Yves Gaudin

Virus membrane fusion

Membrane fusion of enveloped viruses with cellular membranes is mediated by viral glycoproteins (GP). Interaction of GP with cellular receptors alone or coupled to exposure to the acidic environment of endosomes induces extensive conformational changes in the fusion protein which pull two membranes into close enough proximity to trigger bilayer fusion. The refolding process provides the energy for fusion and repositions both membrane anchors, the transmembrane and the fusion peptide regions, at the same end of an elongated hairpin structure in all fusion protein structures known to date. The fusion process follows several lipidic intermediate states, which are generated by the refolding process. Although the major principles of viral fusion are understood, the structures of fusion protein intermediates and their mode of lipid bilayer interaction, the structures and functions of the membrane anchors and the number of fusion proteins required for fusion, necessitate further investigations.

Rabies virus glycoprotein is a trimer.

Abstract The oligomerization state of the rabies virus envelope glycoprotein (G protein) was determined using electron microscopy and sedimentation analysis of detergent solubilized G. Most of the detergents used in this study solubilized G in a 4 S monomeric form. However, when CHAPS was used, G had a sedimentation coefficient of 9 S. This high sedimentation coefficient allowed its further separation from M1 and M2. Using electron microscopy of negatively stained samples, we studied the morphology of G on virus and after detergent extraction. End-on views of G on virus clearly showed triangles consisting of three dots indicating the trimeric nature of native G. End-on views of CHAPS-isolated G showed very similar triangles confirming that, using this detergent, G was solubilized in its native trimeric structure. Electron microscopy also showed that G had a “head” and a “stalk” and provided the basis for a low-resolution model of the glycoprotein structure.

Low-pH induced conformational changes in viral fusion proteins: implications for the fusion mechanism

Introduction. Entry of enveloped viruses into host cells requires binding of the virus to one or more receptors present at the host cell surface followed by fusion of the viral envelope with a cellular membrane. After binding, viruses such as paramyxoviruses, retroviruses and herpesviruses are thought to fuse directly with the plasma membrane. For other viruses, including the alpha-, rhabdo- and ortho-myxoviruses, binding does not directly lead to fusion. Instead, the bound virus particles are first internalized and then, at the low-pH within this compartment, fuse with the endosomal membrane. In this review, we will focus on this latter class of viruses. For alpha-, rhabdo- and orthomyxoviruses, a glycoprotein is responsible for both virus attachment and fusion. In the acidic environment of the endosome, the ectodomain portion of the glycoprotein undergoes a major structural rearrangement to generate a fusion-competent state.

Functional Characterization of Negri Bodies (NBs) in Rabies Virus-Infected Cells: Evidence that NBs Are Sites of Viral Transcription and Replication

ABSTRACT Rabies virus infection induces the formation of cytoplasmic inclusion bodies that resemble Negri bodies found in the cytoplasm of some infected nerve cells. We have studied the morphogenesis and the role of these Negri body-like structures (NBLs) during viral infection. The results indicate that these spherical structures (one or two per cell in the initial stage of infection), composed of the viral N and P proteins, grow during the virus cycle before appearing as smaller structures at late stages of infection. We have shown that the microtubule network is not necessary for the formation of these inclusion bodies but is involved in their dynamics. In contrast, the actin network does not play any detectable role in these processes. These inclusion bodies contain Hsp70 and ubiquitinylated proteins, but they are not misfolded protein aggregates. NBLs, in fact, appear to be functional structures involved in the viral life cycle. Specifically, using in situ fluorescent hybridization techniques, we show that all viral RNAs (genome, antigenome, and every mRNA) are located inside the inclusion bodies. Significantly, short-term RNA labeling in the presence of BrUTP strongly suggests that the NBLs are the sites where viral transcription and replication take place.

IN VIVO INTERACTION OF RABIES VIRUS PHOSPHOPROTEIN (P) AND NUCLEOPROTEIN (N) : EXISTENCE OF TWO N-BINDING SITES ON P PROTEIN

The rabies virus phosphoprotein (P) and nucleoprotein (N) are involved in transcription and replication of the viral genome. Interaction between N and P was studied in vivo in transfected cells expressing both proteins. Co-immunoprecipitation assays revealed that the N-P complex is present in cells expressing both proteins as well as in infected cells. Furthermore, immunostaining showed that coexpression of N and P was sufficient to induce the formation of cytoplasmic inclusions similar to those found in infected cells. In addition, deletion mutant analysis of P was performed to identify the regions of P interacting with N. The results indicate that at least two independent N-binding sites exist on P protein: one is located in the carboxy-terminal part of the protein and another between amino acids 69 and 177. The formation of cytoplasmic inclusions seems to require the presence of both N-binding sites on P protein.

