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    • The translation of the HCV genomic

    2022-01-28

    The translation of the HCV genomic RNA is initiated in a different manner, referred to as internal initiation: the 40S subunit directly forms a binary complex with the HCV genomic RNA via an internal RNA sequence called IRES, which is located in the 5′-UTR (Pestova et al., 1998, Tsukiyama-Kohara et al., 1992). Extensive biochemical and structural studies have revealed that the HCV IRES consists of three structural and functional entities: “body,” “long arm,” and “short arm” (Figure 1A; Figure S1A). The HCV IRES body, consisting of domain III except for subdomain IIIb, serves as the minimal structural motif for binding to the 40S subunit (Kieft et al., 2001, Quade et al., 2015, Yamamoto et al., 2015). The long and short arms, corresponding to domain II and subdomain IIIb, respectively, harbor the regulatory functions of the HCV IRES. The long arm guides its downstream mRNA onto the 40S subunit and supports the start codon recognition by the TC and the subsequent joining of the 60S subunit (Otto and Puglisi, 2004, Quade et al., 2015, Yamamoto et al., 2015). Simultaneous binding of the HCV IRES and eIF3 to the ribosome is allowed, even though they compete for overlapping others nervous on the 40S subunit (Hashem et al., 2013a, Quade et al., 2015). This is because the short arm of the HCV IRES acts as another binding site for eIF3 (Hashem et al., 2013b). Based on these findings, many studies have hypothesized that the HCV IRES first binds to the free 40S ribosomal subunit, before the 60S subunit joins. However, to our knowledge, no direct validation of this scheme has been provided.
    Results
    Discussion Considering the present results, we perceive how the HCV genomic RNA hijacks the translational machinery (Figure 7B). When the HCV genomic RNA is released into the reaction pool, the HCV IRES binds to the 40S subunit, regardless of whether it exists alone (state VI in Figure 7B) or as part of the translating 80S ribosome (state I in Figure 7B), but only if the ribosome is free of eIF3. The translation initiation factor eIF3 tends to leave the translating 80S ribosomes, as compared with the 40S subunit (Asano et al., 1997, Mengod and Trachsel, 1985), and therefore the HCV IRES may encounter the eIF3-free 80S ribosomes with high probability. Once the HCV IRES body captures the translating 80S ribosome, the HCV genomic RNA is likely to remain bound to the 40S subunit through the HCV IRES body until the ongoing translation terminates (state II in Figure 7B). After the translation termination (state III in Figure 7B), the HCV IRES long arm approaches the E site and inserts its downstream RNA into the mRNA path on the 40S subunit (state IV in Figure 7B) to initiate its own translation (states IV to V in Figure 7B). Considering that the concurrent cap-dependent translation enhances the HCV IRES-dependent translation, an unknown mechanism that facilitates the HCV IRES-dependent translation might exist between states II and V. Furthermore, the HCV IRES body can retain the captured 40S subunit of the 80S ribosome while its own downstream viral polypeptide is translated (state V in Figure 7B). The HCV genomic RNA could potentially repeat the translation of the viral polypeptide on the same 40S subunit in a manner referred to as “reinitiation” (states V to III in Figure 7B). Further experiments are necessary to confirm whether the currently proposed mechanism actually occurs in the cell. However, the HCV IRES is likely to utilize a sophisticated process to hijack the host translational machinery while translation is ongoing. The 3′-UTR of the HCV genomic RNA also reportedly binds to the 40S subunit (Bai et al., 2013), and thus the behavior and function of the 3′-UTR during translation are also interesting. Consequently, the HCV genomic RNA, holding the IRES in the 5′-UTR, is efficiently translated into the HCV viral polypeptide. The resultant efficiently produced viral proteins eventually support the replication of the HCV genomic RNA. Although the currently proposed ribosome-hijacking mechanism seems to be unique to the IRESs from HCV, its closely related pestiviruses (Pestova and Hellen, 1999, Pestova et al., 1998), and some picornaviruses (Bakhshesh et al., 2008, de Breyne et al., 2008, Pisarev et al., 2004, Willcocks et al., 2011), a similar mechanism might be employed by other kinds of IRESs. We hope that this study provides an opportunity to further investigate the common and specific mechanisms of the IRES functions by multidisciplinary approaches.