Evidence that Rabies Virus Forms Different Kinds of Fusion Machines with Different pH Thresholds for Fusion

Interferon-inducible transmembrane proteins (IFITMs) can restrict the entry of a wide range of viruses. IFITM3 localizes to endosomes and can potently restrict the replication of influenza A viruses (IAV) and several other viruses that also enter host cells through the endocytic pathway. Here, we investigate whether IFITMs are involved in protection in ducks, the natural host of influenza virus. We identify and sequence duck IFITM1, IFITM2, IFITM3, and IFITM5. Using quantitative PCR (qPCR), we demonstrate the upregulation of these genes in lung tissue in response to highly pathogenic IAV infection by 400-fold, 30-fold, 30-fold, and 5-fold, respectively. We express each IFITM in chicken DF-1 cells and show duck IFITM1 localizes to the cell surface, while IFITM3 localizes to LAMP1-containing compartments. DF-1 cells stably expressing duck IFITM3 (but not IFITM1 or IFITM2) show increased restriction of replication of H1N1, H6N2, and H11N9 IAV strains but not vesicular stomatitis virus. Although duck and human IFITM3 share only 38% identity, critical residues for viral restriction are conserved. We generate chimeric and mutant IFITM3 proteins and show duck IFITM3 does not require its N-terminal domain for endosomal localization or antiviral function; however, this N-terminal end confers endosomal localization and antiviral function on IFITM1. In contrast to mammalian IFITM3, the conserved YXX endocytosis signal sequence in the N-terminal domain of duck IFITM3 is not essential for correct endosomal localization. Despite significant structural and amino acid divergence, presumably due to hostvirus coevolution, duck IFITM3 is functional against IAV. IMPORTANCEImmune IFITM genes are poorly conserved across species, suggesting that selective pressure from host-specific viruses has driven this divergence. We wondered whether coevolution between viruses and their natural host would result in the evasion of IFITM restriction. Ducks are the natural host of avian influenza A viruses and display few or no disease symptoms upon infection with most strains, including highly pathogenic avian influenza. We have characterized the duck IFITM locus and identified IFITM3 as an important restrictor of several influenza A viruses, including avian strains. With only 38% amino acid identity to human IFITM3, duck IFITM3 possesses antiviral function against influenza virus. Thus, despite long coevolution of virus and host effectors in the natural host, influenza virus evasion of IFITM3 restriction in ducks is not apparent.T ype I interferons are produced in response to viral infection and rapidly upregulate hundreds of interferon-stimulated genes (ISGs) in surrounding cells to induce an antiviral state. The interferon-inducible transmembrane proteins (IFITMs) are recently recognized ISGs with antiviral activity against a broad range of viruses (1, 2). Originally identified in a cDNA screen of genes upregulated by type I interferons, their antiviral properties have since been well characterized in vitro (3, 4). IFITMs inhibit the ent...

Section: Discussionmentioning

confidence: 82%

Duck Interferon-Inducible Transmembrane Protein 3 Mediates Restriction of Influenza Viruses

Blyth

Chan

Webster

et al. 2016

“…This would suggest that the A117F poisoning of fusion may require intertrimer interactions. For rabies virus, it has been suggested that the refolding of as many as 16 trimers are required to drive membrane fusion (11,12,32). If a similarly high threshold is required for VSIV G, this may necessitate a significant number of A117F homotrimers to poison wild-type fusion.…”

Section: Discussionmentioning

confidence: 99%

A Recombinant Vesicular Stomatitis Virus Bearing a Lethal Mutation in the Glycoprotein Gene Uncovers a Second Site Suppressor That Restores Fusion

