Abstract
Recruitment of the matrix protein M1 to the assembly site of the influenza virus is thought to be mediated by interactions with the cytoplasmic tail of hemagglutinin (HA). Based on a comprehensive sequence comparison of all sequences present in the database, we analyzed the effect of mutating conserved residues in the cytosol-facing part of the transmembrane region and cytoplasmic tail of HA (A/WSN/33 (H1N1) strain) on virus replication and morphology of virions. Removal of the two cytoplasmic acylation sites and substitution of a neighboring isoleucine by glutamine prevented rescue of infectious virions. In contrast, a conservative exchange of the same isoleucine, non-conservative exchanges of glycine and glutamine, deletion of the acylation site at the end of the transmembrane region and shifting it into the tail did not affect virus morphology and had only subtle effects on virus growth and on the incorporation of M1 and Ribo-Nucleoprotein Particles (RNPs). Thus, assuming that essential amino acids are conserved between HA subtypes we suggest that, besides the two cytoplasmic acylation sites (including adjacent hydrophobic residues), no other amino acids in the cytoplasmic tail of HA are indispensable for virus assembly and budding.
Highlights
Most models of influenza virus assembly postulate a direct interaction between the viral matrix protein M1 and the cytoplasmic domains of the envelope glycoproteins hemagglutinin (HA) and/or neuraminidase (NA) as a driving force for virus assembly [1,2,3]
Whereas the crystal structure of the ectodomains of various HA subtypes has been determined, very little is known about the remaining part of the molecule, the cytoplasmic tail, the transmembrane domain (TMD) and the linker region that connects the TMD to the ectodomain
We have shown here that the cytoplasmic tail of H1 subtype HA is essential for virus replication since WSN virus with tailless HA could not be generated
Summary
Most models of influenza virus assembly postulate a direct interaction between the viral matrix protein M1 and the cytoplasmic domains of the envelope glycoproteins hemagglutinin (HA) and/or neuraminidase (NA) as a driving force for virus assembly [1,2,3]. Such an interaction has never been demonstrated biochemically, for example by co-precipitation approaches. Detergent extraction at low temperature was used to demonstrate the specific interaction of M1 with HA and NA [8] Both HA and NA fractionate into detergent resistant membranes (DRMs), whereas M1 expressed alone was soluble, but became resistant when co-expressed with HA and NA. The data are consistent with a model in which the glycoproteins control virus budding by sorting to lipid raft nanodomains and recruiting the internal viral core components
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