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.
The matrix protein (M) of vesicular stomatitis virus (VSV) is involved in virus assembly, budding, gene regulation, and cellular pathogenesis. Using a yeast two-hybrid system, the M globular domain ...was shown to interact with LMP2, a catalytic subunit of the immunoproteasome (which replaces the standard proteasome catalytic subunit PSMB6). The interaction was validated by coimmunoprecipitation of M and LMP2 in VSV-infected cells. The sites of interaction were characterized. A single mutation of M (I96A) which significantly impairs the interaction between M and LMP2 was identified. We also show that M preferentially binds to the inactive precursor of LMP2 (bearing an N-terminal propeptide which is cleaved upon LMP2 maturation). Furthermore, taking advantage of a sequence alignment between LMP2 and its proteasome homolog, PSMB6 (which does not bind to M), we identified a mutation (L45R) in the S1 pocket where the protein substrate binds prior to cleavage and a second one (D17A) of a conserved residue essential for the catalytic activity, resulting in a reduction of the level of binding to M. The combination of both mutations abolishes the interaction. Taken together, our data indicate that M binds to LMP2 before its incorporation into the immunoproteasome. As the immunoproteasome promotes the generation of major histocompatibility complex (MHC) class I-compatible peptides, a feature which favors the recognition and the elimination of infected cells by CD8 T cells, we suggest that M, by interfering with the immunoproteasome assembly, has evolved a mechanism that allows infected cells to escape detection and elimination by the immune system.
The immunoproteasome promotes the generation of MHC class I-compatible peptides, a feature which favors the recognition and the elimination of infected cells by CD8 T cells. Here, we report on the association of vesicular stomatitis virus (VSV) matrix protein (M) with LMP2, one of the immunoproteasome-specific catalytic subunits. M preferentially binds to the LMP2 inactive precursor. The M-binding site on LMP2 is facing inwards in the immunoproteasome and is therefore not accessible to M after its assembly. Hence, M binds to LMP2 before its incorporation into the immunoproteasome. We suggest that VSV M, by interfering with the immunoproteasome assembly, has evolved a mechanism that allows infected cells to escape detection and elimination by the immune system. Modulating this M-induced immunoproteasome impairment might be relevant in order to optimize VSV for oncolytic virotherapy.
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.
Vesicular stomatitis virus (VSV; the prototype rhabdovirus) fusion is triggered at low pH and mediated by glycoprotein G, which undergoes a low-pH-induced structural transition. A unique feature of ...rhabdovirus G is that its conformational change is reversible. This allows G to recover its native prefusion state at the viral surface after its transport through the acidic Golgi compartments. The crystal structures of G pre- and postfusion states have been elucidated, leading to the identification of several acidic amino acid residues, clustered in the postfusion trimer, as potential pH-sensitive switches controlling the transition back toward the prefusion state. We mutated these residues and produced a panel of single and double mutants whose fusion properties, conformational change characteristics, and ability to pseudotype a virus lacking the glycoprotein gene were assayed. Some of these mutations were also introduced in the genome of recombinant viruses which were further characterized. We show that D268, located in the segment consisting of residues 264 to 273, which refolds into postfusion helix F during G structural transition, is the major pH sensor while D274, D395, and D393 have additional contributions. Furthermore, a single passage of recombinant virus bearing the mutation D268L (which was demonstrated to stabilize the G postfusion state) resulted in a pseudorevertant with a compensatory second mutation, L271P. This revealed that the propensity of the segment of residues 264 to 273 to refold into helix F has to be finely tuned since either an increase (mutation D268L alone) or a decrease (mutation L271P alone) of this propensity is detrimental to the virus.
Vesicular stomatitis virus enters cells via endocytosis. Endosome acidification induces a structural transition of its unique glycoprotein (G), which mediates fusion between viral and endosomal membranes. G conformational change is reversible upon increases in pH. This allows G to recover its native prefusion state at the viral surface after its transport through the acidic Golgi compartments. We mutated five acidic residues, proposed to be pH-sensitive switches controlling the structural transition back toward the prefusion state. Our results indicate that residue D268 is the major pH sensor, while other acidic residues have additional contributions, and reveal that the propensity of the segment consisting of residues 264 to 273 to adopt a helical conformation is finely regulated. This segment might be a good target for antiviral compounds.
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.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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 toward a ...trimeric post‐fusion state via monomeric intermediates. Here, we present the structure of the G fusion protein at intermediate pH for two vesiculoviruses, vesicular stomatitis virus (VSV) and Chandipura virus (CHAV), which is responsible for deadly encephalopathies. First, a CHAV G crystal structure shows two intermediate conformations forming a flat dimer of heterodimers. On virions, electron microscopy (EM) and tomography reveal monomeric spikes similar to one of the crystal conformations. In solution, mass spectrometry shows dimers of G. Finally, mutations at a dimer interface, involving fusion domains associated in an antiparallel manner to form an intermolecular β‐sheet, affect G fusion properties. The location of the compensatory mutations restoring fusion activity strongly suggests that this interface is functionally relevant. This work reveals the range of G structural changes and suggests that G monomers can re‐associate, through antiparallel interactions between fusion domains, into dimers that play a role at some early stage of the fusion process.
Synopsis
Structural determination of two intermediate conformations of a vesiculovirus glycoprotein reveals the chronological order of fusion‐associated structural changes, and that antiparallel interactions between fusion domains are required at some early stage of the fusion process.
New crystal forms of Chandipura virus G fusion protein show two intermediate conformations during fusion‐associated conformational changes.
Electron microscopy reveals monomeric spikes at the surface of vesicular stomatitis virus, resembling one of the crystal conformations.
Antiparallel interaction between fusion domains, leading to the formation of an intermolecular β‐sheet, plays a role at some early stage of the fusion process.
Distinct conformations of Chandipura virus glycoprotein reveal the chronological order of fusion‐associated structural changes, including an early‐stage requirement for antiparallel fusion domain interactions.
Vesicular stomatitis virus (VSV) matrix protein (M) has a flexible amino-terminal part that recruits cellular partners. It contains a dynamin-binding site that is required for efficient virus ...assembly, and two motifs, 24PPPY27 and 37PSAP40, that constitute potential late domains. Late domains are present in proteins of several enveloped viruses and are involved in the ultimate step of the budding process (i.e. fission between viral and cellular membranes). In baby hamster kidney (BHK)-21 cells, it has been demonstrated that the 24PPPY27 motif binds the Nedd4 (neuronal precursor cell-expressed developmentally downregulated 4) E3 ubiquitin ligase for efficient virus budding and that the 37PSAP40 motif, although conserved among M proteins of vesiculoviruses, does not possess late-domain activity. In this study, we have re-examined the contribution of the PSAP motif to VSV budding. First, we demonstrate that VSV M indeed binds TSG101 tumour susceptibility gene 101; a component of the ESCRT1 (endosomal sorting complex required for transport 1) through its PSAP motif. Second, we analysed the phenotype of several recombinant mutants. We show that a double mutant with point mutations in both the PSAP and the PPPY motifs is impaired compared with a single mutant in the PPPY motif, indicating that the PSAP motif partially compensates for the lack of the PPPY motif. Mutants’ phenotypes depend on cell lines: in CERA (chicken embryo-related, Alger clone) cells, a recombinant virus with a single mutation in the PSAP motif was impaired compared with the wild type, and a mutant with a single mutation in the dynamin-binding motif was much less impaired in Vero cells than in BSR (clones of BHK-21) cells. These results have implications for the VSV budding pathway that will be discussed.
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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