Influenza virus assembly: Effect of influenza virus glycoproteins on the membrane association of M1 protein

Influenza virus matrix protein (M1), a critical protein required for virus assembly and budding, is presumed to interact with viral glycoproteins on t he outer side and viral ribonucleoprotein on the inner side. However, becau se of the inherent membrane-binding ability of M1 protein, it has been diff icult to demonstrate the specific interaction of hll protein with hemagglut inin (HA) or neuraminidase (NA), the influenza virus envelope glycoproteins . Using Triton X-100 (TX-100) detergent treatment of membrane fractions and floatation in sucrose gradients, we observed that the membrane-bound M1 pr otein expressed alone or coexpressed with heterologous Sendai virus F was t otally TX-100 soluble but the membrane-bound M1 protein expressed in the pr esence of HA and NA was predominantly detergent resistant and floated to th e top of the density gradient. Furthermore, both the cytoplasmic tail and t he transmembrane domain of HA facilitated binding of M1 to detergent-resist ant membranes. Analysis of the membrane association of M1 in the early and late phases of the influenza virus infectious cycle revealed that the inter action of hll with mature glycoproteins which associated with the detergent -resistant lipid rafts was responsible for the detergent resistance of memb rane-bound M1. Immunofluorescence analysis by confocal microscopy also demo nstrated that, in influenza virus-infected cells, a fraction of M1 protein colocalized with HA and associated with the HA in transit to the plasma mem brane via the exocytic pathway. Similar results for colocalization were obt ained when M1 and HA were coexpressed and HA transport was blocked by monen sin treatment. These studies indicate that both HA and NA interact with inf luenza virus M1 and that HA associates with M1 via its cytoplasmic tail and transmembrane domain.