Visualization of conformational changes and membrane remodeling leading to genome delivery by viral class-II fusion machinery

Chikungunya virus (CHIKV) is a human pathogen that delivers its genome to the host cell cytoplasm through endocytic low pH-activated membrane fusion mediated by class-II fusion proteins. Though structures of prefusion, icosahedral CHIKV are available, structural characterization of virion interaction with membranes has been limited. Here, we have used cryo-electron tomography to visualize CHIKV's complete membrane fusion pathway, identifying key intermediary glycoprotein conformations coupled to membrane remodeling events. Using sub-tomogram averaging, we elucidate features of the low pH-exposed virion, nucleocapsid and full-length E1-glycoprotein's post-fusion structure. Contrary to class-I fusion systems, CHIKV achieves membrane apposition by protrusion of extended E1-glycoprotein homotrimers into the target membrane. The fusion process also features a large hemifusion diaphragm that transitions to a wide pore for intact nucleocapsid delivery. Our analyses provide comprehensive ultrastructural insights into the class-II virus fusion system function and direct mechanistic characterization of the fundamental process of protein-mediated membrane fusion

Structural studies on the rubella virus capsid protein and its organization in the virion

Rubella virus is a leading cause of birth defects due to infectious agents. When contracted during pregnancy, rubella infection leads to severe damage in fetuses. Despite its medical importance, very little is known about the structure of the pleomorphic rubella virus as compared to its alphavirus relatives. The rubella capsid protein is a critical structural component of virions as well as a key factor in virus-host interactions. Three crystal structures of the structural domain of the rubella capsid protein have been described here. The polypeptide fold of the capsid protomer has not been observed previously. The capsid protein structure, along with cryo-electron tomograms of rubella virus particles and mutational studies on the capsid protein, provides a low resolution structure of the virus particle. Nucleocapsid assembly studies on the rubella capsid protein has given additional information on the factors affecting formation of the virion cores. Together, these studies enumerate critical differences between rubella virus and alphaviruses that might affect their specific pathogenicities

Visualization of conformational changes and membrane remodeling leading to genome delivery by viral class-II fusion machinery

Chikungunya virus (CHIKV) is a human pathogen that delivers its genome to the host cell cytoplasm through endocytic low pH-activated membrane fusion mediated by class-II fusion proteins. Though structures of prefusion, icosahedral CHIKV are available, structural characterization of virion interaction with membranes has been limited. Here, we have used cryo-electron tomography to visualize CHIKV's complete membrane fusion pathway, identifying key intermediary glycoprotein conformations coupled to membrane remodeling events. Using sub-tomogram averaging, we elucidate features of the low pH-exposed virion, nucleocapsid and full-length E1-glycoprotein's post-fusion structure. Contrary to class-I fusion systems, CHIKV achieves membrane apposition by protrusion of extended E1-glycoprotein homotrimers into the target membrane. The fusion process also features a large hemifusion diaphragm that transitions to a wide pore for intact nucleocapsid delivery. Our analyses provide comprehensive ultrastructural insights into the class-II virus fusion system function and direct mechanistic characterization of the fundamental process of protein-mediated membrane fusion.</p

Assembly, maturation and three-dimensional helical structure of the teratogenic rubella virus

<div><p>Viral infections during pregnancy are a significant cause of infant morbidity and mortality. Of these, rubella virus infection is a well-substantiated example that leads to miscarriages or severe fetal defects. However, structural information about the rubella virus has been lacking due to the pleomorphic nature of the virions. Here we report a helical structure of rubella virions using cryo-electron tomography. Sub-tomogram averaging of the surface spikes established the relative positions of the viral glycoproteins, which differed from the earlier icosahedral models of the virus. Tomographic analyses of <i>in vitro</i> assembled nucleocapsids and virions provide a template for viral assembly. Comparisons of immature and mature virions show large rearrangements in the glycoproteins that may be essential for forming the infectious virions. These results present the first known example of a helical membrane-enveloped virus, while also providing a structural basis for its assembly and maturation pathway.</p></div

Nucleocapsid organization in rubella virions.

<p>(A) Cross-section at the nucleocapsid surface of a rubella virion tomogram, showing a grid-like pattern of the nucleocapsid units (dashed red box). Scale bar is 50 Å long. (B) and (C) Left panel shows a tomogram section at the surface of the rubella virions; the right panel shows a section at the nucleocapsid surface. Red arrows indicate the glycoprotein rows and the corresponding nucleocapsid rows. Scale bar corresponds to a length of 100 Å. Black represents high density. A ball and stick model for the glycoprotein and nucleocapsid organization is given in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006377#ppat.1006377.s005" target="_blank">S4 Fig</a>.</p

Helical organization of the rubella virus glycoproteins.

