A peptide derived from the rotavirus outer capsid protein VP7 permeabilizes artificial membranes.

Biological membranes represent a physical barrier that most viruses have to cross for replication. While enveloped viruses cross membranes through a well-characterized membrane fusion mechanism, non-enveloped viruses, such as rotaviruses, require the destabilization of the host cell membrane by processes that are still poorly understood. We have identified, in the C-terminal region of the rotavirus glycoprotein VP7, a peptide that was predicted to contain a membrane domain and to fold into an amphipathic α-helix. Its structure was confirmed by circular dichroism in media mimicking the hydrophobic environment of the membrane at both acidic and neutral pHs. The helical folding of the peptide was corroborated by ATR-FTIR spectroscopy, which suggested a transmembrane orientation of the peptide. The interaction of this peptide with artificial membranes and its affinity were assessed by plasmon waveguide resonance. We have found that the peptide was able to insert into membranes and permeabilize them while the native protein VP7 did not. Finally, NMR studies revealed that in a hydrophobic environment, this helix has amphipathic properties characteristic of membrane-perforating peptides. Surprisingly, its structure varies from that of its counterpart in the structure of the native protein VP7, as was determined by X-ray. All together, our results show that a peptide released from VP7 is capable of changing its conformation and destabilizing artificial membranes. Such peptides could play an important role by facilitating membrane crossing by non-enveloped viruses during cell infection.

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 (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.

The picobirnavirus crystal structure provides functional insights into virion assembly and cell entry.

Double-stranded (ds) RNA virus particles are organized around a central icosahedral core capsid made of 120 identical subunits. This core capsid is unable to invade cells from outside, and animal dsRNA viruses have acquired surrounding capsid layers that are used to deliver a transcriptionally active core particle across the membrane during cell entry. In contrast, dsRNA viruses infecting primitive eukaryotes have only a simple core capsid, and as a consequence are transmitted only vertically. Here, we report the 3.4 A X-ray structure of a picobirnavirus--an animal dsRNA virus associated with diarrhoea and gastroenteritis in humans. The structure shows a simple core capsid with a distinctive icosahedral arrangement, displaying 60 two-fold symmetric dimers of a coat protein (CP) with a new 3D-fold. We show that, as many non-enveloped animal viruses, CP undergoes an autoproteolytic cleavage, releasing a post-translationally modified peptide that remains associated with nucleic acid within the capsid. Our data also show that picobirnavirus particles are capable of disrupting biological membranes in vitro, indicating that its simple 120-subunits capsid has evolved animal cell invasion properties.

NMR structure of a viral peptide inserted in artificial membranes: a view on the early steps of the birnavirus entry process.

Nonenveloped virus must penetrate the cellular membrane to access the cytoplasm without the benefit of membrane fusion. For birnavirus, one of the peptides present in the virus capsid, pep46 for infectious bursal disease virus, is able to induce pores into membranes as an intermediate step of the birnavirus-penetration pathway. Using osmotic protection experiments, we demonstrate here that pep46 and its pore-forming N-terminal moiety (pep22) form pores of different diameters, 5-8 and 2-4 nm, respectively, showing that both pep46 moieties participate to pore formation. The solution structures of pep46, pep22, and pep24 (the pep46 C-terminal moiety) in different hydrophobic environments and micelles determined by (1)H NMR studies provide structural insights of the pep46 domain interaction. In CDCl(3)/CD(3)OH mixture and in dodecylphosphocholine micelles, the N-terminal domain of pep46 is structured in a long kinked helix, although the C terminus is structured in one or two helices depending upon the solvents used. We also show that the folding and the proline isomerization status of pep46 depend on the type of hydrophobic environment. NMR spectroscopy with labeled phospholipid micelles, differential scanning calorimetry, and plasmon waveguide resonance studies show the peptides lie parallel to the lipid-water interface, perturbing the fatty acid chain packing. All these data lead to a model in which the two domains of pep46 interact with the membrane to form pores.

