Cargando…

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

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 w...

Descripción completa

Detalles Bibliográficos
Autores principales: Patel, Ankoor, Treffers, Emmely E., Meier, Markus, Patel, Trushar R., Stetefeld, Jörg, Snijder, Eric J., Mark, Brian L.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: American Society for Biochemistry and Molecular Biology 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7939443/
https://www.ncbi.nlm.nih.gov/pubmed/33127640
http://dx.doi.org/10.1074/jbc.RA120.016105
_version_ 1783661750241984512
author Patel, Ankoor
Treffers, Emmely E.
Meier, Markus
Patel, Trushar R.
Stetefeld, Jörg
Snijder, Eric J.
Mark, Brian L.
author_facet Patel, Ankoor
Treffers, Emmely E.
Meier, Markus
Patel, Trushar R.
Stetefeld, Jörg
Snijder, Eric J.
Mark, Brian L.
author_sort Patel, Ankoor
collection PubMed
description 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.
format Online
Article
Text
id pubmed-7939443
institution National Center for Biotechnology Information
language English
publishDate 2021
publisher American Society for Biochemistry and Molecular Biology
record_format MEDLINE/PubMed
spelling pubmed-79394432021-06-08 Molecular characterization of the RNA-protein complex directing −2/−1 programmed ribosomal frameshifting during arterivirus replicase expression Patel, Ankoor Treffers, Emmely E. Meier, Markus Patel, Trushar R. Stetefeld, Jörg Snijder, Eric J. Mark, Brian L. J Biol Chem Editors' Picks 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. American Society for Biochemistry and Molecular Biology 2021-01-13 /pmc/articles/PMC7939443/ /pubmed/33127640 http://dx.doi.org/10.1074/jbc.RA120.016105 Text en © 2020 © 2020 Patel et al. https://creativecommons.org/licenses/by/4.0/This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
spellingShingle Editors' Picks
Patel, Ankoor
Treffers, Emmely E.
Meier, Markus
Patel, Trushar R.
Stetefeld, Jörg
Snijder, Eric J.
Mark, Brian L.
Molecular characterization of the RNA-protein complex directing −2/−1 programmed ribosomal frameshifting during arterivirus replicase expression
title Molecular characterization of the RNA-protein complex directing −2/−1 programmed ribosomal frameshifting during arterivirus replicase expression
title_full Molecular characterization of the RNA-protein complex directing −2/−1 programmed ribosomal frameshifting during arterivirus replicase expression
title_fullStr Molecular characterization of the RNA-protein complex directing −2/−1 programmed ribosomal frameshifting during arterivirus replicase expression
title_full_unstemmed Molecular characterization of the RNA-protein complex directing −2/−1 programmed ribosomal frameshifting during arterivirus replicase expression
title_short Molecular characterization of the RNA-protein complex directing −2/−1 programmed ribosomal frameshifting during arterivirus replicase expression
title_sort molecular characterization of the rna-protein complex directing −2/−1 programmed ribosomal frameshifting during arterivirus replicase expression
topic Editors' Picks
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7939443/
https://www.ncbi.nlm.nih.gov/pubmed/33127640
http://dx.doi.org/10.1074/jbc.RA120.016105
work_keys_str_mv AT patelankoor molecularcharacterizationofthernaproteincomplexdirecting21programmedribosomalframeshiftingduringarterivirusreplicaseexpression
AT treffersemmelye molecularcharacterizationofthernaproteincomplexdirecting21programmedribosomalframeshiftingduringarterivirusreplicaseexpression
AT meiermarkus molecularcharacterizationofthernaproteincomplexdirecting21programmedribosomalframeshiftingduringarterivirusreplicaseexpression
AT pateltrusharr molecularcharacterizationofthernaproteincomplexdirecting21programmedribosomalframeshiftingduringarterivirusreplicaseexpression
AT stetefeldjorg molecularcharacterizationofthernaproteincomplexdirecting21programmedribosomalframeshiftingduringarterivirusreplicaseexpression
AT snijderericj molecularcharacterizationofthernaproteincomplexdirecting21programmedribosomalframeshiftingduringarterivirusreplicaseexpression
AT markbrianl molecularcharacterizationofthernaproteincomplexdirecting21programmedribosomalframeshiftingduringarterivirusreplicaseexpression