Unravelling the Tripartite Interactions Among Hepatitis E Virus RNA, miR-140 and hnRNP K

• Published in: Journal of molecular biology Vol. 435; no. 10; p. 168050
• Main Authors: Patil, Rajashree, Salunke, Pooja, Karpe, Yogesh A.
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier Ltd 15-05-2023
• Subjects: Hepatitis E virus; Hepatitis E virus - genetics; Heterogeneous-Nuclear Ribonucleoprotein K - genetics; Heterogeneous-Nuclear Ribonucleoprotein K - metabolism; hnRNP K; Humans; microRNA; MicroRNAs - genetics; MicroRNAs - metabolism; miR-140; RNA, Viral - metabolism; Virus Replication - genetics; microRNA; miR-140; hnRNP K; Hepatitis E virus
• ISSN: 0022-2836; 1089-8638
• DOI: 10.1016/j.jmb.2023.168050

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•The miR-140 binding site is highly conserved among all HEV genotypes and related RNA viruses.•miR-140 interacts directly at conserved miR-140 binding site on HEV genome.•Host factor-miR-140 and intact miR-140 binding site on HEV genome is obligatory for HEV replication.•Other than Ago2, miR-140 exhibits novel interaction with hnRNP K at the endogenous level as well as during HEV replication.•An intact miR-140 binding site forms a scaffold for the recruitment of hnRNP K, only in the presence of miR-140.
In the present investigation, we have identified the functional significance of the highly conserved miR-140 binding site on the Hepatitis E Virus (HEV) genome. Multiple sequence alignment of the viral genome sequences along with RNA folding prediction indicated that the putative miR-140 binding site has significant conservation for sequence and secondary RNA structure among HEV genotypes. Site-directed mutagenesis and reporter assays indicated that an intact sequence of the miR-140 binding site is essential for HEV translation. Provision of mutant miR-140 oligos carrying same mutation as on mutant HEV successfully rescued mutant HEV replication. In vitro cell-based assays with modified oligos proved that host factor-miR-140 is a critical requirement for HEV replication. Biotinylated RNA pulldown and RNA immunoprecipitation assays proved that the predicted secondary RNA structure of the miR-140 binding site allows the recruitment of hnRNP K, which is a key protein of the HEV replication complex. We predicted the model from the obtained results that the miR-140 binding site can serve as a platform for recruitment of hnRNP K and other proteins of HEV replication complex only in the presence of miR-140.