A Glu-Glu-Tyr sequence in the cytoplasmic tail of the M2 protein renders IAV susceptible to restriction of HA-M2 association in primary human macrophages

Abstract

Influenza A virus (IAV) assembly at the plasma membrane is orchestrated by at least five viral components including hemagglutinin (HA), neuraminidase (NA), matrix (M1), the ion channel M2, and viral ribonucleoprotein (vRNP) complexes although particle formation itself requires only HA and/or NA. While these five viral components are expressed efficiently in primary human monocyte-derived macrophages (MDM) upon IAV infection, this cell type does not support efficient HA-M2 association and IAV particle assembly at the plasma membrane. The defects in HA-M2 association and particle assembly are specific to MDM and not observed in a monocytic cell line differentiated into macrophage-like cells. Notably, both these defects can be reversed upon disruption of the actin cytoskeleton. In the current study, we sought to examine whether M2 contributes to particle assembly in MDM and to identify a viral determinant involved in the MDM-specific and actin-dependent suppression of IAV assembly. An analysis using correlative fluorescence and scanning electron microscopy showed that an M2-deficient virus fails to form budding structures at the cell surface even after F-actin is disrupted, indicating that M2 is essential for virus particle formation at the MDM surface. Notably, proximity ligation analysis revealed that single amino acid substitution in a Glu-Glu-Tyr sequence (residues 74-76) in the M2 cytoplasmic tail allows HA-M2 association to occur efficiently even in MDM with intact actin cytoskeleton. This phenotype did not correlate with known phenotypes of the M2 substitution mutants regarding M1 interaction or vRNP packaging in epithelial cells. Overall, our study identifies a viral determinant for susceptibility to cytoskeleton-dependent regulation in MDM and hence, sheds light on the molecular mechanism behind the MDM-specific restriction of IAV assembly. ImportanceNon-permissive cell types that are unable to support viral replication serve as important tools for identification of host factors that either block viral replication (restriction factors) or support viral replication in permissive cell types (co-factors). We previously identified the MDM as a cell type that is non-permissive to IAV assembly, likely due to a block in HA-M2 association. In the current study, we determined that the IAV M2 protein is necessary for virus particle formation in MDM but also renders the virus susceptible to the MDM-specific suppression of virus assembly. We identified a specific amino acid motif in the M2 cytoplasmic tail, disruption of which allows M2 to associate with HA even in MDM. Our findings strongly support the possibility that the MDM-specific defect in HA-M2 association is due to the presence of a restriction factor(s) in MDM, which likely interacts directly with the M2 cytoplasmic domain, rather than indirectly through other internal viral components, and thereby prevents M2 from associating with HA.