Interferon-Induced Cell Membrane Proteins, IFITM3 and Tetherin, Inhibit Vesicular Stomatitis Virus Infection via Distinct Mechanisms

Abstract

Tetherin and IFITM3 are recently identified interferon-induced cellular proteins that restrict infections by retroviruses and filoviruses and of influenza virus and flaviviruses, respectively. In our efforts to further explore their antiviral activities against other viruses and determine their antiviral mechanisms, we found that the two antiviral proteins potently inhibit the infection of vesicular stomatitis virus (VSV), a prototype member of the Rhabdoviridae family. Taking advantage of this well-studied virus infection system, we show that although both tetherin and IFITM3 are plasma membrane proteins, tetherin inhibits virion particle release from infected cells, while IFITM3 disrupts an early event after endocytosis of virion particles but before primary transcription of incoming viral genomes. Furthermore, we demonstrate that both the N-terminal 21 amino acid residues and C-terminal transmembrane region of IFITM3 are required for its antiviral activity. Collectively, our work sheds light on the mechanisms by which tetherin and IFITM3 restrict infection with rhabdoviruses and possibly other pathogenic viruses.The interferon (IFN) system is the first line of defense against virus infection in vertebrates. Infection of cells by viruses can be detected by host cellular pattern recognition receptors (PRRs), such as toll-like receptors and RIG-I-like receptors (1,33,42). Engagement of the PRRs with virusassociated molecular patterns, such as 5Ј-triphosphate and/or double-stranded viral RNA, triggers signaling cascades that lead to the synthesis and secretion of type I IFNs, represented by IFN-␣ and IFN-␤ (25). Type I IFNs bind to their cognate receptors on the cell surface and activate the receptor-associated Janus kinase 1 (JAK1) and tyrosine kinase 2 (Tyk2) through tyrosine phosphorylation, which in turn stimulates the tyrosine phosphorylation of STAT1 and STAT2. Phosphorylated STAT1 and STAT2, in combination with IRF9, form a trimeric ISGF3 transcription factor that translocates into the nucleus and activates the expression of IFN-stimulated genes (ISGs), whose products limit viral replication, regulate cell proliferation, and/or modulate host innate and adaptive immune responses (34,36,39).Although IFN treatment of cells induces the expression of hundreds of ISGs, only a few ISGs have thus far been demonstrated to instigate an antiviral state in cultured cells, and only four of them, including ISG15 (IFN-stimulated protein of 15 kDa), myxovirus resistance 1 (Mx1), RNase L, and doublestranded RNA-activated protein kinase (PKR), have been shown to play a role in mediating the IFN antiviral response in vivo with gene knockout mouse models (36). While it is most likely that inhibition of the infection of any given virus by IFNs is through induction of multiple ISGs that work cooperatively to disrupt multiple steps of viral replication, identification of individual antiviral ISGs and subsequent elucidation of their modes of action are essential to uncover the antiviral mechanism of IFNs and viral pathogenesis (2...

Interferon-Induced Tetherin Restricts Vesicular Stomatitis Virus Release in Neurons

“…After knocking down the expression of tetherin, medium-treated neuronal cells were found to yield more than 100-fold more virus after VSV infection. Our observation was consistent with the results from Ju-Tao Guo's laboratory obtained from a BHK-21 FLP-IN T Rex-derived stable tetherin knockdown cell line (Weidner et al, 2010); they did not assess the role of IFN treatment on the tetherindeficient line. We found that there was no significant difference between the viral yield in medium-treated tetherin shRNA cells and IFN-b-treated tetherin-silenced neuronal cells (Fig.…”

“…Tetherin forms a bridge between virions and the plasma membrane (Sato et al, 2009). Tetherin contributes to antiviral activity against various enveloped viruses, including Ebola, Marburg, Lassa, KSHV, HIV-1, HIV-2, SIV, MLV, E1AV, VSV, HTLV-1, XMRV, and spumaviruses ( Jouvenet et al, 2009;Kaletsky et al, 2009;Le Tortorec and Neil, 2009;Mansouri et al, 2009;Miyakawa et al, 2009;Sakuma et al, 2009;Groom et al, 2010;Weidner et al, 2010;Xu et al, 2011).…”

“…1A and 2A). Several previous studies have demonstrated IFN-b induction of tetherin in many different cell types (Ohtomo et al, 1999;Blasius et al, 2006;Neil et al, 2007;Kawai et al, 2008;Miyagi et al, 2009;Weidner et al, 2010;Dietrich et al, 2011). To determine whether type I (IFNb) or type II (IFN-g) regulate tetherin mRNA expression in murine neuroblastoma cells, we treated the cells overnight with IFN-b (400 U/mL) or for 72 h with IFN-g (20 ng/mL).…”

How Samhd1 changes our view of viral restriction

“…Therefore, because HIV-1 cannot counteract SAMHD1, it seems that, in contrast to other restriction factors, overcoming SAMHD1 is dispensable for efficient HIV-1 spread. This is in contrast, for example, to BST-2, because BST-2 nonspecifically targets the lipidic outlayer of enveloped viruses and has a broad impact on viral release [33][34][35][36][37][38][39][40][41]. Consequently, viruses have evolved distinct means to counteract this hurdle to viral budding.…”

Tetherin antagonism by Vpu protects HIV-infected cells from antibody-dependent cell-mediated cytotoxicity

“…This activity can be explained by the unusual topology of tetherin, which includes an N-terminal transmembrane domain and a C-terminal glycosyl-phosphatidylinositol tail that allow both ends of the molecule to be anchored in lipid membranes (4). Although tetherin was initially identified as the cellular gene product that accounts for a late-stage defect in the release of vpu-deleted HIV-1 from restrictive cells (5,6), it is now recognized to have antiviral activity against diverse families of enveloped viruses (7)(8)(9)(10)(11).…”