Hepatitis E virus (HEV) is a positive-sense single-stranded RNA virus that is a common cause of acute viral hepatitis worldwide. HEV-associated chronic hepatitis with extrahepatic symptoms has also been reported in immunocompromised patients [1]. So far, 8 HEV genotypes have been identified; however, only four of them are very well defined. HEV genotypes 1 and 2 cause infection in resource-limited countries like Africa, India, and Asia [2]. These two genotypes infect via the fecal-oral route and are frequently associated with extended outbreaks or sporadic cases, with a high death rate in pregnant women. HEV infection is responsible for 10,500 maternal deaths in Southern Asia per year [3]. HEV genotypes 3 and 4 infect people via animals and are common in both developing and developed countries 4, 5. Unfortunately, despite several investigations on HEV, the mechanisms of HEV-associated maternal mortality and HEV-associated extrahepatic clinical manifestations remain unknown. This is due to a lack of thorough information on the molecular mechanism of the HEV infection cycle, which is essential for the development of effective therapeutics and vaccines.

It is imperative to investigate the interactions of host components with HEV to comprehend the molecular mechanism of the HEV replication cycle. Because viruses bearing genomes with limited coding capability depend entirely on the host factors for energy, viral particle synthesis, viral genome stability, regulation, transport, and assembly, viruses are able to exploit the host’s cellular functions by relying on host cellular factors [6]. HEV is a positive-sense single-stranded RNA virus with a protein-coding ORFs, untranslated (UTR) region, 5'-m7G cap, and 3'polyA tail that can mimic mRNA following infection [7]. Cellular mRNAs always have dynamic RNA-RNA or RNA-protein interactions to accomplish the majority of a cell’s critical functions in all organisms [8]. Positive-sense RNA virus genomes are also associated with viral or host RNA Binding Proteins (RBPs) and other cellular RNAs. RBPs perform their functions either by interacting with specific sequences, patterns, or specific RNA structures. RNA structures, also known as cis-acting elements, are required for the replication of various RNA viruses 9, 10 Cellular RBPs regulate numerous aspects of positive-sense RNA virus replication [11]. Host RBPs influence the permissiveness of specific cell types as well as the pathophysiology of viral infection. Developing a deeper understanding of host RBP functions might provide new ways to control viral diseases and enhance our understanding of viral life cycles [12]. We previously reported the role of RBPs-heterogenous nuclear ribonuclear proteins (hnRNPs) in the HEV life cycle, including the proviral roles of hnRNP K [13]. In various gene regulation processes, hnRNP K is a highly conserved, abundantly expressed nucleocytoplasmic shuttling RBP. There are three repeats of the K homology domain in hnRNP K, with ∼ 65-70 highly conserved amino acid sequences. hnRNP K favorably binds to poly-C sequences of target RNA. As part of RNA virus replication, hnRNPK is involved in diverse functions like RNA processing, splicing, transcription, and translation 14, 15, 16 This study explores further insights into the interaction of hnRNP K in HEV replication.

miRNAs are an evolutionarily conserved class of regulatory small non-coding RNAs. miRNAs are post-transcriptional regulatory molecules that are 21-24 nucleotides long [17] miRNAs bind to target mRNAs and regulate their expressions by degrading mRNA and/or impeding translation [18] This master gene regulator has grabbed much attention from researchers as one miRNA can post-transcriptionally control around 1000 gene expressions. miRNAs have been studied in diverse cellular processes like cell proliferation, differentiation, apoptosis, immunity, and development 19, 20, 21, 22 Several reports have also shown a strong link between miRNAs and various diseases such as cancer, diabetes, cardiovascular disease, neurodegenerative diseases, and viral diseases 23, 24, 25, 26 miRNA has been found to play unconventional roles in viral infection. Direct interaction of miR-122 enhances HCV-RNA genome stability and prevents HCV RNA from exoribonuclease activity instead of degrading target viral RNA. In the replication of HEV, human immunodeficiency virus, bovine viral diarrhea virus, and eastern equine encephalitis virus, such novel roles of positive regulation of miRNAs have been investigated 27, 28, 29 There is evidence that the putative miR-140 binding site is conserved among all the HEV genotypes and other closely related RNA viruses. The RNA-dependent RNA polymerase found in RNA viruses frequently exhibits error-prone viral genome replication; hence, viral genome mutations occur frequently. Still, the conservation of miRNA binding site is retained among HEV and related RNA virus genomes, indicating its significance. Hence, the current study aims to investigate the functional significance of putative miR-140 binding site on HEV genome in viral replication.

In the present study, we identified that highly conserved intact miR-140 binding site (MBS) and host factor has-miR-140-3p are the critical requirements for HEV replication. MBS might form a secondary RNA structure that allows the recruitment of hnRNP K, which is a key protein of the HEV replication complex. In the process of HEV replication, MBS can serve as a binding platform for hnRNP K only in the presence of hsa-miR-140-3p. In addition, we also identified the novel interaction of hnRNP K and miR-140 endogenously as well as during HEV replication.