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      Molecular characterization of the RNA-protein complex directing −2/−1 programmed ribosomal frameshifting during arterivirus replicase expression

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          Abstract

          Programmed ribosomal frameshifting (PRF) is a mechanism used by arteriviruses like porcine reproductive and respiratory syndrome virus (PRRSV) to generate multiple proteins from overlapping reading frames within its RNA genome. PRRSV employs −1 PRF directed by RNA secondary and tertiary structures within its viral genome (canonical PRF), as well as a noncanonical −1 and −2 PRF that are stimulated by the interactions of PRRSV nonstructural protein 1β (nsp1β) and host protein poly(C)-binding protein (PCBP) 1 or 2 with the viral genome. Together, nsp1β and one of the PCBPs act as transactivators that bind a C-rich motif near the shift site to stimulate −1 and −2 PRF, thereby enabling the ribosome to generate two frameshift products that are implicated in viral immune evasion. How nsp1β and PCBP associate with the viral RNA genome remains unclear. Here, we describe the purification of the nsp1β:PCBP2:viral RNA complex on a scale sufficient for structural analysis using small-angle X-ray scattering and stochiometric analysis by analytical ultracentrifugation. The proteins associate with the RNA C-rich motif as a 1:1:1 complex. The monomeric form of nsp1β within the complex differs from previously reported homodimer identified by X-ray crystallography. Functional analysis of the complex via mutational analysis combined with RNA-binding assays and cell-based frameshifting reporter assays reveal a number of key residues within nsp1β and PCBP2 that are involved in complex formation and function. Our results suggest that nsp1β and PCBP2 both interact directly with viral RNA during formation of the complex to coordinate this unusual PRF mechanism.

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          Matplotlib: A 2D Graphics Environment

           John Hunter (2007)
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            The EMBL-EBI search and sequence analysis tools APIs in 2019

            Abstract The EMBL-EBI provides free access to popular bioinformatics sequence analysis applications as well as to a full-featured text search engine with powerful cross-referencing and data retrieval capabilities. Access to these services is provided via user-friendly web interfaces and via established RESTful and SOAP Web Services APIs (https://www.ebi.ac.uk/seqdb/confluence/display/JDSAT/EMBL-EBI+Web+Services+APIs+-+Data+Retrieval). Both systems have been developed with the same core principles that allow them to integrate an ever-increasing volume of biological data, making them an integral part of many popular data resources provided at the EMBL-EBI. Here, we describe the latest improvements made to the frameworks which enhance the interconnectivity between public EMBL-EBI resources and ultimately enhance biological data discoverability, accessibility, interoperability and reusability.
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              Inference of macromolecular assemblies from crystalline state.

              We discuss basic physical-chemical principles underlying the formation of stable macromolecular complexes, which in many cases are likely to be the biological units performing a certain physiological function. We also consider available theoretical approaches to the calculation of macromolecular affinity and entropy of complexation. The latter is shown to play an important role and make a major effect on complex size and symmetry. We develop a new method, based on chemical thermodynamics, for automatic detection of macromolecular assemblies in the Protein Data Bank (PDB) entries that are the results of X-ray diffraction experiments. As found, biological units may be recovered at 80-90% success rate, which makes X-ray crystallography an important source of experimental data on macromolecular complexes and protein-protein interactions. The method is implemented as a public WWW service.
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                Author and article information

                Contributors
                Journal
                J Biol Chem
                J Biol Chem
                The Journal of Biological Chemistry
                American Society for Biochemistry and Molecular Biology
                0021-9258
                1083-351X
                13 January 2021
                25 December 2020
                13 January 2021
                : 295
                : 52
                : 17904-17921
                Affiliations
                [1 ]Department of Microbiology, University of Manitoba, Winnipeg, Manitoba, Canada
                [2 ]Molecular Virology Laboratory, Department of Medical Microbiology, Leiden University Medical Center, Leiden, The Netherlands
                [3 ]Department of Chemistry, University of Manitoba, Winnipeg, Manitoba, Canada
                [4 ]Alberta RNA Research and Training Institute, Department of Chemistry and Biochemistry, University of Lethbridge, Lethbridge, Alberta, Canada
                Author notes
                [* ]For correspondence: Brian L. Mark brian.mark@ 123456umanitoba.ca
                Article
                S0021-9258(17)50671-7
                10.1074/jbc.RA120.016105
                7939443
                33127640
                © 2020 © 2020 Patel et al.

                This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

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