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 RNA viruses, similar phosphorylation-dependent mechanisms may exist for other virus pathogens that require cytoplasmic virus factories for replication.M any viral pathogens, including both DNA viruses (poxviruses) and RNA viruses [rotavirus (RV), orthoreovirus, hepatitis C virus, dengue virus, Marburg virus] replicate in cytoplasmic compartments that are physical scaffolds composed of both viral and cellular proteins (1-3). These compartments, excluded from the rest of the cytoplasm, are sites where the virus genome is replicated and progeny particles are assembled. Such replication compartments are alternately known as virus factories, virus replication centers or complexes, and viroplasms. Exactly how these virus factories form is largely unknown, so it is intriguing to consider that there may be common mechanisms that initiate their formation and regulate their assembly.RV-induced severe, dehydrating gastroenteritis remains a leading cause of morbidity and mortality in infants and children less than 5 y of age worldwide and accounts for greater than 200,000 deaths annually (4). To begin to understand the mechanism of replication factory assembly, we studied the assembly of RV replication complexes known as viroplasms. Viroplasm formation requires the interaction of two nonstructural proteins, NSP2 and NSP5, and expression of NSP2 with NSP5 in cells lacking all other viral proteins results in the formation of viroplasm-like structures (VLS) (5). Loss of either NSP2 or NSP5 abolishes viroplasm formation and rotavirus replication (6-9). While information about the individual characteristics of NSP2 and NSP5 and some knowledge of how they physically interact are available, exactly...

Conserved Rotavirus NSP5 and VP2 Domains Interact and Affect Viroplasm

“…Our conclusion then provides additional insights into the molecular mechanism previously proposed (44,58) for the formation of viroplasms and subsequent NSP5 hyperphosphorylation. This mechanistic interpretation is consistent with previous observations that viroplasm formation requires a phosphorylation cascade triggered by NSP2, which depends on CK1-alpha (43,44,59). It also is consistent with the fact that NSP5 phosphorylation on S67 by CK1-alpha is a step after the interaction with VP2 (7 and this study) and NSP2 (19,44,58).…”

A Calcium Sensor Discovered in Bluetongue Virus Nonstructural Protein 2 Is Critical for Virus Replication

“…Specifically, different members of the Reoviridae family also form similar VIB-like structures (referred to as viral factories or viroplasms in rotavirus/reovirus), although each have their own unique features (33,34). One of the important regulatory factors critical for VIB/viroplasm assembly is phosphorylation of viral proteins, such as NSP2 of rotavirus and NS2 of BTV, which controls self-oligomerization of VIB-forming viral proteins (2,33). Thus, our study provides further insights on the mechanism behind the regulation of this phosphorylation of VIB-forming protein NS2 in BTV.…”

Differential Localization of Structural and Non-Structural Proteins during the Bluetongue Virus Replication Cycle

“…While it has been established that the virus factories or VIBs utilized by members of the Reoviridae family are responsible for replication and the assembly of virus particles [30][31][32], crucial stages of these processes remain ill-defined. This includes the egress of virus cores from VIBs, and in the context of BTV, their subsequent associating with the outer capsid proteins VP5 and VP2 to generate mature infectious virus particles that are ready for egress from the cell.…”