Phosphorylation at the Homotypic Interface Regulates Nucleoprotein Oligomerization and Assembly of the Influenza Virus Replication Machinery

The maintenance of skeletal muscle mass is essential for health and quality of life. It is well recognized that maximal-intensity contractions, such as those which occur during resistance exercise, promote an increase in muscle mass. Yet, the molecules that sense the mechanical information and convert it into the signalling events (e.g. phosphorylation) that drive the increase in muscle mass remain undefined. Here we describe a phosphoproteomics workflow to examine the effects of electrically evoked maximal-intensity contractions (MICs) on protein phosphorylation in mouse skeletal muscle. While a preliminary phosphoproteomics experiment successfully identified a number of MIC-regulated phosphorylation events, a large proportion of these identifications were present on highly abundant myofibrillar proteins. We subsequently incorporated a centrifugation-based fractionation step to deplete the highly abundant myofibrillar proteins and performed a second phosphoproteomics experiment. In total, we identified 5983 unique phosphorylation sites of which 663 were found to be regulated by MIC. GO term enrichment, phosphorylation motif analyses, and kinase-substrate predictions indicated that the MIC-regulated phosphorylation sites were chiefly modified by mTOR, as well as multiple isoforms of the MAPKs and CAMKs. Moreover, a high proportion of the regulated phosphorylation sites were found on proteins that are associated with the Z-disc, with over 74% of the Z-disc proteins experiencing robust changes in phosphorylation. Finally, our analyses revealed that the phosphorylation state of two Z-disc kinases (striated muscle-specific serine/threonine protein kinase and obscurin) was dramatically altered by MIC, and we propose ways these kinases could play a fundamental role in skeletal muscle mechanotransduction.

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“…; Mondal et al . ; Riley & Coon, ). Importantly, skeletal muscles are composed of abundant contractile proteins (e.g.…”

Section: Introductionmentioning

confidence: 99%

A map of the phosphoproteomic alterations that occur after a bout of maximal‐intensity contractions

Potts

McNally

Blanco

et al. 2017

The Journal of Physiology

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“…Phosphorylation can dramatically change the conformation of proteins, thus controlling access to catalytically active sites and regulating protein interactions by obscuring or revealing interacting domains, particularly with respect to kinases (35,36). Phosphorylation of viral proteins is increasingly recognized as a regulator of RNA virus replication (37)(38)(39).…”

Section: Discussionmentioning

confidence: 99%

Phosphorylation cascade regulates the formation and maturation of rotaviral replication factories

Criglar

Anish

et al. 2018

Proc. Natl. Acad. Sci. U.S.A.

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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...

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“…RNP assembly in the presence or absence of ANP32A was measured as previously described (Mondal et al, 2015). To test protein interactions during infection, wild-type A549 cells or those stably expressing ANP32A were inoculated with WSN PB2-FLAG or PB2-K627E-FLAG (MOI 0.2).…”

Section: Star⋆methodsmentioning

confidence: 99%

Differential Splicing of ANP32A in Birds Alters Its Ability to Stimulate RNA Synthesis by Restricted Influenza Polymerase

2018

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SUMMARY Adaptation of viruses to their hosts can result in specialization and a restricted host range. Species specific polymorphisms in the influenza virus polymerase restrict its host range during transmission from birds to mammals. ANP32A was recently identified as a cellular co-factor affecting polymerase adaption and activity. Avian influenza polymerases require ANP32A containing an insertion resulting from an exon duplication uniquely encoded in birds. Here we find that natural splice variants surrounding this exon create avian ANP32A proteins with distinct effects on polymerase activity. We demonstrate species-independent direct interactions between all ANP32A variants and the PB2 polymerase subunit. This interaction is enhanced in the presence of viral genomic RNA. In contrast, only avian ANP32A restored ribonucleoprotein complex assembly for a restricted polymerase by enhancing RNA synthesis. Our data suggest that ANP32A splicing variation among birds differentially affects viral replication, polymerase adaption, and the potential of avian hosts to be reservoirs.

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Phosphorylation at the Homotypic Interface Regulates Nucleoprotein Oligomerization and Assembly of the Influenza Virus Replication Machinery

Cited by 55 publications

References 68 publications

A map of the phosphoproteomic alterations that occur after a bout of maximal‐intensity contractions

A map of the phosphoproteomic alterations that occur after a bout of maximal‐intensity contractions

Phosphorylation cascade regulates the formation and maturation of rotaviral replication factories

Differential Splicing of ANP32A in Birds Alters Its Ability to Stimulate RNA Synthesis by Restricted Influenza Polymerase

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