Stability of local secondary structure determines selectivity of viral RNA chaperones

To maintain genome integrity, segmented double-stranded RNA viruses of the Reoviridae family must accurately select and package a complete set of up to a dozen distinct genomic RNAs. It is thought that the high fidelity segmented genome assembly involves multiple sequence-specific RNA–RNA interactions between single-stranded RNA segment precursors. These are mediated by virus-encoded non-structural proteins with RNA chaperone-like activities, such as rotavirus (RV) NSP2 and avian reovirus σNS. Here, we compared the abilities of NSP2 and σNS to mediate sequence-specific interactions between RV genomic segment precursors. Despite their similar activities, NSP2 successfully promotes inter-segment association, while σNS fails to do so. To understand the mechanisms underlying such selectivity in promoting inter-molecular duplex formation, we compared RNA-binding and helix-unwinding activities of both proteins. We demonstrate that octameric NSP2 binds structured RNAs with high affinity, resulting in efficient intramolecular RNA helix disruption. Hexameric σNS oligomerizes into an octamer that binds two RNAs, yet it exhibits only limited RNA-unwinding activity compared to NSP2. Thus, the formation of intersegment RNA–RNA interactions is governed by both helix-unwinding capacity of the chaperones and stability of RNA structure. We propose that this protein-mediated RNA selection mechanism may underpin the high fidelity assembly of multi-segmented RNA genomes in Reoviridae

Visualization of membrane protein domains by cryo-electron microscopy of dengue virus

Improved technology for reconstructing cryo-electron microscopy (cryo-EM) images has now made it possible to determine secondary structural features of membrane proteins in enveloped viruses. The structure of mature dengue virus particles was determined to a resolution of 9.5 Å by cryo-EM and image reconstruction techniques, establishing the secondary structural disposition of the 180 envelope (E) and 180 membrane (M) proteins in the lipid envelope. The ɑ-helical 'stem' regions of the E molecules, as well as part of the N-terminal section of the M proteins, are buried in the outer leaflet of the viral membrane. The 'anchor' regions of E and the M proteins each form antiparallel E-E and M-M transmembrane alpha-helices, leaving their C termini on the exterior of the viral membrane, consistent with the predicted topology of the unprocessed polyprotein. This is one of only a few determinations of the disposition of transmembrane proteins in situ and shows that the nucleocapsid core and envelope proteins do not have a direct interaction in the mature virus

Erratum: Genomic RNA folding mediates assembly of human parechovirus.

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Insights into Kinesin-1 Activation from the Crystal Structure of KLC2 Bound to JIP3

Kinesin-1 transports numerous cellular cargoes along microtubules. The kinesin-1 light chain (KLC) mediates cargo binding and regulates kinesin-1 motility. To investigate the molecular basis for kinesin-1 recruitment and activation by cargoes, we solved the crystal structure of the KLC2 tetratricopeptide repeat (TPR) domain bound to the cargo JIP3. This, combined with biophysical and molecular evolutionary analyses, reveals a kinesin-1 cargo binding site, located on KLC TPR1, which is conserved in homologs from sponges to humans. In the complex, JIP3 crosslinks two KLC2 TPR domains via their TPR1s. We show that TPR1 forms a dimer interface that mimics JIP3 binding in all crystal structures of the unbound KLC TPR domain. We propose that cargo-induced dimerization of the KLC TPR domains via TPR1 is a general mechanism for activating kinesin-1. We relate this to activation by tryptophan-acidic cargoes, explaining how different cargoes activate kinesin-1 through related molecular mechanisms