The 24-angstrom structure of respiratory syncytial virus nucleocapsid protein-RNA decameric rings

Respiratory syncytial virus (RSV), a nonsegmented, negative-sense RNA-containing virus, is a common cause of lower respiratory tract disease. Expression of RSV nucleocapsid protein (N) in insect cells using the baculovirus expression system leads to the formation of N-RNA complexes that are morphologically indistinguishable from viral nucleocapsids. When imaged in an electron microscope, three distinct types of structures were observed: tightly wound short-pitch helices, highly extended helices, and rings. Negative stain images of N-RNA rings were used to calculate a three-dimensional reconstruction at 24 A resolution, revealing features similar to those observed in nucleocapsids from other viruses of the order Mononegavirales. The reconstructed N-RNA rings comprise 10 N monomers and have an external radius of 83 A and an internal radius of 40 A. Comparison of this structure with crystallographic data from rabies virus and vesicular stomatitis virus N-RNA rings reveals striking morphological similarities.     

Structure of recombinant rabies virus nucleoprotein-RNA complex and identification of the phosphoprotein binding site

Rabies virus nucleoprotein (N) was produced in insect cells, in which it forms nucleoprotein-RNA (N-RNA) complexes that are biochemically and biophysically indistinguishable from rabies virus N-RNA. We selected recombinant N-RNA complexes that were bound to short insect cellular RNAs which formed small rings containing 9 to 11 N monomers. We also produced recombinant N-RNA rings and viral N-RNA that were treated with trypsin and that had lost the C-terminal quarter of the nucleoprotein. Trypsin-treated N-RNA no longer bound to recombinant rabies virus phosphoprotein (the viral polymerase cofactor), so the presence of the C-terminal part of N is needed for binding of the phosphoprotein. Both intact and trypsin-treated recombinant N-RNA rings were analyzed with cryoelectron microscopy, and three-dimensional models were calculated from single-particle image analysis combined with back projection. Nucleoprotein has a bilobed shape, and each monomer has two sites of interaction with each neighbor. Trypsin treatment cuts off part of one of the lobes without shortening the protein or changing other structural parameters. Using negative-stain electron microscopy, we visualized phosphoprotein bound to the tips of the N-RNA rings, most likely at the site that can be removed by trypsin. Based on the shape of N determined here and on structural parameters derived from electron microscopy on free rabies virus N-RNA and from nucleocapsid in virus, we propose a low-resolution model for rabies virus N-RNA in the virus.     

Nucleoprotein-RNA orientation in the measles virus nucleocapsid by three-dimensional electron microscopy

Recombinant measles virus nucleoprotein-RNA (N-RNA) helices were analyzed by negative-stain electron microscopy. Three-dimensional reconstructions of trypsin-digested and intact nucleocapsids coupled to the docking of the atomic structure of the respiratory syncytial virus (RSV) N-RNA subunit into the electron microscopy density map support a model that places the RNA at the exterior of the helix and the disordered C-terminal domain toward the helix interior, and they suggest the position of the six nucleotides with respect to the measles N protomer.     

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.     

Three-dimensional reconstruction of a recombinant influenza virus ribonucleoprotein particle

A three-dimensional structural model of an influenza virus ribonucleoprotein particle reconstituted in vivo from recombinant proteins and a model genomic vRNA has been generated by electron microscopy. It shows a circular shape and contains nine nucleoprotein monomers, two of which are connected with the polymerase complex. The nucleoprotein monomers show a curvature that may be responsible for the formation of helical structures in the full-size viral ribonucleoproteins. The monomers show distinct contact boundaries at the two sides of the particle, suggesting that the genomic RNA may be located in association with the nucleoprotein at the base of the ribonucleoprotein complex. Sections of the three-dimensional model show a trilobular morphology in the polymerase complex that is consistent with the presence of its three subunits.     

Mechanism of RNA synthesis initiation by the vesicular stomatitis virus polymerase

The minimal RNA synthesis machinery of non-segmented negative-strand RNA viruses comprises a genomic RNA encased within a nucleocapsid protein (N-RNA), and associated with the RNA-dependent RNA polymerase (RdRP). The RdRP is contained within a viral large (L) protein, which associates with N-RNA through a phosphoprotein (P). Here, we define that vesicular stomatitis virus L initiates synthesis via a de-novo mechanism that does not require N or P, but depends on a high concentration of the first two nucleotides and specific template requirements. Purified L copies a template devoid of N, and P stimulates L initiation and processivity. Full processivity of the polymerase requires the template-associated N protein. This work provides new mechanistic insights into the workings of a minimal RNA synthesis machine shared by a broad group of important human, animal and plant pathogens, and defines a mechanism by which specific inhibitors of RNA synthesis function.     

