Transport of viral proteins to the apical membranes and interaction of matrix protein with glycoproteins in the assembly of influenza viruses

Influenza virus assembly and morphogenesis require transport of viral compo nents to the assembly site at the apical plasma membrane of polarized epith elial cells and interaction among the viral components. In this report we h ave discussed the apical determinants present in the transmembrane domain ( TMD) of influenza virus hemagglutinin (HA) and neuraminidase (NA), and the interaction of M1 with influenza virus HA and NA. Earlier studies have show n that the NA and HA TMDs possess determinant(s) for apical sorting and raf t-association (Kundu et al., 1996. J. Virol 70, 6508-6515; Lin et al., 1998 . J. Cell Biol. 142, 51-57). Analysis of chimeric constructs between NA and TR (human transferring receptor) TMDs and the mutations in the NA and HA T MD sequences showed that the COOH terminus of the NA TMD and NH2 terminus o f the HA TMD encompassing the exoplasmic leaflet of the lipid bilayers were significantly involved in lipid raft-association and that apical determina nts were not discrete sequences but rather dispersed within the TMD of HA a nd NA. These analyses also showed that although both signals for apical sor ting and raft -association resided in the NA TMD, they were not identical a nd varied independently. Interactions of M1 protein with HA or NA, the infl uenza virus envelope glycoproteins. were investigated by TX-100 detergent t reatment of membrane fractions and floatation in sucrose gradients. Results from these analyses showed that the interaction of M1 with mature HA and N A, which associated with the detergent-resistant lipid rafts caused an incr eased detergent- resistance of the membrane-bound M1 and that M1 interacted with HA and NA both in influenza virus-infected cells as well as in recomb inant vaccinia virus-infected cells coexpressing M1 with HA and/or NA. Furt hermore, both the cytoplasmic tail and the TMD of HA caused an increased de tergent-resistance of the membrane-bound M1 supporting their interaction wi th M1. Immunofluorescence analysis by confocal microscopy also showed coloc alization supporting the interaction of M1 with HA and NA at the cell surfa ce and during exocytic transport both in influenza virus-infected cells as well as in coexpressing cells. (C) 2001 Elsevier Science B.V. All rights re served.