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

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.

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.

Rabies virus transcription and replication.

Rabies virus (RABV) is a negative-stranded RNA virus. Its genome is tightly encapsidated by the viral nucleoprotein (N) and this RNA-N complex is the template for transcription and replication by the viral RNA-dependent RNA polymerase (L) and its cofactor, the phosphoprotein (P). We present molecular, structural, and cellular aspects of RABV transcription and replication. We first summarize the characteristics and molecular biology of both RNA synthesis processes. We then discuss biochemical and structural data on the viral proteins (N, P, and L) and their interactions with regard to their role in viral transcription and replication. Finally, we review evidence that rabies viral transcription and replication take place in cytoplasmic inclusion bodies formed in RABV-infected cells and discuss the role of this cellular compartmentalization.

Distinct structural rearrangements of the VSV glycoprotein drive membrane fusion.

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.

Binding of rabies virus polymerase cofactor to recombinant circular nucleoprotein-RNA complexes.

In rabies virus, the attachment of the L polymerase (L) to the viral nucleocapsids (NCs)-a nucleoprotein (N)-RNA complex that serves as template for RNA transcription and replication-is mediated by the polymerase cofactor, the phosphoprotein (P). P forms dimers (P(2)) that bind through their C-terminal domains (P(CTD)) to the C-terminal region of the N. Recombinant circular N(m)-RNA complexes containing 9 to 12 protomers of N (hereafter, the subscript m denotes the number of N protomers) served here as model systems for studying the binding of P to NC-like N(m)-RNA complexes. Titration experiments show that there are only two equivalent and independent binding sites for P dimers on the N(m)-RNA rings and that each P dimer binds through a single P(CTD). A dissociation constant in the nanomolar range (160+/-20 nM) was measured by surface plasmon resonance, indicating a strong interaction between the two partners. Small-angle X-ray scattering (SAXS) data and small-angle neutron scattering data showed that binding of two P(CTD) had almost no effect on the size and shape of the N(m)-RNA rings, whereas binding of two P(2) significantly increased the size of the complexes. SAXS data and molecular modeling were used to add flexible loops (N(NTD) loop, amino acids 105-118; N(CTD) loop, amino acids 376-397) missing in the recently solved crystal structure of the circular N(11)-RNA complex and to build a model for the N(10)-RNA complex. Structural models for the N(m)-RNA-(P(CTD))(2) complexes were then built by docking the known P(CTD) structure onto the completed structures of the circular N(10)-RNA and N(11)-RNA complexes. A multiple-stage flexible docking procedure was used to generate decoys, and SAXS and biochemical data were used for filtering the models. In the refined model, the P(CTD) is bound to the C-terminal top of one N protomer (N(i)), with the C-terminal helix (alpha(6)) of P(CTD) lying on helix alpha(14) of N(i). By an induced-fit mechanism, the N(CTD) loop of the same protomer (N(i)) and that of the adjacent one (N(i)(-1)) mold around the P(CTD), making extensive protein-protein contacts that could explain the strong affinity of P for its template. The structural model is in agreement with available biochemical data and provides new insights on the mechanism of attachment of the polymerase complex to the NC template.

Unphosphorylated rhabdoviridae phosphoproteins form elongated dimers in solution.

The phosphoprotein (P) is an essential component of the replication machinery of rabies virus (RV) and vesicular stomatitis virus (VSV), and the oligomerization of P, potentially controlled by phosphorylation, is required for its function. Up to now the stoichiometry of phosphoprotein oligomers has been controversial. Size exclusion chromatography combined with detection by multiangle laser light scattering shows that the recombinant unphosphorylated phosphoproteins from VSV and from RV exist as dimers in solution. Hydrodynamic analysis indicates that the dimers are highly asymmetric, with a Stokes radius of 4.8-5.3 nm and a frictional ratio larger than 1.7. Small-angle neutron scattering experiments confirm the dimeric state and the asymmetry of the structure and yield a radius of gyration of about 5.3 nm and a cross-sectional radius of gyration of about 1.6-1.8 nm. Similar hydrodynamic properties and molecular dimensions were obtained with a variant of VSV phosphoprotein in which Ser60 and Thr62 are substituted by Asp residues and which has been reported previously to mimic phosphorylation by inducing oligomerization and activating transcription. Here, we show that this mutant also forms a dimer with hydrodynamic properties and molecular dimensions similar to those of the wild type protein. However, incubation at 30 degrees C for several hours induced self-assembly of both wild type and mutant proteins, leading to the formation of irregular filamentous structures.

Isolation and crystallization of a unique size category of recombinant Rabies virus Nucleoprotein-RNA rings.

In order to study the packaging of rabies virus RNA inside the viral nucleocapsid, rabies nucleoprotein was expressed in insect cells. In the cells, it binds to cellular RNA to form long, helical or short circular complexes, depending on the length of the bound RNA. The circular complexes contained from 9 up to 13 N-protomers per ring. Separation of the rings into defined size classes was impossible through regular column chromatographies or gradient centrifugation. The size classes could be separated by native polyacrylamide gel electrophoresis. A large-scale separation was achieved with a 4% native gel using a preparative electrophoresis apparatus. Crystallization trials were set up with N-RNA rings from three size classes and crystals were obtained in all cases. The best diffracting crystals, diffracting up to 6A, contained rings with 11 N-protomers plus an RNA molecule of 99 nucleotides. The diffraction limit was improved to 3.5A by air dehydration prior to flash freezing.

Crystal structure of the rabies virus nucleoprotein-RNA complex.

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.