Characterization of nucleocapsid binding by the measles virus and mumps virus phosphoproteins

We report an analysis of the interaction between the P protein and the RNA-associated N protein (N-RNA) for both measles and mumps viruses with proteins produced in a bacterial expression system. During this study, we verified that the C-terminal tail of the N protein is not required for nucleocapsid formation. For both measles and mumps virus N, truncated proteins encompassing amino acids 1 to 375 assemble into nucleocapsid-like particles within the bacterial cell. For measles virus N, the binding site for the P protein maps to residues 477 to 505 within the tail of the molecule, a sequence relatively conserved among the morbilliviruses. For mumps virus N, a binding site for the P protein maps to the assembly domain of N (residues 1 to 398), while no strong binding of the P protein to the tail of N was detected. These results suggest that the site of attachment for the polymerase varies among the paramyxoviruses. Pulldown experiments demonstrate that the last 50 amino acids of both measles virus and mumps virus P (measles virus P, 457 to 507; mumps virus P, 343 to 391) by themselves constitute the nucleocapsid-binding domain (NBD). Spectroscopic studies show that the NBD is predominantly alpha-helical in both viruses. However, only in measles virus P is the NBD stable and folded, having a lesser degree of tertiary organization in mumps virus P. With isothermal titration calorimetry, we demonstrate that the measles virus P NBD binds to residues 477 to 505 of measles virus N with 1:1 stoichiometry. The dissociation constant (K(d)) was determined to be 13 microM at 20 degrees C and 35 microM at 37 degrees C. Our data are consistent with a model in which an alpha-helical nucleocapsid binding domain, located at the C terminus of P, is responsible for tethering the viral polymerase to its template yet also suggest that, in detail, polymerase binding in morbilliviruses and rubulaviruses differs significantly.     

Virucidal activity of a scorpion venom peptide variant mucroporin-M1 against measles, SARS-CoV and influenza H5N1 viruses

Outbreaks of SARS-CoV, influenza A (H5N1, H1N1) and measles viruses in recent years have raised serious concerns about the measures available to control emerging and re-emerging infectious viral diseases. Effective antiviral agents are lacking that specifically target RNA viruses such as measles, SARS-CoV and influenza H5N1 viruses, and available vaccinations have demonstrated variable efficacy. Therefore, the development of novel antiviral agents is needed to close the vaccination gap and silence outbreaks. We previously indentified mucroporin, a cationic host defense peptide from scorpion venom, which can effectively inhibit standard bacteria. The optimized mucroporin-M1 can inhibit gram-positive bacteria at low concentrations and antibiotic-resistant pathogens. In this investigation, we further tested mucroporin and the optimized mucroporin-M1 for their antiviral activity. Surprisingly, we found that the antiviral activities of mucroporin-M1 against measles, SARS-CoV and influenza H5N1 viruses were notably increased with an EC₅₀ of 7.15 μg/ml (3.52 μM) and a CC₅₀ of 70.46 μg/ml (34.70 μM) against measles virus, an EC₅₀ of 14.46 μg/ml (7.12 μM) against SARS-CoV and an EC₅₀ of 2.10 μg/ml (1.03 μM) against H5N1, while the original peptide mucroporin showed no antiviral activity against any of these three viruses. The inhibition model could be via a direct interaction with the virus envelope, thereby decreasing the infectivity of virus. This report provides evidence that host defense peptides from scorpion venom can be modified for antiviral activity by rational design and represents a practical approach for developing broad-spectrum antiviral agents, especially against RNA viruses.     

