not result in MeV-like infectious center formation. Finally, we assessed whether the MeV-related paramyxovirus Nipah (NiV) attachment and fusion proteins (G and F, respectively) support cell-to-cell spread. Co-transduction of HAE with Ad-NiV-G and Ad-NiV-F did not result in infectious center formation, suggesting that decoupling of the membrane fusion apparatus function from replication is a unique feature of the MeV envelope glycoproteins. IMPORTANCE Measles virus is a highly contagious pathogen. We characterized a mechanism by which measles virus spreads rapidly through the cells that line the human airway in an ex vivo model. We show that the proteins of the viral membrane fusion apparatus, hemagglutinin (H) and fusion (F), move independently from and reach newly infected cells before the proteins of the replicative complex. Thus, viral protein spread is assembled and arranged in order (i.e., marshaled ). Using adenovirus-based vectors, we show that co-expression of H and F proteins promotes rapid sequential spread to multiple cells and the formation of infectious centers. Further elucidation of the mechanism of measles virus amplification and cell-to-cell spread within the lung may help explain its highly contagious nature.