Role of M1 Self-Organization in Influenza Virus Assembly: A Combined Rics and AFM Study

Abstract

The matrix protein (M1) of influenza virus is generally considered to be the key organizer in the budding of new virions from the plasma membrane (PM) of infected cells. In fact, this protein interacts with viral genetic material and envelope proteins, while binding to the inner leaflet of the PM. Its oligomerization plays a pivotal role in viral organization and function. Of interest, the molecular details of M1 oligomerization or its interaction with lipids and other viral proteins are not fully understood.In order to clarify the role of M1 in influenza virus assembly, we applied a combination of several quantitative microscopy approaches. We first characterized protein multimerization upon interaction with other viral proteins at the PM of living cells, using Number&Brightness (N&B) microscopy. Second, we used controlled biophysical models of the PM (e.g. supported bilayers) to delve into the details of M1-lipid and M1-M1 interaction, using a combination of Raster Image Correlation Spectroscopy (RICS) and Atomic Force Microscopy (AFM). Our results show that M1 oligomer formation is strongly concentration-dependent and does not necessarily require the presence of other viral proteins. Furthermore, we identified several novel lipid binding partners for M1, including phosphatidic acid and phosphatidylinositol phosphates. Finally, we show that specific interaction with the PM does not influence the oligomerization process but rather modulates the overall M1 binding to the membrane.