Abstract
Many viruses strongly prefer to infect certain cell types, a phenomenon known as “tropism.” Understanding tropism’s molecular basis is important for the design of vaccines and antiviral therapy. A common mechanism involves viral protein interactions with cell-specific surface receptors, but intracellular mechanisms involving translation have also been described. In this report, we focus on Hepatitis A Virus (HAV) tissue tropism from the standpoint of the translational machinery. HAV genomic RNA, like other positive stranded RNA viruses, is devoid of a cap structure and its translation is driven by highly structured RNA sequences termed internal ribosome entry site (IRES) in the 5′ untranslated region (UTR). Unlike most viral IRESs, HAV IRES-mediated translation requires eIF4E and the 3′ end of HAV RNA is polyadenylated. However, the molecular mechanism of HAV IRES-mediated translation initiation remains poorly understood. We analyzed HAV-IRES-mediated translation in a cell-free system derived from either non-hepatic cells (HeLa) or hepatoma cells (Huh-7) that enables investigation of the contribution of the cap and the poly(A) tail. This revealed that HAV IRES-mediated translation activity in hepatoma cell extracts is higher as compared to extracts derived from a non-hepatic line. Our data suggest that HAV IRES-mediated translation is upregulated by a hepatic cell-specific activator in a poly(A) tail-independent manner.
Highlights
In eukaryotes, the vast majority of cellular mRNAs are capped at the 5 end and polyadenylated at the 3 end
By measuring Hepatitis A Virus (HAV) IRESmediated translation activity in Huh-7 and human cervical carcinoma cell (HeLa) cell extracts, we show that an HAV internal ribosome entry site (IRES) reporter mRNA is translated more efficiently in Huh-7 cell extracts than in HeLa cells
To assess our in vitro translation system, we measured Nluc activity from reporter mRNAs with an m7GpppG cap, analogous to the physiological cap, or a non-physiological ApppG cap analog (Acap) that is not recognized by eIF4E and with or without poly (A) tail
Summary
The vast majority of cellular mRNAs are capped at the 5 end and polyadenylated at the 3 end. Translation initiation on these mRNAs follows a well-defined pathway (Green et al, 2016) involving multiple stages governed by a large number of eIFs interacting with the mRNA and the ribosome (Jackson et al, 2010). Hepatic Cell-Specific Translation of HAV (eIF2–GTP–Met–tRNAi), several eIFs including eIF1, 1A, 3, and 5, and the 40S small ribosomal subunit, is recruited to the mRNA via the eIF4F complex.After binding to mRNA, the 43S PIC scans the mRNA 5 UTR in a 5 to 3 direction until the Met– tRNAi recognizes the start AUG codon. Some eIFs are released, and the 60S large ribosome subunit is recruited to form an 80S initiation complex ready to synthesize encoded peptide
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