Geometric mismatches within the concentric layers of rotavirus particles: a potential regulatory switch of viral particle transcription activity.

Rotaviruses are prototypical double-stranded RNA viruses whose triple-layered icosahedral capsid constitutes transcriptional machinery activated by the release of the external layer. To understand the molecular basis of this activation, we studied the structural interplay between the three capsid layers by electron cryo-microscopy and digital image processing. Two viral particles and four virus-like particles containing various combinations of inner (VP2)-, middle (VP6)-, and outer (VP7)-layer proteins were studied. We observed that the absence of the VP2 layer increases the particle diameter and changes the type of quasi-equivalent icosahedral symmetry, as described by the shift in triangulation number (T) of the VP6 layer (from T = 13 to T = 19 or more). By fitting X-ray models of VP6 into each reconstruction, we determined the quasi-atomic structures of the middle layers. These models showed that the VP6 lattices, i.e., curvature and trimer contacts, are characteristic of the particle composition. The different functional states of VP6 thus appear as being characterized by trimers having similar conformations but establishing different intertrimeric contacts. Remarkably, the external protein VP7 reorients the VP6 trimers located around the fivefold axes of the icosahedral capsid, thereby shrinking the channel through which mRNA exits the transcribing rotavirus particle. We conclude that the constraints arising from the different geometries imposed by the external and internal layers of the rotavirus capsid constitute a potential switch regulating the transcription activity of the viral particles.

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.

Infectious bursal disease virus, a non-enveloped virus, possesses a capsid-associated peptide that deforms and perforates biological membranes.

Double-stranded RNA (dsRNA) virions constitute transcriptionally competent machines that must translocate across cell membranes to function within the cytoplasm. The entry mechanism of such non-enveloped viruses is not well described. Birnaviruses are unique among dsRNA viruses because they possess a single shell competent for entry. We hereby report how infectious bursal disease virus, an avian birnavirus, can disrupt cell membranes and enter into its target cells. One of its four structural peptides, pep46 (a 46-amino acid amphiphilic peptide) deforms synthetic membranes and induces pores visualized by electron cryomicroscopy, having a diameter of less than 10 nm. Using both biological and synthetic membranes, the pore-forming domain of pep46 was identified as its N terminus moiety (pep22). The N and C termini of pep22 are shown to be accessible during membrane destabilization and pore formation. NMR studies show that pep46 inserted into micelles displays a cis-trans proline isomerization at position 16 that we propose to be associated to the pore formation process. Reverse genetic experiments confirm that the amphiphilicity and proline isomerization of pep46 are both essential to the viral cycle. Furthermore, we show that virus infectivity and its membrane activity (probably because of the release of pep46 from virions) are controlled differently by calcium concentration, suggesting that entry is performed in two steps, endocytosis followed by endosome permeabilization. Our findings reveal a possible entry pathway of infectious bursal disease virus: in endosomes containing viruses, the lowering of the calcium concentration promotes the release of pep46 that induces the formation of pores in the endosomal membrane.

Genetic variation in the VP7 gene of human rotavirus isolated in Montevideo-Uruguay from 1996-1999.

The gene encoding the protein VP7 that induces the major neutralizing response has been sequenced from 34 human rotaviruses isolated from children with acute diarrhea in Montevideo (Uruguay) over a 4-year period (1996-1999). These sequences were analyzed and compared to representative corresponding sequences available on databases. In most years, serotype G1 was present as the single serotype, except in 1999 when serotypes G1 and G4 were present simultaneously. Two G1 VP7 lineages were identified. Serotype G2 was present in 1997. The G4 isolates are grouped with Argentine strains and emerged during 1998 in a recently defined sublineage. Neither serotype G3 nor the emerging serotype G9 were isolated during the study. Antigenic domains of isolates and of representative reference strains of each serotype were compared. Sequences of strains isolated during the same year, showed a high degree of homology among strains belonging to the same serotype.