Stanifer

Cureton

Whelan

2011

Vesicular stomatitis virus (VSV), a prototype of the Rhabdoviridae family, contains a single surface glycoprotein (G) that is responsible for attachment to cells and mediates membrane fusion. Working with the Indiana serotype of VSV, we employed a reverse genetic approach to produce fully authentic recombinant viral particles bearing lethal mutations in the G gene. By altering the hydrophobicity of the two fusion loops within G, we produced a panel of mutants, W72A, Y73A, Y116A, and A117F, that were nonfusogenic. Propagation of viruses bearing those lethal mutations in G completely depended on complementation by expression of the glycoprotein from the heterologous New Jersey serotype of VSV. The nonfusogenic G proteins oligomerize and are transported normally to the cell surface but fail to mediate acid pH-triggered membrane fusion. The nonfusogenic G proteins also interfered with the ability of wild-type G to mediate fusion, either by formation of mixed trimers or by inhibition of trimer function during fusion. Passage of one recombinant virus, A117F, identified a second site suppressor of the fusion block, E76K. When analyzed in the absence of the A117F substitution, E76K rendered G more sensitive to acid pH-triggered fusion, suggesting that this compensatory mutation is destabilizing. Our work provides a set of authentic recombinant VSV particles bearing lethal mutations in G, confirms that the hydrophobic fusion loops of VSV G protein are critical for membrane fusion, and underscores the importance of the sequence elements surrounding the hydrophobic tips of the fusion loops in driving fusion. This study has implications for understanding dominant targets for inhibition of G-mediated fusion. Moreover, the recombinant viral particles generated here will likely be useful in dissecting the mechanism of G-catalyzed fusion as well as study steps of viral assembly.Enveloped viral fusion machines fall into one of three classes: class I, II, or III. These classes are respectively exemplified by influenza A virus hemagglutinin (HA), flavivirus envelope (E) proteins, and the rhabdovirus glycoproteins (G) (18). All three classes of fusion proteins share basic characteristics. In response to specific triggers, conformational rearrangements in the protein lead to the insertion of a hydrophobic region(s) into the target cell membrane, followed by the refolding of the protein to bring the viral and cellular membrane in close proximity. Class I and II viral fusion machines undergo a priming cleavage event, and the conformational changes that occur on exposure to the appropriate trigger are irreversible. By contrast, the class III viral fusion machines do not undergo such a priming cleavage (as reviewed in reference 18). For rhabdoviruses, the trigger for conformational change in G is exposure to acidic pH. Uniquely, the pH-triggered conformational changes in class III fusion proteins are fully reversible, such that reexposure of viral particles to neutral pH results in G refolding into the prefusion conformation (4, 13, 31)...

“…Like other rhabdoviruses, RABV gains access to the cellular interior by endocytosis and subsequent low pH-dependent fusion (5)(6)(7). Electron micrographs of viral particles in vesicles with electron-dense coats suggest that clathrin-coated pits mediate the uptake of RABV in both neuronal and nonneuronal cells (8,9).…”

mentioning

confidence: 99%

Uptake of Rabies Virus into Epithelial Cells by Clathrin-Mediated Endocytosis Depends upon Actin

Piccinotti

Kirchhausen

Whelan

2013

dRabies virus (RABV) causes a fatal zoonotic encephalitis. Disease symptoms require replication and spread of the virus within neuronal cells; however, in infected animals as well as in cell culture the virus replicates in a broad range of cell types. Here we use a single-cycle RABV and a recombinant vesicular stomatitis virus (rVSV) in which the glycoprotein (G) was replaced with that of RABV (rVSV RABV G) to examine RABV uptake into the African green monkey kidney cell line BS-C-1. Combining biochemical studies and real-time spinning-disk confocal fluorescence microscopy, we show that the predominant entry pathway of RABV particles into BS-C-1 cells is clathrin dependent. Viral particles enter cells in pits with elongated structures and incomplete clathrin coats which depend upon actin to complete the internalization process. By measuring the time of internalization and the abundance of the clathrin adaptor protein AP2, we further show that the pits that internalize RABV particles are similar to those that internalize VSV particles. Pharmacological perturbations of dynamin or of actin polymerization inhibit productive infection, linking our observations on particle uptake with viral infectivity. This work extends to RABV particles the finding that clathrin-mediated endocytosis of rhabdoviruses proceeds through incompletely coated pits which depend upon actin. R abies virus (RABV) is the prototypical member of the zoonotic lyssavirus genus responsible for fatal encephalitis in animals and humans. A single-stranded negative-sense RNA virus, RABV encases its RNA genome in a bullet-shaped, enveloped particle that incorporates a single surface glycoprotein (G). RABV G mediates all internalization steps from cell binding to membrane fusion. In addition, G is a major determinant of RABV neurotropism (1). Conjugation or pseudotyping with the ectodomain of pathogenic RABV G or peptides derived from receptor-binding regions allows retargeting of biologically active molecules to the central nervous system (CNS) for drug delivery or as neurotracers (2, 3). Since a large part of RABV pathogenesis is reliant on the virus garnering access to neurons and the CNS, G is also a determining factor in RABV virulence. The pathogenicity of attenuated strains can be effectively increased by replacing the glycoprotein with one from a neurotropic, virulent strain (4).Like other rhabdoviruses, RABV gains access to the cellular interior by endocytosis and subsequent low pH-dependent fusion (5-7). Electron micrographs of viral particles in vesicles with electron-dense coats suggest that clathrin-coated pits mediate the uptake of RABV in both neuronal and nonneuronal cells (8, 9). However, static images cannot inform on the fate of such particles or the relevance of these interactions for subsequent infection. High-resolution live-imaging techniques permit tracking of viral uptake into coated pits (10-15). Fluorescence tagging of coatedpit components and quantitative analysis methods have revealed differences for the pits engaging fluoresc...