IFITM3 Inhibits Influenza A Virus Infection by Preventing Cytosolic Entry

Ragon Institute of Massachusetts General Hospital, Massachusetts Institute of Technology and Harvard University, Charlestown, Massachusetts, United States of America.
PLoS Pathogens (Impact Factor: 7.56). 10/2011; 7(10):e1002337. DOI: 10.1371/journal.ppat.1002337
Source: PubMed


To replicate, viruses must gain access to the host cell's resources. Interferon (IFN) regulates the actions of a large complement of interferon effector genes (IEGs) that prevent viral replication. The interferon inducible transmembrane protein family members, IFITM1, 2 and 3, are IEGs required for inhibition of influenza A virus, dengue virus, and West Nile virus replication in vitro. Here we report that IFN prevents emergence of viral genomes from the endosomal pathway, and that IFITM3 is both necessary and sufficient for this function. Notably, viral pseudoparticles were inhibited from transferring their contents into the host cell cytosol by IFN, and IFITM3 was required and sufficient for this action. We further demonstrate that IFN expands Rab7 and LAMP1-containing structures, and that IFITM3 overexpression is sufficient for this phenotype. Moreover, IFITM3 partially resides in late endosomal and lysosomal structures, placing it in the path of invading viruses. Collectively our data are consistent with the prediction that viruses that fuse in the late endosomes or lysosomes are vulnerable to IFITM3's actions, while viruses that enter at the cell surface or in the early endosomes may avoid inhibition. Multiple viruses enter host cells through the late endocytic pathway, and many of these invaders are attenuated by IFN. Therefore these findings are likely to have significance for the intrinsic immune system's neutralization of a diverse array of threats.

