Abstract
Influenza A virus (IAV) propagates efficiently in epithelial cells, its primary target in the respiratory tract. In contrast, 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 even compared to a monocytic cell line differentiated to macrophage-like cells, primary human monocyte-derived macrophages (MDM) are inefficient in IAV production, 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 a step in IAV particle assembly is blocked in MDM. Using an in situ proximity ligation assay, we further determined that HA associates with neuraminidase (NA) but fails to associate with another viral transmembrane protein, M2, at the MDM plasma membrane. Notably, the defects in HA-M2 association and particle assembly in MDM were reversed upon cytochalasin D treatment that inhibits actin polymerization. These results suggest that HA-M2 association on the plasma membrane is a discrete step in IAV production, which is susceptible to suppression by actin cytoskeleton in MDM. Virus release remained inefficient in MDM upon cytochalasin D treatment, suggesting the presence of an additional defect(s) in virus release in this cell type. Overall, our study revealed the presence of multiple cell-type-specific mechanisms negatively regulating IAV production at the plasma membrane in MDM.IMPORTANCE Identification of host cell determinants promoting or suppressing replication of 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, which are not permissive to IAV replication, fail to support virus particle formation. 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 and particle budding, but not virus release, in MDM are rescued by disruption of actin cytoskeleton, revealing a previously unknown, negative role for actin, which specifically targets an early step in the multistep IAV production. Overall, our study uncovered a host-mediated restriction of association between viral transmembrane components during IAV assembly.
Highlights
Influenza A virus (IAV) propagates efficiently in epithelial cells, its primary target in the respiratory tract
We have shown that despite efficient trafficking of the viral glycoproteins to the cell surface (Fig. 3), infectious virus particle formation at the plasma membrane is inefficient in human monocyte-derived macrophages (MDM) (Fig. 2 and 4)
The current study further identified HA-M2 association as an IAV assembly step suppressed in MDM (Fig. 5)
Summary
MDM are inefficient in supporting productive IAV infection relative to differentiated THP1 cells. Even though MDM showed higher numbers of HA-positive puncta on the cell surface than dTHP1 cells, the numbers of virus buds were drastically reduced in MDM relative to dTHP1 cells (Fig. 4B) Overall, these results indicate that virus particle assembly and budding are inefficient in MDM despite efficient trafficking of HA, NA, and M2 to the plasma membrane. The increase in HA-M2 PLA signal upon Cyto D treatment of MDM was not due to an increase in surface expression of HA and M2 in drug-treated cells, as shown by the flow cytometry analysis (Fig. S4B and C) These results suggest that actin polymerization suppresses HA-M2 association in MDM. Very few such budding structures corresponding to the size of IAV particles were observed on the surface of untreated or Cyto D-treated, mockinfected dTHP1 cells and MDM (Fig. 7B) These data suggest that disruption of the actin cytoskeleton promotes IAV particle assembly in MDM. VRNA release efficiency was 4- to 5-fold reduced in vehicle-treated MDM cultures relative to vehiclembio.asm.org 9
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