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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 CK1?, a ubiquitous cellular kinase, hyperphosphorylates NSP5, but requires NSP2 for reasons that are unclear. Here we show that silencing CK1? 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 CK1? directly affects NSP2. Accordingly, an in vitro kinase assay showed that CK1? 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 CK1? controls viroplasm assembly. Considering that CK1? 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.

SUBMITTER: Criglar JM

PROVIDER: S-EPMC6304940 | biostudies-literature | 2018 Dec

REPOSITORIES: biostudies-literature

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Phosphorylation cascade regulates the formation and maturation of rotaviral replication factories.

Criglar Jeanette M JM Anish Ramakrishnan R Hu Liya L Crawford Sue E SE Sankaran Banumathi B Prasad B V Venkataram BVV Estes Mary K MK

Proceedings of the National Academy of Sciences of the United States of America 20181203 51

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 cellu ...[more]

PMID: 30509975

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Project description:Replication and packaging of the rotavirus genome occur in cytoplasmic compartments called viroplasms, which form during virus infection. These processes are orchestrated by yet-to-be-understood complex networks of interactions involving nonstructural proteins (NSPs) 2, 5, and 6 and structural proteins (VPs) 1, 2, 3, and 6. The multifunctional enzyme NSP2, an octamer with RNA binding activity, is critical for viroplasm formation with its binding partner, NSP5, and for genome replication/packaging through its interactions with replicating RNA, the viral polymerase VP1, and the inner core protein VP2. Using isothermal calorimetry, biolayer interferometry, and peptide array screening, we examined the interactions between NSP2, VP1, VP2, NSP5, and NSP6. These studies provide the first evidence that NSP2 can directly bind to VP1, VP2, and NSP6, in addition to the previously known binding to NSP5. The interacting sites identified from reciprocal peptide arrays were found to be in close proximity to the RNA template entry and double-stranded RNA (dsRNA) exit tunnels of VP1 and near the catalytic cleft and RNA-binding grooves of NSP2; these sites are consistent with the proposed role of NSP2 in facilitating dsRNA synthesis by VP1. Peptide screening of VP2 identified NSP2-binding sites in the regions close to the intersubunit junctions, suggesting that NSP2 binding could be a regulatory mechanism for preventing the premature self-assembly of VP2. The binding sites on NSP2 for NSP6 were found to overlap that of VP1, and the NSP5-binding sites overlap those of VP2 and VP1, suggesting that interaction of these proteins with NSP2 is likely spatially and/or temporally regulated.Replication and packaging of the rotavirus genome occur in cytoplasmic compartments called viroplasms that form during virus infection and are orchestrated by complex networks of interactions involving nonstructural proteins (NSPs) and structural proteins (VPs). A multifunctional RNA-binding NSP2 octamer with nucleotidyl phosphatase activity is central to viroplasm formation and RNA replication. Here we provide the first evidence that NSP2 can directly bind to VP1, VP2, and NSP6, in addition to the previously known binding to NSP5. The interacting sites identified from peptide arrays are consistent with the proposed role of NSP2 in facilitating dsRNA synthesis by VP1 and also point to NSP2's possible role in preventing the premature self-assembly of VP2 cores. Our findings lead us to propose that the NSP2 octamer with multiple enzymatic activities is a principal regulator of viroplasm formation, recruitment of viral proteins into the viroplasms, and possibly genome replication.

Dataset Information

Phosphorylation cascade regulates the formation and maturation of rotaviral replication factories.

Publications

Phosphorylation cascade regulates the formation and maturation of rotaviral replication factories.

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