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Title: Phosphorylation cascade regulates the formation and maturation of rotaviral replication factories

Abstract

The rotavirus (RV) genome is replicated and packaged into virus progeny in cytoplasmic inclusions called viroplasms, which require interactions between RV nonstructural proteins NSP2 and NSP5. How viroplasms form remains unknown. We previously found two forms of NSP2 in RV-infected cells: a cytoplasmically dispersed dNSP2, which interacts with hypophosphorylated NSP5; and a viroplasm-specific vNSP2, which interacts with hyperphosphorylated NSP5. Other studies report that CK1α, a ubiquitous cellular kinase, hyperphosphorylates NSP5, but requires NSP2 for reasons that are unclear. Here we show that silencing CK1α in cells before RV infection resulted in ( i ) >90% decrease in RV replication, ( ii ) disrupted vNSP2 and NSP5 interaction, ( iii ) dispersion of vNSP2 throughout the cytoplasm, and ( iv ) reduced vNSP2 protein levels. Together, these data indicate that CK1α directly affects NSP2. Accordingly, an in vitro kinase assay showed that CK1α phosphorylates serine 313 of NSP2 and triggers NSP2 octamers to form a lattice structure as demonstrated by crystallographic analysis. Additionally, a dual-specificity autokinase activity for NSP2 was identified and confirmed by mass spectrometry. Together, our studies show that phosphorylation of NSP2 involving CK1α controls viroplasm assembly. Considering that CK1α plays a role in the replication of other RNAmore » viruses, similar phosphorylation-dependent mechanisms may exist for other virus pathogens that require cytoplasmic virus factories for replication.« less

Authors:
; ; ; ; ; ;
Publication Date:
Sponsoring Org.:
USDOE
OSTI Identifier:
1484388
Grant/Contract Number:  
AC02-05CH11231
Resource Type:
Journal Article: Published Article
Journal Name:
Proceedings of the National Academy of Sciences of the United States of America
Additional Journal Information:
Journal Name: Proceedings of the National Academy of Sciences of the United States of America Journal Volume: 115 Journal Issue: 51; Journal ID: ISSN 0027-8424
Publisher:
Proceedings of the National Academy of Sciences
Country of Publication:
United States
Language:
English

Citation Formats

Criglar, Jeanette M., Anish, Ramakrishnan, Hu, Liya, Crawford, Sue E., Sankaran, Banumathi, Prasad, B. V. Venkataram, and Estes, Mary K. Phosphorylation cascade regulates the formation and maturation of rotaviral replication factories. United States: N. p., 2018. Web. doi:10.1073/pnas.1717944115.
Criglar, Jeanette M., Anish, Ramakrishnan, Hu, Liya, Crawford, Sue E., Sankaran, Banumathi, Prasad, B. V. Venkataram, & Estes, Mary K. Phosphorylation cascade regulates the formation and maturation of rotaviral replication factories. United States. doi:10.1073/pnas.1717944115.
Criglar, Jeanette M., Anish, Ramakrishnan, Hu, Liya, Crawford, Sue E., Sankaran, Banumathi, Prasad, B. V. Venkataram, and Estes, Mary K. Mon . "Phosphorylation cascade regulates the formation and maturation of rotaviral replication factories". United States. doi:10.1073/pnas.1717944115.
@article{osti_1484388,
title = {Phosphorylation cascade regulates the formation and maturation of rotaviral replication factories},
author = {Criglar, Jeanette M. and Anish, Ramakrishnan and Hu, Liya and Crawford, Sue E. and Sankaran, Banumathi and Prasad, B. V. Venkataram and Estes, Mary K.},
abstractNote = {The rotavirus (RV) genome is replicated and packaged into virus progeny in cytoplasmic inclusions called viroplasms, which require interactions between RV nonstructural proteins NSP2 and NSP5. How viroplasms form remains unknown. We previously found two forms of NSP2 in RV-infected cells: a cytoplasmically dispersed dNSP2, which interacts with hypophosphorylated NSP5; and a viroplasm-specific vNSP2, which interacts with hyperphosphorylated NSP5. Other studies report that CK1α, a ubiquitous cellular kinase, hyperphosphorylates NSP5, but requires NSP2 for reasons that are unclear. Here we show that silencing CK1α in cells before RV infection resulted in ( i ) >90% decrease in RV replication, ( ii ) disrupted vNSP2 and NSP5 interaction, ( iii ) dispersion of vNSP2 throughout the cytoplasm, and ( iv ) reduced vNSP2 protein levels. Together, these data indicate that CK1α directly affects NSP2. Accordingly, an in vitro kinase assay showed that CK1α phosphorylates serine 313 of NSP2 and triggers NSP2 octamers to form a lattice structure as demonstrated by crystallographic analysis. Additionally, a dual-specificity autokinase activity for NSP2 was identified and confirmed by mass spectrometry. Together, our studies show that phosphorylation of NSP2 involving CK1α controls viroplasm assembly. Considering that CK1α plays a role in the replication of other RNA viruses, similar phosphorylation-dependent mechanisms may exist for other virus pathogens that require cytoplasmic virus factories for replication.},
doi = {10.1073/pnas.1717944115},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
number = 51,
volume = 115,
place = {United States},
year = {Mon Dec 03 00:00:00 EST 2018},
month = {Mon Dec 03 00:00:00 EST 2018}
}

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Works referenced in this record:

The CCP4 suite programs for protein crystallography
journal, September 1994


PHENIX: a comprehensive Python-based system for macromolecular structure solution
journal, January 2010

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