52 Reads
  • Source
    • "IAV-infected cells were immunostained for HA expression using either an anti-HA monoclonal (WSN/33 Wistar Institute; John et al., 2013) or anti-NP antibodies (H3N2, Millipore clone H16-L10-4R5 anti-IAV virus antibody). Pseudoparticles were produced using the noted envelopes as previously described (Feeley et al., 2011). "
    [Show abstract] [Hide abstract]
    ABSTRACT: Human rhinovirus (HRV) causes upper respiratory infections and asthma exacerbations. We screened multiple orthologous RNAi reagents and identified host proteins that modulate HRV replication. Here, we show that RNASEK, a transmembrane protein, was needed for the replication of HRV, influenza A virus, and dengue virus. RNASEK localizes to the cell surface and endosomal pathway and closely associates with the vacuolar ATPase (V-ATPase) proton pump. RNASEK is required for endocytosis, and its depletion produces enlarged clathrin-coated pits (CCPs) at the cell surface. These enlarged CCPs contain endocytic cargo and are bound by the scissioning GTPase, DNM2. Loss of RNASEK alters the localization of multiple V-ATPase subunits and lowers the levels of the ATP6AP1 subunit. Together, our results show that RNASEK closely associates with the V-ATPase and is required for its function; its loss prevents the early events of endocytosis and the replication of multiple pathogenic viruses. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.
    Cell Reports 07/2015; 12(5). DOI:10.1016/j.celrep.2015.06.076 · 8.36 Impact Factor
  • Source
    • "For some of the ISGs, the anti-viral function has been described, e.g. MX1, EIFAK2 (PKR, PRKR), IFITM3, ISG15, RSAD2 (Viperin), and OAS1 [28], [29], [30], [31], [32], [33], [34], [35], [36]. However, the functional role of the majority of ISGs remains to be elucidated. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Interferons represent one of the first and essential host defense mechanisms after infection, and the activation of the IFN-pathway results in the transcriptional activation of hundreds of interferon-stimulated genes. The alpha-inducible protein 27 like 2A (Ifi27l2a) gene (human synonym: ISG12) is strongly up-regulated in the lung after influenza A infection in mice and has been shown in gene expression studies to be highly correlated to other activated genes. Therefore, we investigated the role of Ifi27l2a for the host defense to influenza A infections in more detail. RT-PCR analyses in non-infected mice demonstrated that Ifi27l2a was expressed in several tissues, including the lung. Detailed analyses of reporter gene expression in lungs from Ifi27l2a-LacZ mice revealed that Ifi27l2a was expressed in macrophages and lymphocytes but not in alveolar cells or bronchiolar epithelium cells. The number of macrophages and lymphocyte strongly increased in the lung after infection, but no significant increase in expression levels of the LacZ reporter gene was found within individual immune cells. Also, no reporter gene expression was found in bronchiolar epithelial cells, alveolar cells or infiltrating neutrophils after infection. Thus, up-regulation of Ifi27l2a in infected lungs is mainly due to the infiltration of macrophages and lymphocytes. Most surprisingly, deletion of Ifi27l2a in mouse knock-out lines did not result in increased susceptibility to infections with H1N1 or H7N7 influenza A virus compared to wild type C57BL/6N mice, suggesting a less important role of the gene for the host response to influenza infections than for bacterial infections.
    PLoS ONE 09/2014; 9(9):e106392. DOI:10.1371/journal.pone.0106392 · 3.23 Impact Factor
  • Source
    • "Of note, we tried to perform siRNA assays to knockdown IFITM3 in macrophages; however, knockdown efficiency varied severely from donor to donor, precluding further mechanistic analyzes based on this strategy. Nevertheless, as it has been shown that IFITM3 restricts influenza life cycle by preventing arrival of viral ribonucleoproteins (RNPs) to the host cell nucleus [54], we measured the levels of A(H1N1pdm)09 bound to plasma membrane and the amount of viral RNPs that reached the nucleus of HIV-1-infected macrophages at early and late time points, by synchronizing influenza infection and measuring influenza RNA levels in the macrophage nuclear and non-nuclear fractions (see Material and Methods for details). Consistently with the mechanism of action of IFITM3, A(H1N1)pdm09 penetration into the nucleus was reduced by 80%, in HIV-1-infected macrophages (Figure 7B; pellet fraction from the early lysis). "
    [Show abstract] [Hide abstract]
    ABSTRACT: HIV-1-infected patients co-infected with A(H1N1)pdm09 surprisingly presented benign clinical outcome. The knowledge that HIV-1 changes the host homeostatic equilibrium, which may favor the patient resistance to some co-pathogens, prompted us to investigate whether HIV-1 infection could influence A(H1N1)pdm09 life cycle in vitro. We show here that exposure of A(H1N1)pdm09-infected epithelial cells to HIV-1 viral particles or its gp120 enhanced by 25% the IFITM3 content, resulting in a decrease in influenza replication. This event was dependent on toll-like receptor 2 and 4. Moreover, knockdown of IFITM3 prevented HIV-1 ability to inhibit A(H1N1)pdm09 replication. HIV-1 infection also increased IFITM3 levels in human primary macrophages by almost 100%. Consequently, the arrival of influenza ribonucleoproteins (RNPs) to nucleus of macrophages was inhibited, as evaluated by different approaches. Reduction of influenza RNPs entry into the nucleus tolled A(H1N1)pdm09 life cycle in macrophages earlier than usual, limiting influenza's ability to induce TNF-α. As judged by analysis of the influenza hemagglutin (HA) gene from in vitro experiments and from samples of HIV-1/A(H1N1)pdm09 co-infected individuals, the HIV-1-induced reduction of influenza replication resulted in delayed viral evolution. Our results may provide insights on the mechanisms that may have attenuated the clinical course of Influenza in HIV-1/A(H1N1)pdm09 co-infected patients during the recent influenza form 2009/2010.
    PLoS ONE 06/2014; 9(6):e101056. DOI:10.1371/journal.pone.0101056 · 3.23 Impact Factor
Show more