Abstract
Genome replication and virion assembly of segmented RNA viruses are highly coordinated events, tightly regulated by sequence and structural elements in the UTRs of viral RNA. This process is poorly defined and likely requires the participation of host proteins in concert with viral proteins. In this study, we employed a proteomics-based approach, named RNA-protein interaction detection (RaPID), to comprehensively screen for host proteins that bind to a conserved motif within the rotavirus (RV) 3' terminus. Using this assay, we identified ATP5B, a core subunit of the mitochondrial ATP synthase, as having high affinity to the RV 3'UTR consensus sequences. During RV infection, ATP5B bound to the RV 3'UTR and co-localized with viral RNA and viroplasm. Functionally, siRNA-mediated genetic depletion of ATP5B or other ATP synthase subunits such as ATP5A1 and ATP5O reduced the production of infectious viral progeny without significant alteration of intracellular viral RNA levels or RNA translation. Chemical inhibition of ATP synthase diminished RV yield in both conventional cell culture and in human intestinal enteroids, indicating that ATP5B positively regulates late-stage RV maturation in primary intestinal epithelial cells. Collectively, our results shed light on the role of host proteins in RV genome assembly and particle formation and identify ATP5B as a novel pro-RV RNA-binding protein, contributing to our understanding of how host ATP synthases may galvanize virus growth and pathogenesis.
Highlights
Genome replication and virion assembly of segmented RNA viruses are highly coordinated events, tightly regulated by sequence and structural elements in the untranslated regions (UTRs) of viral RNA
Proteomic analysis reveals novel host proteins that interact with RV 3UTR consensus sequences
As a proof of principle, we first tested the interaction between a pair of positive controls: a 15-nucleotide UG–rich EDEN15 motif (UGUUUGUUUGUUUGU) that is reported to bind to the CUG triplet repeat RNA-binding protein 1 (CUG-BP1) [38]
Summary
Proteomic analysis reveals novel host proteins that interact with RV 3UTR consensus sequences. In a multiple-round infection assay, at 24 h postinfection, knockdown of ATP5B led to 40% reduction in the mRNA levels of NSP5, an RV gene transcript representative of intracellular RV RNA synthesis (Fig. 4C), and we observed an ϳ40% decrease in the amount of infectious RVs in the supernatant of the ATP5B knockdown cells (Fig. 4D), suggesting that ATP5B facilitates RV replication in host cells. We measured viral RNA by quantitative PCR and protein levels using polyclonal antibody that recognizes RV double-layered particles [44] at 8 h post-infection within a single virus replication cycle We found that both levels were comparable between control and ATP5B siRNA-transfected cells (Fig. 5, C and D). Our results support an important function of the ATP synthase complex in promoting efficient RV replication by enhancing viral genome assembly
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