Regulation of Hepatitis C Virus Genome Replication by Xrn1 and MicroRNA-122 Binding to Individual Sites in the 5′ Untranslated Region

Abstract

miR-122 is a liver-specific microRNA (miRNA) that binds to two sites (S1 and S2) on the 5= untranslated region (UTR) of the hepatitis C virus (HCV) genome and promotes the viral life cycle. It positively affects viral RNA stability, translation, and replication, but the mechanism is not well understood. To unravel the roles of miR-122 binding at each site alone or in combination, we employed miR-122 binding site mutant viral RNAs, Hep3B cells (which lack detectable miR-122), and complementation with wild-type miR-122, an miR-122 with the matching mutation, or both. We found that miR-122 binding at either site alone increased replication equally, while binding at both sites had a cooperative effect. Xrn1 depletion rescued miR-122-unbound fulllength RNA replication to detectable levels but not to miR-122-bound levels, confirming that miR-122 protects HCV RNA from Xrn1, a cytoplasmic 5=-to-3= exoribonuclease, but also has additional functions. In cells depleted of Xrn1, replication levels of S1-bound HCV RNA were slightly higher than S2-bound RNA levels, suggesting that both sites contribute, but their contributions may be unequal when the need for protection from Xrn1 is reduced. miR-122 binding at S1 or S2 also increased translation equally, but the effect was abolished by Xrn1 knockdown, suggesting that the influence of miR-122 on HCV translation reflects protection from Xrn1 degradation. Our results show that occupation of each miR-122 binding site contributes equally and cooperatively to HCV replication but suggest somewhat unequal contributions of each site to Xrn1 protection and additional functions of miR-122. Hepatitis C virus (HCV) is a hepatotropic virus that infects an estimated 150 million humans worldwide, a significant portion of whom do not know their status due to the largely asymptomatic nature of the infection (1). The virus is transmitted by blood-to-blood contact, and humans are the only known reservoir. Chronic infection occurs in approximately 70% of cases and can lead to sequelae such as metabolic disease, steatosis, hepatocellular carcinoma, and decompensated liver disease late in infection (2).One of the major determinants of the virus' hepatotropism is its requirement for the liver-specific, liver-abundant miR-122 microRNA (miRNA) (3, 4). miR-122 binds to two sites at the 5= end of the virus' positive-sense RNA genome and has been shown to directly enhance viral RNA accumulation, since mutation of the miR-122 binding sites abolishes RNA accumulation, and the provision of exogenous miR-122 sequences that have compensatory mutations to restore binding also reinstates RNA accumulation (4-10). Argonaute-2, one of the key effector proteins in the microRNA pathway and a component of the RNA-induced silencing complex (RISC), binds in association with miR-122 and is required to increase HCV replication, while several other proteins in the microRNA pathway and RISC have been implicated in either the biogenesis or activity of miR-122 (5, 11-14). Although miR-122 uses canonical microRNA se...

Cellular DEAD-box RNA helicase DDX6 modulates interaction of miR-122 with the 5′ untranslated region of hepatitis C virus RNA

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“…Translation and replication of the HCV genome is directed by RNA elements within both UTRs. Interestingly, specific interactions of the liver-specific microRNA miR-122 with two binding sites in the 5′ UTR mask the 5′ end of the genome from Xrn1 and Xrn2 5′-to-3′ exonuclease digestion (Li et al, 2013, 2015; Sedano and Sarnow, 2014; Thibault et al, 2015), and thus contribute to maintaining HCV RNA abundance. While the binding of miR-122 to both sites 1 and 2 is critical (Jopling et al, 2008), the exact nature of these interactions remains elusive (Garcia-Sastre and Evans, 2013).…”

Human MicroRNA-548p Decreases Hepatic Apolipoprotein B Secretion and Lipid Synthesis

“…Further, significant attempts have been made to test potential use of miRs as therapeutic agents. MiR-122, the most abundant miR expressed in the liver, binds to the 5′-UTR of the Hepatitis C virus genome to positively affect RNA stability and replication (3). Therefore, inhibition of endogenous miR-122 shows efficacy in treating chronic Hepatitis C infection.…”

The roles of the exoribonucleases DIS3L2 and XRN1 in human disease

“…miR-122 is a major determinant of viral RNA replication, as it inhibits XRN1 from degrading the viral RNA, allowing accumulation, increased replication, and increased translation of viral RNA. Studies have shown that miR-122 only needs to be bound at one of the two sites for it to have this inhibitory effect, and binding to both sites maintains a co-operative effect, rather than increasing the effectiveness [52]. In addition to the miR-122 binding site, the 5'UTR HCV viral RNA includes four stem loops of which three (loops 2-4) appear to function as a highly structured internal ribosome entry site, allowing translation initiation of the viral RNA [50].…”

“…Recent publications show that 5'-3' degradation of HCV transcripts is stalled by expression of miR-122 in the host cell, which binds via its miRNA seed sequence to the 5'UTR of the viral transcript ( Figure 2C). The association of miR-122/RISC, including Ago2 [51], at two sites in the 5' UTR, protects it from degradation by XRN1 [52]. miR-122 is a major determinant of viral RNA replication, as it inhibits XRN1 from degrading the viral RNA, allowing accumulation, increased replication, and increased translation of viral RNA.…”