Vesicular stomatitis virus (VSV) is an oncolytic rhabdovirus and its glycoprotein G is widely used to pseudotype other viruses for gene therapy. Low-density lipoprotein receptor (LDL-R) serves as a ...major entry receptor for VSV. Here we report two crystal structures of VSV G in complex with two distinct cysteine-rich domains (CR2 and CR3) of LDL-R, showing that their binding sites on G are identical. We identify two basic residues on G, which are essential for its interaction with CR2 and CR3. Mutating these residues abolishes VSV infectivity even though VSV can use alternative receptors, indicating that all VSV receptors are members of the LDL-R family. Collectively, our data suggest that VSV G has specifically evolved to interact with receptor CR domains. These structural insights into the interaction between VSV G and host cell receptors provide a basis for the design of recombinant viruses with an altered tropism.
Negative-strand RNA viruses condense their genome into a helical nucleoprotein-RNA complex, the nucleocapsid, which is packed into virions and serves as a template for the RNA-dependent RNA ...polymerase complex. The crystal structure of a recombinant rabies virus nucleoprotein-RNA complex, organized in an undecameric ring, has been determined at 3.5 angstrom resolution. Polymerization of the nucleoprotein is achieved by domain exchange between protomers, with flexible hinges allowing nucleocapsid formation. The two core domains of the nucleoprotein clamp around the RNA at their interface and shield it from the environment. RNA sequestering by nucleoproteins is likely a common mechanism used by negative-strand RNA viruses to protect their genomes from the innate immune response directed against viral RNA in human host cells at certain stages of an infectious cycle.
Viruses reshape the organization of the cell interior to achieve different steps of their cellular cycle. Particularly, viral replication and assembly often take place in viral factories where ...specific viral and cellular proteins as well as nucleic acids concentrate. Viral factories can be either membrane-delimited or devoid of any cellular membranes. In the latter case, they are referred as membrane-less replication compartments. The most emblematic ones are the Negri bodies, which are inclusion bodies that constitute the hallmark of rabies virus infection. Interestingly, Negri bodies and several other viral replication compartments have been shown to arise from a liquid-liquid phase separation process and, thus, constitute a new class of liquid organelles. This is a paradigm shift in the field of virus replication. Here, we review the different aspects of membrane-less virus replication compartments with a focus on the Mononegavirales order and discuss their interactions with the host cell machineries and the cytoskeleton. We particularly examine the interplay between viral factories and the cellular innate immune response, of which several components also form membrane-less condensates in infected cells.
•Viral factories harbor essential steps of the viral cycle.•Some viral factories are devoid of membranes and have liquid organelle properties.•Liquid viral factories concentrate cellular factors required for replication.•Innate immunity sensors and several ISGs are found in membrane-less condensates.•A subtle interplay exists between liquid viral factories and innate immunity actors.
•Class III fusion proteins share a common fold but have distinct modes of activation.•Distinct fusion loops organizations suggest distinct membrane-interaction modes.•For VSV G, pH sensitive ...molecular switches have been identified and characterized.•Comparing close homologs reveals constraints acting on class III fusion proteins.
Enveloped viruses enter the cell by fusing their envelope with a cellular membrane. Fusion is catalyzed by conformational changes of viral glycoproteins from pre-fusion to post-fusion states. Structural studies have defined three classes of viral fusion glycoproteins. Class III comprises the fusion glycoproteins from rhabdoviruses (G), herpesviruses (gB), and baculoviruses (GP64). Although sharing the same fold, those glycoproteins exhibit striking differences in their modes of activation and interaction with the target membrane. Furthermore, for gB and GP64, only the post-fusion structure is known and the extent of their conformational change is still an unresolved issue. Further structural studies are therefore required to get a detailed insight in the working of those fusion machines.
VSV fusion machinery, like that of many other enveloped viruses, is triggered at low pH in endosomes after virion endocytosis. It was suggested that some histidines could play the role of ...pH-sensitive switches. By mutating histidine residues H22, H60, H132, H162, H389, H397, H407, and H409, we demonstrate that residues H389 and D280, facing each other in the six-helix bundle of the post-fusion state, and more prominently H407, located at the interface between the C-terminal part of the ectodomain and the fusion domain, are crucial for fusion. Passages of recombinant viruses bearing mutant G resulted in the selection of compensatory mutations. Thus, the H407A mutation in G resulted in two independent compensatory mutants, L396I and S422I. Together with a crystal structure of G, presented here, which extends our knowledge of G pre-fusion structure, this indicates that the conformational transition is initiated by refolding of the C-terminal part of the G ectodomain.
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•A VSV-G pre-fusion crystal structure reveals its C-terminal part organization•H407A mutation affects G fusion properties by destabilizing the G pre-fusion state•Mutants L396I and S422I compensate H407A by stabilizing the G pre-fusion state•G structural transition is initiated in the C-terminal part of the ectodomain
Via a systematic mutational analysis, Beilstein et al. identify histidine residues acting as pH-sensitive switches during VSV-G conformational change. Selection of compensatory mutations and a crystal structure of the G pre-fusion state indicate that the conformational transition is initiated by the refolding of the C-terminal part of the G ectodomain.
Rhabdoviruses enter the cell via the endocytic pathway and subsequently fuse with a cellular membrane within the acidic environment of the endosome. Both receptor recognition and membrane fusion are ...mediated by a single transmembrane viral glycoprotein (G). Fusion is triggered via a low-pH induced structural rearrangement. G is an atypical fusion protein as there is a pH-dependent equilibrium between its pre- and post-fusion conformations. The elucidation of the atomic structures of these two conformations for the vesicular stomatitis virus (VSV) G has revealed that it is different from the previously characterized class I and class II fusion proteins. In this review, the pre- and post-fusion VSV G structures are presented in detail demonstrating that G combines the features of the class I and class II fusion proteins. In addition to these similarities, these G structures also reveal some particularities that expand our understanding of the working of fusion machineries. Combined with data from recent studies that revealed the cellular aspects of the initial stages of rhabdovirus infection, all these data give an integrated view of the entry pathway of rhabdoviruses into their host cell.
