Molecular and Cellular Aspects of Rhabdovirus Entry

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

Characterization of Monomeric Intermediates during VSV Glycoprotein Structural Transition

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 (Gth, 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, Gth 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

STRUCTURAL BASIS OF RHABDOVIRUS ENTRY

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Structural and cellular biology of rhabdovirus entry

International audienceRhabdoviruses are enveloped viruses with a negative-sense single strand RNA genome and are widespread among a great variety of organisms. In their membrane, they have a single glycoprotein (G) that mediates both virus attachment to cellular receptors and fusion between viral and endosomal membranes allowing viral genome release in the cytoplasm. We present structural and cellular aspects of Rhabdovirus entry into their host cell with a focus on vesicular stomatitis virus (VSV) and rabies virus (RABV) for which the early events of the viral cycle have been extensively studied. Recent data have shown that the only VSV receptors are the members of the LDL-R family. This is in contrast with RABV for which multiple receptors belonging to unrelated families have been identified. Despite having different receptors, after attachment, rhabdovirus internalization occurs through clathrin-mediated endocytosis (CME) in an actin-dependent manner. There are still debates about the exact endocytic pathway of VSV in the cell and on RABV transport in the neuronal axon. In any case, fusion is triggered in the endosomal vesicle via a low-pH induced structural rearrangement of G from its pre- to its postfusion conformation. Vesiculovirus G is one of the best characterized fusion glycoproteins as the previously reported crystal structures of the pre- and postfusion states have been recently completed by those of intermediates during the structural transition. Understanding the entry pathway of rhabdoviruses may have strong impact in biotechnologies as, for example, VSV G is used for pseudotyping lentiviruses to promote efficient transduction, and VSV is a promising oncolytic virus

Recent mechanistic and structural insights on class III viral fusion glycoproteins.

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Negri bodies and other virus membrane-less replication compartments

International audienceViruses 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

Monomeric Intermediates Formed by Vesiculovirus Glycoprotein during Its Low-pH-induced Structural Transition

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Crystal structure of Mokola virus glycoprotein in its post-fusion conformation

International audienceMokola virus (MOKV) belongs to the lyssavirus genus. As other genus members-including rabies virus (RABV)-it causes deadly encephalitis in mammals. MOKV entry into host cells is mediated by its transmembrane glycoprotein G. First, G binds cellular receptors, triggering virion endocytosis. Then, in the acidic endosomal environment, G undergoes a confor-mational change from its pre-toward its post-fusion state that catalyzes the merger of the viral and endosomal membranes. Here, we have determined the crystal structure of a soluble MOKV G ectodomain in which the hydrophobic fusion loops have been replaced by more hydrophilic sequences. The crystal structure corresponds to a monomer that is similar to the protomer of the trimeric post-fusion state of vesicular stomatitis virus (VSV) G. However , by electron microscopy, we show that, at low pH, at the surface of pseudotyped VSV, MOKV spikes adopt the trimeric post-fusion conformation and have a tendency to reorganize into regular arrays. Sequence alignment between MOKV G and RABV G allows a precise location of RABV G antigenic sites. Repositioning MOKV G domains on VSV G pre-fusion structure reveals that antigenic sites are located in the most exposed part of the molecule in its pre-fusion conformation and are therefore very accessible to antibodies. Furthermore , the structure allows the identification of pH-sensitive molecular switches. Specifically, the long helix, which constitutes the core of the post-fusion trimer for class III fusion glyco-proteins, contains many acidic residues located at the trimeric interface. Several of them, aligned along the helix, point toward the trimer axis. They have to be protonated for the post-fusion trimer to be stable. At high pH, when they are negatively charged, they destabilize the interface, which explains the conformational change reversibility. Finally, the present structure will be of great help to perform rational mutagenesis on lyssavirus glycoproteins

Structural basis for the recognition of LDL-receptor family members by VSV glycoprotein

Glycoprotein G of vesicular stomatitis virus (VSV) enables viral entry by binding to the major VSV receptor LDL-R. Here the authors present crystal structures of G in complex with two distinct CR domains of LDL-R, identifying structural determinants for VSV infectivity in mammalian and insect cells

Structure of the glycoprotein of Mokola virus in its post-fusion conformation

International audienceMokola virus (MOKV) is a rhabdovirus belonging to the lyssavirus genus close to the rabies virus (RABV). MOKV, like all members of this genus, causes fatal encephalitis in mammals. Entry of MOKV into host cells is mediated by its transmembrane glycoprotein G. First, G binds cellular receptors, triggering endocytosis of the virus. Then, in the acidic environment of the endosome, G undergoes a conformational change from a pre- to a post-fusion state, which catalyzes the fusion of the viral and endosomal membranes. We solved the crystalline structure of MOKV G ectodomain. In the crystal, MOKV G monomer is similar to the protomer of the G trimeric post-fusion state of vesicular stomatitis virus (VSV). Electron microscopy observations on VSV particles pseudotyped with MOKV G show that MOKV G can adopt the trimeric post-fusion conformation on the viral surface. We also showed that MOKV G can reorganize into regular arrays at acidic pH. Sequence alignment of MOKV G with RABV G allows the localization of RABV G antigenic sites on MOKV G. Repositioning of MOKV G domains on the pre-fusion structure of VSV G reveals that the antigenic sites are located in the most exposed part of G and are therefore highly accessible to antibodies. Finally, analysis of MOKV G structure allows the identification of pH-sensitive molecular switches. In particular, a long helix, which constitutes the core of the post-fusion trimer for class III fusion glycoproteins, contains several conserved acidic residues localized at the trimeric interface. Most of them are aligned along this helix and point toward the 3-fold axis. These residues must be protonated in the post-fusion trimer of MOKV G to be stable. At high pH, when negatively charged, these acidic residues destabilize the interface, which explains the reversibility of the conformational change. This structure of a rabies-related virus glycoprotein paves the way for the development of a pan-lyssavirus vaccine