Structures of vesicular stomatitis virus glycoprotein: membrane fusion revisited

Abstract.Glycoprotein G of the vesicular stomatitis virus (VSV) is involved in receptor recognition at the host cell surface and then, after endocytosis of the virion, triggers membrane fusion 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 atomic structures of these two conformations reveal that it is homologous to glycoprotein gB of herpesviruses and that it combines features of the previously characterized class I and class II fusion proteins. Comparison of the structures of G pre- and postfusion states shows a dramatic reorganization of the molecule that is reminiscent of that of paramyxovirus fusion protein F. It also allows identification of conserved key residues that constitute pH-sensitive molecular switches. Besides the similarities with other viral fusion machineries, the fusion properties and structures of G also reveal some striking particularities that invite us to reconsider a few dogmas concerning fusion proteins.

Molecular basis for the interaction between rabies virus phosphoprotein P and the dynein light chain LC8: dissociation of dynein-binding properties and transcriptional functionality of P

The lyssavirus phosphoprotein P is a co-factor of the viral RNA polymerase and plays a central role in virus transcription and replication. It has been shown previously that P interacts with the dynein light chain LC8, which is involved in minus end-directed movement of organelles along microtubules. Co-immunoprecipitation experiments and the two-hybrid system were used to map the LC8-binding site to the sequence (139)RSSEDKSTQTTGR(151). Site-directed mutagenesis of residues D(143) and Q(147) to an A residue abolished binding to LC8. The P-LC8 association is not required for virus transcription, since the double mutant was not affected in its transcription ability in a minigenome assay. Based on the crystal structure of LC8 bound to a peptide from neuronal nitric oxide synthase, a model for the complex between the peptide spanning residues 140-150 of P and LC8 is proposed. This model suggests that P binds LC8 in a manner similar to other LC8 cellular partners.

Crystal structure of vesicular stomatitis virus matrix protein

The vesicular stomatitis virus (VSV) matrix protein (M) interacts with cellular membranes, self‐associates and plays a major role in virus assembly and budding. We present the crystallographic structure, determined at 1.96 Å resolution, of a soluble thermolysin resistant core of VSV M. The fold is a new fold shared by the other vesiculovirus matrix proteins. The structure accounts for the loss of stability of M temperature‐sensitive mutants deficient in budding, and reveals a flexible loop protruding from the globular core that is important for self‐assembly. Membrane floatation shows that, together with the M lysine‐rich N‐terminal peptide, a second domain of the protein is involved in membrane binding. Indeed, the structure reveals a hydrophobic surface located close to the hydrophobic loop and surrounded by conserved basic residues that may constitute this domain. Lastly, comparison of the negative‐stranded virus matrix proteins with retrovirus Gag proteins suggests that the flexible link between their major membrane binding domain and the rest of the structure is a common feature shared by these proteins involved in budding and virus assembly.

Fossil Rhabdoviral Sequences Integrated into Arthropod Genomes: Ontogeny, Evolution, and Potential Functionality

Retroelements represent a considerable fraction of many eukaryotic genomes and are considered major drives for adaptive genetic innovations. Recent discoveries showed that despite not normally using DNA intermediates like retroviruses do, Mononegaviruses (i.e., viruses with nonsegmented, negative-sense RNA genomes) can integrate gene fragments into the genomes of their hosts. This was shown for Bornaviridae and Filoviridae, the sequences of which have been found integrated into the germ line cells of many vertebrate hosts. Here, we show that Rhabdoviridae sequences, the major Mononegavirales family, have integrated only into the genomes of arthropod species. We identified 185 integrated rhabdoviral elements (IREs) coding for nucleoproteins, glycoproteins, or RNA-dependent RNA polymerases; they were mostly found in the genomes of the mosquito Aedes aegypti and the blacklegged tick Ixodes scapularis. Phylogenetic analyses showed that most IREs in A. aegypti derived from multiple independent integration events. Since RNA viruses are submitted to much higher substitution rates as compared with their hosts, IREs thus represent fossil traces of the diversity of extinct Rhabdoviruses. Furthermore, analyses of orthologous IREs in A. aegypti field mosquitoes sampled worldwide identified an integrated polymerase IRE fragment that appeared under purifying selection within several million years, which supports a functional role in the hosts biology. These results show that A. aegypti was subjected to repeated Rhabdovirus infectious episodes during its evolution history, which led to the accumulation of many integrated sequences. They also suggest that like retroviruses, integrated rhabdoviral sequences may participate actively in the evolution of their hosts.

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.