A Defect in Influenza A Virus Particle Assembly Specific to Primary Human Macrophages

Abstract The primary target of Influenza A virus (IAV) is epithelial cells in the respiratory tract. In contrast to epithelial cells, productive infection of most IAV strains is either blocked or highly inefficient in macrophages. The exact nature of the defect in IAV replication in human macrophages remains unknown. In this study, we showed that primary human monocyte-derived macrophages (MDM) are inefficient in IAV release even when compared to a monocytic cell line differentiated to macrophage-like cells, despite comparable levels of expression of viral glycoproteins at the plasma membrane. Correlative fluorescence scanning electron microscopy revealed that formation of budding structures at the cell surface is inefficient in MDM even though clustering of a viral glycoprotein, hemagglutinin (HA), is observed, suggesting that IAV particle assembly is blocked in human MDM. Using an in situ proximity ligation assay, we further determined that association between HA and the viral ion channel protein M2 is defective at the plasma membrane of MDM. In contrast, HA and another glycoprotein neuraminidase (NA) associate with each other on the MDM surface efficiently. Notably, the defect in association between HA and M2 in MDM was reversed upon inhibition of actin polymerization by cytochalasin D. Altogether, these results suggest that HA-M2 association on the plasma membrane is a discrete step in the IAV assembly process, which is separable from the association between HA and NA and susceptible to suppression by actin cytoskeleton. Overall, our study revealed the presence of a cell-type-specific mechanism negatively regulating IAV assembly at the plasma membrane. Importance Identification of host cell determinants promoting or suppressing replication of many viruses has been aided by analyses of host cells that impose inherent blocks on viral replication. In this study, we show that primary human MDM are not permissive to IAV replication due to a defect at the virus particle formation step. This defect is specific to primary human macrophages, since a human monocytic cell line differentiated to macrophage-like cells supports IAV particle formation. We further identified association between two viral transmembrane proteins, HA and M2, on the cell surface as a discrete assembly step, which is defective in MDM. Defective HA-M2 association in MDM is rescued by disruption of the actin cytoskeleton, revealing a previously unknown, negative role for actin polymerization, which is generally thought to play positive roles in IAV assembly. Overall, our study uncovered a host-mediated restriction of association between viral transmembrane components during IAV assembly.