A Glu-Glu-Tyr Sequence in the Cytoplasmic Tail of the M2 Protein Renders Influenza A Virus Susceptible to Restriction of the Hemagglutinin-M2 Association in Primary Human Macrophages.

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 is observed with expression of only HA and/or NA. While these five viral components are expressed efficiently in primary human monocyte-derived macrophages (MDMs) upon IAV infection, this cell type does not support efficient HA-M2 association and IAV particle assembly at the plasma membrane. Both defects are specific to MDMs and can be reversed upon disruption of F-actin. However, the relationship between the two defects is unclear. Here, we examined whether M2 contributes to particle assembly in MDMs and if so, which region of M2 determines the susceptibility to the MDM-specific and actin-dependent suppression. An analysis using correlative fluorescence and scanning electron microscopy showed that an M2-deficient virus failed to form budding structures at the cell surface even after F-actin was disrupted, indicating that M2 is essential for virus particle formation at the MDM surface. Notably, proximity ligation analysis revealed that a single amino acid substitution in a Glu-Glu-Tyr sequence (residues 74 to 76) in the M2 cytoplasmic tail allowed the HA-M2 association to occur efficiently even in MDMs 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 identified M2 as a target of MDM-specific restriction of IAV assembly, which requires the Glu-Glu-Tyr sequence in the cytoplasmic tail. IMPORTANCE Human MDMs represent a cell type that is nonpermissive to particle formation of influenza A virus (IAV). We previously showed that close proximity association between viral HA and M2 proteins is blocked in MDMs. However, whether MDMs express a restriction factor against IAV assembly or whether they lack a dependency factor promoting assembly remained unknown. In the current study, we determined that the M2 protein is necessary for particle formation in MDMs but is also a molecular target of the MDM-specific suppression of assembly. Substitutions in the M2 cytoplasmic tail alleviated the block in both the HA-M2 association and particle production in MDMs. These findings suggest that MDMs express dependency factors necessary for assembly but also express a factor(s) that inhibits HA-M2 association and particle formation. High conservation of the M2 sequence rendering the susceptibility to the assembly block highlights the potential for M2 as a target of antiviral strategies.

Macrophage inflammatory state influences susceptibility to lysosomal damage.

Macrophages possess mechanisms for reinforcing the integrity of their endolysosomes against damage. This property, termed inducible renitence, was previously observed in murine macrophages stimulated with LPS, peptidoglycan, IFNγ, or TNFα, which suggested roles for renitence in macrophage resistance to infection by membrane-damaging pathogens. This study analyzed additional inducers of macrophage differentiation for their ability to increase resistance to lysosomal damage by membrane-damaging particles. Renitence was evident in macrophages activated with LPS plus IFNγ, PGE2 , or adenosine, and in macrophages stimulated with IFN-ß, but not in macrophages activated with IL-4 or IL-10. These responses indicated roles for macrophage subtypes specialized in host defense and suppression of immune responses, but not those involved in wound healing. Consistent with this pattern, renitence could be induced by stimulation with agonists for TLR, which required the signaling adaptors MyD88 and/or TRIF, and by infection with murine norovirus-1. Renitence induced by LPS was dependent on cytokine secretion by macrophages. However, no single secreted factor could explain all the induced responses. Renitence induced by the TLR3 agonist Poly(I:C) was mediated in part by the type I IFN response, but renitence induced by Pam3CSK4 (TLR2/1), LPS (TLR4), IFNγ, or TNFα was independent of type 1 IFN signaling. Thus, multiple pathways for inducing macrophage resistance to membrane damage exist and depend on the particular microbial stimulus sensed.