A membrane fusion apparatus trailblazes measles virus spread in airway cells - PROJECT SUMMARY/ABSTRACT Measles virus (MeV) is a leading cause of vaccine-preventable deaths worldwide. Measles outbreaks outnumber mumps, polio, rubella, and whooping cough combined. We reported that MeV rapidly spreads from cell-to-cell through intercellular pores within well-differentiated primary cultures of airway epithelial cells (HAE). This cell culture model is unpassaged and cultured directly from human donor lungs onto support filters at an air-liquid interface. Cell-to-cell spread of MeV in HAE gives rise to distinct foci termed infectious centers. Infectious centers in HAE are not observed with any other respiratory virus tested to date. Our research suggests that the formation of infectious centers, their subsequent release, and the resulting environmental contamination play a crucial role in the efficient host-to-host spread of MeV. In this R21 proposal, we investigate how MeV forms infectious centers and why other paramyxoviruses don’t. We postulate that MeV uses a shrewd strategy in which a membrane fusion apparatus (MFA), consisting of 3 viral proteins (i.e., matrix (M), fusion (F), and hemagglutinin (H)) opens intercellular pores. Following pore formation, the ribonucleoprotein (RNP) complex that minimally contains the viral RNA genome, nucleoprotein (N), phosphoprotein (P), and polymerase (L), passes through the pore. In this proposal, we term this process “MFA trailblazing.” Our long-term objective is to explain why MeV is more contagious than closely related respiratory viruses. The immediate objective of this proposal is to probe the mechanism of rapid infectious center formation in airway cells. In Aim 1, we propose to use live-cell imaging and two fluorescently tagged viruses to confirm our preliminary immunocytochemistry results that show MFAs precede RNPs in newly infected airway cells. MeV- RNPtracker expresses GFP-tagged P protein (in addition to endogenous P). Similarly, MeV-MFAtracker expresses mCherry-tagged H protein and can track the MFA in real-time. In Aim 2, we demonstrate that infectious center formation can be replicated by expression of only 3 MeV proteins that make up the MFA (i.e., M, F, and H). We use replication deficient adenoviral vectors for delivery. Modular expression of adenoviral expressed viral proteins allows numerous advantages including: 1) rapid generation of recombinant expression vectors; 2) low risk that genetic manipulations of transgenes will impact vector titer; and 3) many combinations of viral protein comparisons are possible. This novel tool will allow for the substitution of proteins with known mutations that will alter complex formation; as well as, substitution of orthologous proteins from other paramyxoviruses. MFA trailblazing is a novel mechanism to explain the rapid formation of infectious centers in HAE and has the potential to uncover crucial interactions between virus and host.