<p>Representation of three different rubella virions (A, B and C) showing the organization of their surface glycoprotein rows. The virions have been extracted and rendered using UCSF Chimera [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006377#ppat.1006377.ref061" target="_blank">61</a>] without any averaging procedures (Materials and methods). The extracted virions have been low pass filtered to 75 Å and hence, the surface glycoprotein rows appear as elevated ridges on the outer membrane surface. Scale bar is 100 Å in length. The surface contour is chosen at 0.81 standard deviations above average. The pitch of the helix in Fig 2A–2C is 533 Å, 390 Å and 0 Å, respectively. Further analysis of the glycoprotein rows using sub-tomogram averaging is shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006377#ppat.1006377.s002" target="_blank">S1 Fig</a>.</p

Structure of rubella virus glycoprotein spikes.

<p>(A) Sub-tomogram averaged structure of the rubella glycoprotein spike (light blue) is shown placed on a membrane surface (yellow). The membrane surface has been modelled by extracting a lipid bilayer portion from a co-purified membrane vesicle in the un-averaged virus tomograms. The left and right panels are rotated 90° with respect to each other. (B) The same figure as in panel <i>A</i>, showing the rubella E1 ectodomain’s atomic structure fitted into the averaged density. The yellow star indicates the location of the rubella E2 ectodomain. The parenthesis in black indicate immunogenic surface regions on E1. Scale bars in panels <i>A</i> and <i>B</i> correspond to a length of 25 Å. Intermediates from the sub-tomogram averaging procedures are shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006377#ppat.1006377.s003" target="_blank">S2 Fig</a>. The Fourier Shell Correlation (FSC) curve calculated to estimate the resolution of the averaged glycoprotein spike map is shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006377#ppat.1006377.s004" target="_blank">S3 Fig</a>. See also <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006377#ppat.1006377.s001" target="_blank">S1 Table</a>. (C) Cross-section of a rubella virion showing a representative glycoprotein row. Left panel shows the original tomogram section. The right panel shows the same section after placing the averaged glycoprotein spike (blue) (8X binned) into the tomogram. (D) Cross-sections showing a top view of the same glycoprotein row as in panel C. Black arrow indicates the glycoprotein row being considered. In panels C and D, scale bar is 50 Å long and black represents high density.</p

mRNA-based VP8* nanoparticle vaccines against rotavirus are highly immunogenic in rodents

Abstract Despite the availability of live-attenuated oral vaccines, rotavirus remains a major cause of severe childhood diarrhea worldwide. Due to the growing demand for parenteral rotavirus vaccines, we developed mRNA-based vaccine candidates targeting the viral spike protein VP8*. Our monomeric P2 (universal T cell epitope)-VP8* mRNA design is equivalent to a protein vaccine currently in clinical development, while LS (lumazine synthase)-P2-VP8* was designed to form nanoparticles. Cyro-electron microscopy and western blotting-based data presented here suggest that proteins derived from LS-P2-VP8* mRNA are secreted in vitro and self-assemble into 60-mer nanoparticles displaying VP8*. mRNA encoded VP8* was immunogenic in rodents and introduced both humoral and cellular responses. LS-P2-VP8* induced superior humoral responses to P2-VP8* in guinea pigs, both as monovalent and trivalent vaccines, with encouraging responses detected against the most prevalent P genotypes. Overall, our data provide evidence that trivalent LS-P2-VP8* represents a promising mRNA-based next-generation rotavirus vaccine candidate

Plasma proteome database as a resource for proteomics research

Plasma is one of the best studied compartments in the human body and serves as an ideal body fluid for the diagnosis of diseases. This report provides a detailed functional annotation of all the plasma proteins identified to date. In all, gene products encoded by 3778 distinct genes were annotated based on proteins previously published in the literature as plasma proteins and the identification of multiple peptides from proteins under HUPO's Plasma Proteome Project. Our analysis revealed that 51% of these genes encoded more than one protein isoform. All single nucleotide polymorphisms involving protein-coding regions were mapped onto the protein sequences. We found a number of examples of isoform-specific subcellular localization as well as tissue expression. This database is an attempt at comprehensive annotation of a complex subproteome and is available on the web at http://www.plasmaproteomedatabase.org