Multiple processes exist in a cell to ensure continuous production of essential proteins either through cap-dependent or cap-independent translation processes. Viruses depend on the host translation machinery for viral protein synthesis. Therefore, viruses have evolved clever strategies to utilize the host translation machinery. Earlier studies have shown that genotype 1-Hepatitis E virus (g1-HEV) utilizes both cap-dependent and cap-independent translation machineries for its replication and proliferation. Cap-independent translation in g1-HEV is driven by an eighty seven nucleotide-long RNA element which acts as a noncanonical, internal ribosome entry site like (IRESl) element. Here, we have identified the RNA-protein interactome of the HEV IRESl element and characterized the functional significance of some of its components. Our study reveals indispensable roles of host ribosomal protein RPL5 and DHX9 (RNA helicase A) in mediating efficient translation from the IRESl element and establish the function of HEV IRESl as a bonafide internal ribosome entry site. Author summary Protein synthesis is a fundamental process for survival and proliferation of all living organisms. Majority of cellular proteins are produced through a cap-dependent translation process. However under conditions of stress and/or when cap-dependent translation is not operational, cells utilize a variety of cap-independent translation processes to synthesize essential proteins. Viruses are the only living organisms which depend on the host cell translation apparatus to synthesize their own proteins. Many viruses utilize both cap-dependent and cap-independent machineries for the synthesis of viral proteins. Hepatitis E virus is a major cause of acute hepatitis worldwide. The viral genome is a capped positive strand RNA. Viral non-structural and structural proteins are synthesized through a cap-dependent translation process from one open reading frame (ORF) present in the genomic RNA and two overlapping ORFs present in the subgenomic RNA, respectively. An earlier study from our laboratory reported the presence of a fourth ORF in the viral genome, which produced the ORF4 protein using a cap-independent internal translation initiation mechanism. Using bicistronic reporter assays and site-directed mutagenesis studies, it was demonstrated that translation of the ORF4 protein was driven by an internal ribosome entry site-like (IRESl) element. In the current study, we identified the host proteins that directly or indirectly associate with the HEV-IRESl RNA and generated the RNA-protein interactome. Subsequent studies identified essential roles of ribosome protein L5 (RPL5) and DHX9 (RNA Helicase A) in mediating the function of the IRESl. Biochemical fractionation studies showed the presence of IRESl and its components in the actively translating polysomes. Collectively, our data proves that HEV-IRESl is a bonafide internal ribosome entry site.
RNA-protein interactome at the Hepatitis E virus internal ribosome entry site