Vesiculoviruses enter cells by membrane fusion, driven by a large, low-pH-induced, conformational change in the fusion glycoprotein (G) that involves transition from a trimeric pre-fusion to a ...trimeric post-fusion state. G is the model of class III fusion glycoproteins which also includes the fusion glycoproteins of herpesviruses (gB) and baculoviruses (gp64). Class III fusion proteins combine features of the previously characterized class I and class II fusion proteins. In this review, we first present and discuss the data that indicate that the Vesiculovirus G structural transition proceeds through monomeric intermediates. Then, we focus on a recently determined crystal structure of the Chandipura virus G ectodomain that contained two monomeric intermediate conformations of the glycoprotein, revealing the chronological order of the structural changes in the protein and offering a detailed pathway for the conformational change, in agreement with electron microscopy data. In the crystal, the intermediates were associated through their fusion domain in an antiparallel manner to form an intermolecular β-sheet. Mutagenesis indicated that this interface is functionally relevant. All those structural data challenge the current model proposed for viral membrane fusion. Therefore, we wonder if this mode of operating is specific to Vesiculovirus G and discuss data indicating that class II fusion glycoproteins are monomeric when they interact with the target membrane but also crystal structures suggesting the existence of non-trimeric intermediates for influenza hemagglutinin which is the prototype of class I fusion proteins.
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•Vesiculovirus G is the prototype of class III viral fusion glycoproteins.•The structures of both G pre- and post-fusion conformation have been determined.•The structure of monomeric intermediates reveals the pathway of the transition.•A fusion-loop-exposing antiparallel dimer may initiate the fusion process.•Those data challenge the current model proposed for viral membrane fusion.
Chandipura virus (CHAV), a member of the vesiculovirus genus, is an emerging human pathogen. As for other rhabdoviruses, CHAV entry into susceptible cells is mediated by its single envelope ...glycoprotein G which is both involved in receptor recognition and fusion of viral and cellular membranes. Here, we have characterized the fusion properties of CHAV-G. As for vesicular stomatitis virus (VSV, the prototype of the genus) G, fusion is triggered at low pH below 6.5. We have also analyzed the biochemical properties of a soluble form of CHAV-G ectodomain (CHAV-Gth, generated by thermolysin limited-proteolysis of recombinant VSV particles in which the G gene was replaced by that of CHAV). The overall behavior of CHAV-Gth is similar to that previously reported for VSV-Gth. Particularly, CHAV-Gth pre-fusion trimer is not stable in solution and low-pH-induced membrane association of CHAV-Gth is reversible. Furthermore, CHAV-Gth was crystallized in its low pH post-fusion conformation and its structure was determined at 3.6Å resolution. An overall comparison of this structure with the previously reported VSV-Gth post-fusion conformation, shows a high structural similarity as expected from the comparison of primary structure. Among the three domains of G, the pleckstrin homology domain (PHD) appears to be the most divergent and the largest differences are confined to the secondary structure of the major antigenic site of rhabdoviruses. Finally, local differences indicate that CHAV has evolved alternate structural solutions in hinge regions between PH and fusion domains but also distinct pH sensitive switches. Globally the comparison between the post fusion conformation of CHAV and VSV-G highlights several features essential for the protein's function. It also reveals the remarkable plasticity of G in terms of local structures.
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Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Entry of enveloped viruses requires fusion of viral and cellular membranes, driven by conformational changes of viral glycoproteins. Crystal structures provide static pictures of pre- and post-fusion ...conformations of these proteins but the transition pathway remains elusive. Here, using several biophysical techniques, including analytical ultracentrifugation, circular dichroïsm, electron microscopy and small angle X-ray scattering, we have characterized the low-pH-induced fusogenic structural transition of a soluble form of vesicular stomatitis virus (VSV) glycoprotein G ectodomain (G(th), aa residues 1-422, the fragment that was previously crystallized). While the post-fusion trimer is the major species detected at low pH, the pre-fusion trimer is not detected in solution. Rather, at high pH, G(th) is a flexible monomer that explores a large conformational space. The monomeric population exhibits a marked pH-dependence and adopts more elongated conformations when pH decreases. Furthermore, large relative movements of domains are detected in absence of significant secondary structure modification. Solution studies are complemented by electron micrographs of negatively stained viral particles in which monomeric ectodomains of G are observed at the viral surface at both pH 7.5 and pH 6.7. We propose that the monomers are intermediates during the conformational change and thus that VSV G trimers dissociate at the viral surface during the structural transition.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The entry of enveloped viruses into cells requires the fusion of viral and cellular membranes, driven by conformational changes in viral glycoproteins. Many studies have shown that fusion involves ...the cooperative action of a large number of these glycoproteins, but the underlying mechanisms are unknown. We used electron microscopy and tomography to study the low pH—induced fusion reaction catalyzed by vesicular stomatitis virus glycoprotein (G). Pre- and post-fusion crystal structures were observed on virions at high and low pH, respectively. Individual fusion events with liposomes were also visualized. Fusion appears to be driven by two successive structural rearrangements of G at different sites on the virion. Fusion is initiated at the flat base of the particle. Glycoproteins located outside the contact zone between virions and liposomes then reorganize into regular arrays. We suggest that the formation of these arrays, which have been shown to be an intrinsic property of the G ectodomain, induces membrane constraints, achieving the fusion reaction.
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