@article {65277, title = {Structure of the vesicular stomatitis virus L protein in complex with Its phosphoprotein cofactor.}, journal = {Cell Reports}, volume = {30}, year = {2020}, month = {2020 Jan 07}, pages = {53-60.e5}, abstract = {

The large (L) proteins of non-segmented, negative-strand RNA viruses are multifunctional enzymes that produce capped, methylated, and polyadenylated mRNA and replicate the viral genome. A phosphoprotein (P), required for efficient RNA-dependent RNA polymerization from the viral ribonucleoprotein (RNP) template, regulates the function and conformation of the L protein. We report the structure of vesicular stomatitis virus L in complex with its P cofactor determined by electron cryomicroscopy at 3.0\ {\r A} resolution, enabling us to visualize bound segments of P. The contacts of three P segments with multiple L domains show how P induces a closed, compact, initiation-competent conformation. Binding of P to L positions its N-terminal domain adjacent to a putative RNA exit channel for efficient encapsidation of newly synthesized genomes with the nucleoprotein and orients its C-terminal domain to interact with an RNP template. The model shows that a conserved tryptophan in the priming loop can support the initiating 5{\textquoteright} nucleotide.

}, issn = {2211-1247}, doi = {10.1016/j.celrep.2019.12.024}, author = {Jenni, Simon and Bloyet, Louis-Marie and Diaz-Avalos, Ruben and Liang, Bo and Whelan, Sean P J and Grigorieff, Nikolaus and Harrison, Stephen C} } @article {65024, title = {In situ structure of rotavirus VP1 RNA-dependent RNA polymerase.}, journal = {Journal of Molecular Biology}, volume = {431}, year = {2019}, month = {2019 Jun 21}, pages = {3124-38}, abstract = {

Rotaviruses, like other non-enveloped, double-strand RNA (dsRNA) viruses, package an RNA-dependent RNA polymerase (RdRp) with each duplex of their segmented genomes. Rotavirus cell entry results in loss of an outer protein layer and delivery into the cytosol of an intact, inner capsid particle (the {\textquotedblleft}double-layer particle{\textquotedblright} or DLP). The RdRp, designated VP1, is active inside the DLP; each VP1 achieves many rounds of mRNA transcription from its associated genome segment. Previous work has shown that one VP1 molecule lies close to each fivefold axis of the icosahedrally symmetric DLP, just beneath the inner surface of its protein shell, embedded in tightly packed RNA. We have determined a high-resolution structure for the rotavirus VP1 RdRp\ in situ, by local reconstruction of density around individual fivefold positions. We have analyzed intact virions ({\textquotedblleft}triple-layer particles{\textquotedblright} or TLPs), non-transcribing DLPs and transcribing DLPs. Outer layer dissociation enables the DLP to synthesize RNA,\ in vitro\ as well as\ in vivo, but appears not to induce any detectable structural change in the RdRp. Addition of NTPs, Mg2+, and S-adenosyl methionine, which allows active transcription, results in conformational rearrangements, in both VP1 and the DLP capsid shell protein, that allow a transcript to exit the polymerase and the particle. The position of VP1 (among the five symmetrically related alternatives) at one vertex does not correlate with its position at other vertices. This stochastic distribution of site occupancies limits long-range order in the 11-segment, dsRNA genome.

}, doi = {10.1016/j.jmb.2019.06.016}, author = {Jenni, Simon and Salgado, Eric N. and Herrmann, Tobias and Li, Zongli and Grant, Timothy and Grigorieff, Nikolaus and Trapani, Stefano and Estrozi, Leandro F. and Harrison, Stephen C.} } @article {48489, title = {CryoEM structure of an influenza virus receptor-binding site antibody-antigen interface.}, journal = {Journal of Molecular Biology}, volume = {429}, year = {2017}, month = {2017 May 12}, pages = {1829-39}, abstract = {

Structure-based vaccine design depends on extensive structural analyses of antigen-antibody complexes. Single-particle electron cryomicroscopy (cryoEM) can circumvent some of the problems of x-ray crystallography as a pipeline for obtaining the required structures. We have examined the potential of single-particle cryoEM for determining the structure of influenza-virus hemagglutinin (HA):single-chain Fv (scFv) complexes, by studying a complex we failed to crystallize in pursuing an extended project of the human immune response to influenza vaccines. The result shows that a combination of cryoEM and molecular modeling can yield details of the antigen:antibody interface, although small variation in the twist of the rod-like HA trimer limited the overall resolution to about 4.5{\r A}. Comparison of principal 3D classes suggests ways to modify the HA trimer to overcome this limitation. A closely related antibody from the same donor did yield crystals when bound with the same HA, giving us an independent validation of the cryoEM results The two structures also augment our understanding of receptor-binding site recognition by antibodies that neutralize a wide range of influenza-virus variants.

}, issn = {1089-8638}, doi = {10.1016/j.jmb.2017.05.011}, author = {Liu, Yuhang and Pan, Junhua and Jenni, Simon and Raymond, Donald D and Caradonna, Tim and Do, Khoi T and Schmidt, Aaron G and Harrison, Stephen C and Grigorieff, Nikolaus} } @article {48316, title = {Structure of the L protein of vesicular stomatitis virus from electron cryomicroscopy.}, journal = {Cell}, volume = {162}, year = {2015}, month = {2015 Jul 16}, pages = {314-27}, abstract = {

The large (L) proteins of non-segmented, negative-strand RNA viruses, a group that includes Ebola and rabies viruses, catalyze RNA-dependent RNA polymerization with viral ribonucleoprotein as template, a non-canonical sequence of capping and methylation reactions, and polyadenylation of viral messages. We have determined by electron cryomicroscopy the structure of the vesicular stomatitis virus (VSV) L protein. The density map, at a resolution of 3.8 {\r A}, has led to an atomic model for nearly all of the 2109-residue polypeptide chain, which comprises three enzymatic domains (RNA-dependent RNA polymerase [RdRp], polyribonucleotidyl transferase [PRNTase], and methyltransferase) and two structural domains. The RdRp resembles the corresponding enzymatic regions of dsRNA virus polymerases and influenza virus polymerase. A loop from the PRNTase (capping) domain projects into the catalytic site of the RdRp, where it appears to have the role of a priming loop and to couple product elongation to large-scale conformational changes in L.

}, keywords = {Cryoelectron Microscopy, DNA-Directed RNA Polymerases, Models, Molecular, Protein Structure, Tertiary, Sequence Homology, Amino Acid, Transcription, Genetic, Vesicular stomatitis Indiana virus, Viral Proteins}, issn = {1097-4172}, doi = {10.1016/j.cell.2015.06.018}, author = {Liang, Bo and Li, Zongli and Jenni, Simon and Rahmeh, Amal A and Morin, Benjamin M and Grant, Timothy and Grigorieff, Nikolaus and Harrison, Stephen C and Whelan, Sean P J} }