Influenza A is an acute viral infection that spreads easily from person to person, can affect any age group and causes recurrent seasonal epidemics and global pandemics. Fundamental information on the influenza virus life cycle is still being discovered. In particular, there have been recent developments that provide new insights into how viruses assemble and then bud at the surface of infected cells. An atomic-level understanding of the viral assembly and budding process could lead to strategies to inhibit the replication of viruses and new tactics for inhibiting viral infectivity. Two of the proteins required for the efficient assembly of infectious virus particles are M1 and M2. Developing a detailed biophysical understanding of the interaction between M1 and M2 is the focus of this proposal. Due to the key role the M1-M2 interaction plays in viral assembly, it has been proposed that the disruption of this interaction could lead to a new class of antiviral drugs. The M1 protein is a 252-amino acid soluble protein and M2 protein is a 97-amino acid membrane-bound protein. Both localize to the plasma membrane of an infected cell prior to viral budding. A strength of the current state of knowledge is that published work on M1 and M2 provides the genetic, biochemical and molecular biology foundations necessary to make biophysical studies tractable. Our proposed work addresses a current weakness in the field ? a lack of atomic level detail on the molecular interaction of these two key players in viral budding. A combination of different magnetic resonance approaches will be used to probe the interaction of M1 with M2. Using site-directed spin-label electron paramagnetic resonance (SDSL-EPR), we will measure the mobility of spin labels, accessibility of spin labels to collision with paramagnetic reagents and distance dependent spin couplings. The PI?s lab can already efficiently biochemically produce and spectroscopically characterize spin-labeled full-length M2 protein reconstituted into liposomes. We have already biochemically overexpressed wild-type M1 and we describe our proposed strategy for spin-labeling M1. We propose to characterize the interaction between M1 and M2 by measuring distances between spin- labeled M1 and spin-labeled M2. We also plan to introduce site-specific 19F labels and measure distances using strategies that rely on the presence of both nuclear and electron spins. Additional biophysical techniques (differential light scattering, circular dichroism and isothermal titration calorimetry) will also be used to characterize the M1-M2 complex. Undergraduate research students supported by this grant proposal will explore an issue of critical public health importance using cutting edge biophysical techniques, participate in established interdisciplinary collaborations, be co-authors on published work and be mentored by experts committed to their long-term career development.
The threat of future influenza pandemics, coupled with the growing resistance to current antiviral drugs, makes the development of new influenza drugs a national healthcare priority. This proposal describes experiments designed to provide an atomic-level understanding of how influenza viruses assemble and then bud from infected cells. This structural information could inform efforts to inhibit the replication of viruses, offering a significant potential for a new generation of anti-flu drugs.
