Cargando…

SARS-CoV-2 nucleocapsid protein undergoes liquid-liquid phase separation stimulated by RNA and partitions into phases of human ribonucleoproteins.

Tightly packed complexes of nucleocapsid protein and genomic RNA form the core of viruses and may assemble within viral factories, dynamic compartments formed within the host cells. Here, we examine the possibility that the multivalent RNA-binding nucleocapsid protein (N) from the severe acute respi...

Descripción completa

Detalles Bibliográficos
Autores principales: Perdikari, Theodora Myrto, Murthy, Anastasia C., Ryan, Veronica H., Watters, Scott, Naik, Mandar T., Fawzi, Nicolas L.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Cold Spring Harbor Laboratory 2020
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7302208/
https://www.ncbi.nlm.nih.gov/pubmed/32577653
http://dx.doi.org/10.1101/2020.06.09.141101
_version_ 1783547802579632128
author Perdikari, Theodora Myrto
Murthy, Anastasia C.
Ryan, Veronica H.
Watters, Scott
Naik, Mandar T.
Fawzi, Nicolas L.
author_facet Perdikari, Theodora Myrto
Murthy, Anastasia C.
Ryan, Veronica H.
Watters, Scott
Naik, Mandar T.
Fawzi, Nicolas L.
author_sort Perdikari, Theodora Myrto
collection PubMed
description Tightly packed complexes of nucleocapsid protein and genomic RNA form the core of viruses and may assemble within viral factories, dynamic compartments formed within the host cells. Here, we examine the possibility that the multivalent RNA-binding nucleocapsid protein (N) from the severe acute respiratory syndrome coronavirus (SARS-CoV-2) compacts RNA via protein-RNA liquid-liquid phase separation (LLPS) and that N interactions with host RNA-binding proteins are mediated by phase separation. To this end, we created a construct expressing recombinant N fused to a N-terminal maltose binding protein tag which helps keep the oligomeric N soluble for purification. Using in vitro phase separation assays, we find that N is assembly-prone and phase separates avidly. Phase separation is modulated by addition of RNA and changes in pH and is disfavored at high concentrations of salt. Furthermore, N enters into in vitro phase separated condensates of full-length human hnRNPs (TDP-43, FUS, and hnRNPA2) and their low complexity domains (LCs). However, N partitioning into the LC of FUS, but not TDP-43 or hnRNPA2, requires cleavage of the solubilizing MBP fusion. Hence, LLPS may be an essential mechanism used for SARS-CoV-2 and other RNA viral genome packing and host protein co-opting, functions necessary for viral replication and hence infectivity.
format Online
Article
Text
id pubmed-7302208
institution National Center for Biotechnology Information
language English
publishDate 2020
publisher Cold Spring Harbor Laboratory
record_format MEDLINE/PubMed
spelling pubmed-73022082020-06-23 SARS-CoV-2 nucleocapsid protein undergoes liquid-liquid phase separation stimulated by RNA and partitions into phases of human ribonucleoproteins. Perdikari, Theodora Myrto Murthy, Anastasia C. Ryan, Veronica H. Watters, Scott Naik, Mandar T. Fawzi, Nicolas L. bioRxiv Article Tightly packed complexes of nucleocapsid protein and genomic RNA form the core of viruses and may assemble within viral factories, dynamic compartments formed within the host cells. Here, we examine the possibility that the multivalent RNA-binding nucleocapsid protein (N) from the severe acute respiratory syndrome coronavirus (SARS-CoV-2) compacts RNA via protein-RNA liquid-liquid phase separation (LLPS) and that N interactions with host RNA-binding proteins are mediated by phase separation. To this end, we created a construct expressing recombinant N fused to a N-terminal maltose binding protein tag which helps keep the oligomeric N soluble for purification. Using in vitro phase separation assays, we find that N is assembly-prone and phase separates avidly. Phase separation is modulated by addition of RNA and changes in pH and is disfavored at high concentrations of salt. Furthermore, N enters into in vitro phase separated condensates of full-length human hnRNPs (TDP-43, FUS, and hnRNPA2) and their low complexity domains (LCs). However, N partitioning into the LC of FUS, but not TDP-43 or hnRNPA2, requires cleavage of the solubilizing MBP fusion. Hence, LLPS may be an essential mechanism used for SARS-CoV-2 and other RNA viral genome packing and host protein co-opting, functions necessary for viral replication and hence infectivity. Cold Spring Harbor Laboratory 2020-06-10 /pmc/articles/PMC7302208/ /pubmed/32577653 http://dx.doi.org/10.1101/2020.06.09.141101 Text en http://creativecommons.org/licenses/by/4.0/It is made available under a CC-BY 4.0 International license (http://creativecommons.org/licenses/by/4.0/) .
spellingShingle Article
Perdikari, Theodora Myrto
Murthy, Anastasia C.
Ryan, Veronica H.
Watters, Scott
Naik, Mandar T.
Fawzi, Nicolas L.
SARS-CoV-2 nucleocapsid protein undergoes liquid-liquid phase separation stimulated by RNA and partitions into phases of human ribonucleoproteins.
title SARS-CoV-2 nucleocapsid protein undergoes liquid-liquid phase separation stimulated by RNA and partitions into phases of human ribonucleoproteins.
title_full SARS-CoV-2 nucleocapsid protein undergoes liquid-liquid phase separation stimulated by RNA and partitions into phases of human ribonucleoproteins.
title_fullStr SARS-CoV-2 nucleocapsid protein undergoes liquid-liquid phase separation stimulated by RNA and partitions into phases of human ribonucleoproteins.
title_full_unstemmed SARS-CoV-2 nucleocapsid protein undergoes liquid-liquid phase separation stimulated by RNA and partitions into phases of human ribonucleoproteins.
title_short SARS-CoV-2 nucleocapsid protein undergoes liquid-liquid phase separation stimulated by RNA and partitions into phases of human ribonucleoproteins.
title_sort sars-cov-2 nucleocapsid protein undergoes liquid-liquid phase separation stimulated by rna and partitions into phases of human ribonucleoproteins.
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7302208/
https://www.ncbi.nlm.nih.gov/pubmed/32577653
http://dx.doi.org/10.1101/2020.06.09.141101
work_keys_str_mv AT perdikaritheodoramyrto sarscov2nucleocapsidproteinundergoesliquidliquidphaseseparationstimulatedbyrnaandpartitionsintophasesofhumanribonucleoproteins
AT murthyanastasiac sarscov2nucleocapsidproteinundergoesliquidliquidphaseseparationstimulatedbyrnaandpartitionsintophasesofhumanribonucleoproteins
AT ryanveronicah sarscov2nucleocapsidproteinundergoesliquidliquidphaseseparationstimulatedbyrnaandpartitionsintophasesofhumanribonucleoproteins
AT wattersscott sarscov2nucleocapsidproteinundergoesliquidliquidphaseseparationstimulatedbyrnaandpartitionsintophasesofhumanribonucleoproteins
AT naikmandart sarscov2nucleocapsidproteinundergoesliquidliquidphaseseparationstimulatedbyrnaandpartitionsintophasesofhumanribonucleoproteins
AT fawzinicolasl sarscov2nucleocapsidproteinundergoesliquidliquidphaseseparationstimulatedbyrnaandpartitionsintophasesofhumanribonucleoproteins