A miRNA-responsive cell-free translation system facilitates isolation of hepatitis C virus miRNP complexes

Abstract

Micro(mi)RNAs are 21-to 23-nt RNAs that regulate multiple biological processes. In association with Argonaute (Ago) proteins and other factors that form the RNA-induced silencing complex (RISC), miRNAs typically bind mRNA 3 ′ untranslated regions (UTRs) and repress protein production through antagonizing translation and transcript stability. For a given mRNA-miRNA interaction, cis-acting RNA elements and trans-acting RNA-binding proteins (RBPs) may influence mRNA fate. This is particularly true of the hepatitis C virus (HCV) genome which interacts with miR-122, an abundant liver miRNA. miR-122 binding to HCV RNA considerably stimulates virus replication in cultured cells and primates, but the mechanism(s) and associated host factors required for enhancement of HCV replication have not been fully elucidated. We recapitulated miR-122-HCV RNA interactions in a cell-free translation system derived from cells that express miR-122. Specifically, lysates produced from HEK-293 cells that inducibly transcribe and process pri-miR-122 were characterized alongside those from isogenic cells lacking miR-122 expression. We observed a stimulatory effect of miR-122 on HCV reporter mRNAs in a manner that depended on expression of miR-122 and intact target sites within the HCV 5 ′ UTR. We took advantage of this system to affinity-purify miR-122-HCV RNP complexes. Similar to functional assays, we found that association of immobilized HCV internal ribosome entry site (IRES) RNA with endogenous Ago2 requires both miR-122 expression and intact miR-122 target sites in cis. This combined approach may be generalizable to affinity purification of miRNP complexes for selected target mRNAs, allowing identification of miRNP components and RBPs that may contribute to regulation.

hnRNP L and NF90 Interact with Hepatitis C Virus 5′-Terminal Untranslated RNA and Promote Efficient Replication

“…PCBP2 has been shown to bind to the SLI region of the HCV 5=UTR and to be required for HCV replication (14,20). IGF2BP1 has also been found to bind to the 5= sequence of HCV RNA and to modulate HCV IRES function (26,27). hnRNP L is a novel factor identified in our assay that has not been shown previously to bind the 5= end of HCV RNA.…”

Synthesis and Assembly of Hepatitis B Virus-Like Particles in a Pichia pastoris Cell-Free System

“…Developing new extracts is still of great interest, unlocking access to advantageous properties of the organisms from which they are derived. The available repertoire of developed CFPS extracts is still expanding and now includes HEK293 (Bradrick et al, 2013) Saccharomyces cerevisiae (Gan and Jewett, 2014), BY-2 tobacco cells (Buntru et al, 2014), Streptomyces venezuelae (Moore et al, 2017), Bacillus megaterium (Moore et al, 2018), Vibrio natriegens (Gan and Jewett, 2014) and most recently Pichia pastoris (Aw and Polizzi, 2019).…”

Messenger RNAs bearing tRNA-like features exemplified by interferon alfa 5 mRNA

“…It is possible that the similarly placed recognition elements for the human RNase P enzyme in these viral 5′ UTR domains including HCV, CSFV, BVDV and cellular IFNA s mRNA, together with key consensus sequence, structural similarities, and unconventional polarity of cleavage by RNase P reflect the presence of a similar RNA signal for the molecules that carry them. In these line of argument, the common presence of Aly protein bound to 5′ UTR of HCV [ 73 ] and to IFNA CAR [ 71 ] might provide a clue of the functional correlate for the structural mimicry found between viral and host elements. Nevertheless, further experiments will be needed to prove this hypothesis.…”