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

dc.contributor.authorPatel, Ankoor
dc.contributor.authorTreffers, Emmely E.
dc.contributor.authorMeier, Markus
dc.contributor.authorPatel, Trushar R.
dc.contributor.authorStetefeld, Jörg
dc.contributor.authorSnijder, Eric J.
dc.contributor.authorMark, Brian L.
dc.date.accessioned2023-02-17T21:29:23Z
dc.date.available2023-02-17T21:29:23Z
dc.date.issued2020
dc.descriptionOpen access article. Creative Commons Attribution 4.0 International (CC BY 4.0) appliesen_US
dc.description.abstractProgrammed 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.en_US
dc.identifier.citationPatel, A., Treffers, E. E., Meier, M., Patel, T. R., Stetefeld, J., Snijder, E. J., & Mark, B. L. (2020). Molecular characterization of the RNA-protein complex directing -2/-1 programmed ribosomal frameshifting during arterivirus replicase expression. Journal of Biological Chemistry, 295(52), 17904-17921. https://doi.org/10.1074/jbc.RA120.016105en_US
dc.identifier.urihttps://hdl.handle.net/10133/6437
dc.language.isoen_USen_US
dc.publisherAmerican Society for Biochemistry and Molecular Biologyen_US
dc.publisher.departmentDepartment of Chemistry and Biochemistry
dc.publisher.facultyArts and Science
dc.publisher.institutionUniversity of Manitoba
dc.publisher.institutionLeiden University Medical Center
dc.publisher.institutionUniversity of Lethbridge
dc.publisher.urlhttps://doi.org/10.1074/jbc.RA120.016105
dc.subjectPRRSVen_US
dc.subjectPoly(C)-binding proteinen_US
dc.subjectPCPB2en_US
dc.subjectNonstructural proteinen_US
dc.subjectSedimentation velocityen_US
dc.subjectRibosomeen_US
dc.subjectTranslationen_US
dc.subjectStructural biologyen_US
dc.subjectViral replicationen_US
dc.subjectRNA virusen_US
dc.subjectRibosome functionen_US
dc.subjectNsp 1betaen_US
dc.subjectPCBPen_US
dc.subjectNidovirus
dc.subject.lcshNidoviruses
dc.subject.lcshCarrier proteins
dc.subject.lcshSmall-angle x-ray scattering
dc.subject.lcshRNA
dc.subject.lcshVirology
dc.subject.lcshRibosomes
dc.subject.lcshViruses--Reproduction
dc.subject.lcshRNA viruses
dc.subject.lcshRibosomes
dc.titleMolecular characterization of the RNA-protein complex directing -2/-1 programmed ribosomal frameshifting during arterivirus replilcase expressionen_US
dc.typeArticleen_US
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