Dimer ribbons in the three-dimensional structure of sarcoplasmic reticulum

The three-dimensional structure of scallop sarcoplasmic reticulum membranes has been determined from electron micrographs of two classes of stain-filled tubules by helical reconstruction methods. These structures are characterized by dimer ribbons of Ca2+-ATPase molecules running diagonally around the tube wall. Deep right-handed grooves separate the ribbons. The elongated, curved units of the dimer (approximately 95 A long in the radial direction; 60 to 70 A axially, and about 30 A wide) are displaced axially by approximately 34 A and are connected at their outer ends by a bridge running nearly parallel to the tube axis. The monomers make a second contact at their inner ends. Adjacent units with the same orientation form a strong contact that is responsible for the ribbon appearance. Comparison of tubules of different diameter shows that one set of connections between the dimer ribbons is conserved: the inner ends of axially displaced dimers appear to make contact along a left-handed path almost perpendicular to the major grooves. The lipid bilayer cannot be clearly identified. The two-dimensional map obtained from flattened tubules is consistent with the three-dimensional reconstruction in showing dimer ribbons connected by a weak contact across the grooves, strongly resembling the inter-dimer bond observed in three dimensions. The two-dimensional map shows a 2-fold axis relating units of the dimer, but the three-dimensional tubes show a slight axial polarity that may arise from the presence of proteins other than the Ca2+-ATPase.     

Assembly of nucleocapsidlike structures in animal cells infected with a vaccinia virus recombinant encoding the measles virus nucleoprotein.

A vaccinia virus recombinant containing the measles virus nucleoprotein gene was shown to induce the synthesis of a 60 kDa phosphorylated nucleoprotein similar to authentic measles virus nucleoprotein. Mammalian or avian cells infected with the recombinant virus displayed tubular structures reminiscent of viral nucleocapsids both in the cytoplasm and in the nucleus. Such structures could be labelled in situ by using an immunogold detection method specific for measles virus proteins. Electron microscopic examination of tubular structures purified from cells infected with the vaccinia virus recombinant indicated that they displayed most of the features of measles virus nucleocapsids, although their length was on the average shorter. These results demonstrate the spontaneous assembly of measles virus nucleocapsids in the absence of viral leader RNA and provide a means for a detailed molecular analysis of the requirements for nucleocapsid assembly. Furthermore, these findings raise the possibility of achieving complete assembly of measles virus particles, devoid of infectious RNA, by using a vaccinia virus vector.     

The intrinsically disordered C-terminal domain of the measles virus nucleoprotein interacts with the C-terminal domain of the phosphoprotein via two distinct sites and remains predominantly unfolded

Measles virus is a negative-sense, single-stranded RNA virus within the Mononegavirales order,which includes several human pathogens, including rabies, Ebola, Nipah, and Hendra viruses. The measles virus nucleoprotein consists of a structured N-terminal domain, and of an intrinsically disordered C-terminal domain, N(TAIL) (aa 401-525), which undergoes induced folding in the presence of the C-terminal domain (XD, aa 459-507) of the viral phosphoprotein. With in N(TAIL), an alpha-helical molecular recognition element (alpha-MoRE, aa 488-499) involved in binding to P and in induced folding was identified and then observed in the crystal structure of XD. Using small-angle X-ray scattering, we have derived a low-resolution structural model of the complex between XD and N(TAIL), which shows that most of N(TAIL) remains disordered in the complex despite P-induced folding within the alpha-MoRE. The model consists of an extended shape accommodating the multiple conformations adopted by the disordered N-terminal region of N(TAIL), and of a bulky globular region, corresponding to XD and to the C terminus of N(TAIL) (aa 486-525). Using surface plasmon resonance, circular dichroism, fluorescence spectroscopy, and heteronuclear magnetic resonance, we show that N(TAIL) has an additional site (aa 517-525) involved in binding to XD but not in the unstructured-to-structured transition. This work provides evidence that intrinsically disordered domains can establish complex interactions with their partners, and can contact them through multiple sites that do not all necessarily gain regular secondary structure.     

The plasticity of a structural motif in RNA: Structural polymorphism of a kink turn as a function of its environment

The k-turn is a widespread structural motif that introduces a tight kink into the helical axis of double-stranded RNA. The adenine bases of consecutive G•A pairs are directed toward the minor groove of the opposing helix, hydrogen bonding in a typical A-minor interaction. We show here that the available structures of k-turns divide into two classes, depending on whether N3 or N1 of the adenine at the 2b position accepts a hydrogen bond from the O2′ at the −1n position. There is a coordinated structural change involving a number of hydrogen bonds between the two classes. We show here that Kt-7 can adopt either the N3 or N1 structures depending on environment. While it has the N1 structure in the ribosome, on engineering it into the SAM-I riboswitch, it changes to the N3 structure, resulting in a significant alteration in the trajectory of the helical arms.