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Mir-122–Based Therapies Select for Three Distinct Resistance Mechanisms Based on Alterations in RNA Structure

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Mir-122–Based Therapies Select for Three Distinct Resistance Mechanisms Based on Alterations in RNA Structure miR-122–based therapies select for three distinct resistance mechanisms based on alterations in RNA structure Jasmin Chahala, Luca F. R. Gebertb, Carolina Camargoa, Ian J. MacRaeb, and Selena M. Sagana,c,1 aDepartment of Microbiology & Immunology, McGill University, Montréal, QC H3G 1Y6, Canada; bDepartment of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, CA 92037; and cDepartment of Biochemistry, McGill University, Montréal, QC H3G 1Y6, Canada Edited by Peter Sarnow, Stanford University School of Medicine, Stanford, CA, and approved July 9, 2021 (received for review February 23, 2021) Hepatitis C virus (HCV) is a positive-sense RNA virus that interacts with subsequent exoribonuclease-mediated decay (12, 14, 19–21). Fi- a liver-specific microRNA called miR-122. miR-122 binds to two sites in nally, the Ago protein bound to site 2 makes direct contact with the 5′ untranslated region of the viral genome and promotes HCV RNA the viral IRES, promoting HCV IRES-mediated translation (16). accumulation. This interaction is important for viral RNA accumulation Due to the importance of miR-122 in the HCV life cycle, two in cell culture, and miR-122 inhibitors have been shown to be effective miR-122 inhibitors (antisense oligonucleotides), the first miRNA- at reducing viral titers in chronic HCV-infected patients. Herein, we based drugs to enter clinical trials, have been developed and used to analyzed resistance-associated variantsthatwereisolatedincellcul- treat chronic HCV infection in the clinic (22, 23). Both Miravirsen ture or from patients who underwent miR-122 inhibitor–based ther- (Santaris Pharma, a/s) and RG-101 (Regulus Therapeutics) miR- apy and discovered three distinct resistance mechanisms all based on 122 inhibitors have completed Phase II or Ib clinical trials, re- changes to the structure of the viral RNA. Specifically, resistance- spectively, to investigate their clinical efficacy in chronic HCV in- associated variants promoted riboswitch activity, genome stability, fection (22, 23). Excitingly, both treatments led to dose-dependent or positive-strand viral RNA synthesis, all in the absence of miR-122. and sustained reductions in viral loads; and, in the latter study, two Taken together, these findings provide insight into the mechanism(s) patients achieved a sustained virological response (at least up to 76 of miR-122–mediated viral RNA accumulation and provide mechanisms wk posttreatment) after receiving a single dose of RG-101 (23). of antiviral resistance mediated by changes in RNA structure. Neither treatment was associated with significant adverse events or MICROBIOLOGY long-term safety issues, suggesting that antisense targeting of miR- hepatitis C virus | miR-122 | resistance-associated variants | riboswitch | 122 may be an effective treatment that could be used in future internal ribosomal entry site combination therapies. Interestingly, while no resistance was ap- parent during treatment, when viral RNA rebounded after the epatitis C virus (HCV) is a positive-sense RNA virus of the cessation of the inhibitor, several resistance-associated variants HFlaviviridae family. The ∼9.6 kb HCV genomic RNA consists (RAVs) were identified in the 5′ UTR of the HCV genome (23). of a single open reading frame, which gives rise to the viral pol- Specifically, C3U (genotype 1) was identified as a RAV in both the yprotein that is processed into the 10 mature viral proteins flanked Miravirsen and RG-101 trials, while the C2GC3U (genotype 3/4) by highly structured 5′ and 3′ untranslated regions (UTRs) (1, 2). RAV was identified in the RG-101 trial alone, with both RAVs As a positive-sense RNA virus, the viral genome itself must serve identified in multiple patients (22, 23). Additional RAVs were as a template for the different stages of the viral life cycle, in- cluding viral translation, replication, and packaging (2). To this Significance end, the viral 5′ and 3′ UTRs contain several cis-acting RNA el- ements that play important roles in directing the various stages of MicroRNA (miRNA)–based drugs are quickly taking the clinic by the viral life cycle (2–4). Specifically, the 5′ UTR contains the viral – storm. Herein, we analyzed resistance-associated variants internal ribosomal entry site (IRES) made up of stem loops (SL) (RAVs) to the first miRNA inhibitors to make it to the clinic, II-IV, which is required for viral translation, while sequences and ′ ′ namely miR-122 inhibitors for chronic hepatitis C virus (HCV) SL structures in both the 5 (SLI-II) and 3 UTRs (variable region, infection. We uncovered three distinct resistance mechanisms polyU/UC-tract, and 96-nt X-tail) are required for viral RNA – ′ based on unique alterations to the structure of the viral RNA. replication (2, 5 8). Additionally, the 5 terminus of the HCV Specifically, RAVs altered the structure of the viral RNA in a genome interacts with the highly abundant, liver-specific human manner that promotes riboswitch activity, genome stability, or microRNA (miRNA), miR-122 (9–12). ∼ positive-strand viral RNA synthesis. Our findings support re- miR-122 is a highly expressed miRNA in the liver with 135,000 cent models of miR-122–mediated HCV RNA accumulation and copies per hepatocyte (9, 13). While miRNAs typically interact provide mechanism(s) of resistance to antiviral therapy. These with the 3′ UTRs of their target messenger RNAs (mRNAs) to ′ early insights into the mechanism(s) of resistance to miRNA- dampen gene expression, miR-122 interacts to two sites in the 5 based therapies may be of importance as more miRNA- UTR (site 1 and site 2) of the viral genome, and these interactions targeted therapies enter into the clinic. promote viral RNA accumulation (9, 10, 12). Several recent studies have led to a new model for miR-122:HCV RNA inter- Author contributions: J.C. and S.M.S. designed research; J.C., L.F.R.G., and C.C. performed actions that suggest that miR-122 plays at least three roles in the research; J.C., L.F.R.G., C.C., I.J.M., and S.M.S. analyzed data; and J.C., L.F.R.G., C.C., I.J.M., HCV life cycle (Fig. 1) (14–17). Firstly, the HCV 5′ UTR is and S.M.S. wrote the paper. thought to initially adopt an energetically favorable conformation The authors declare no competing interest. alt (termed SLII ), which results in the recruitment of an Ago:miR- This article is a PNAS Direct Submission. 122 complex to site 2 of the HCV genome. This results in an RNA This open access article is distributed under Creative Commons Attribution License 4.0 chaperone-like switch in conformation, akin to a bacterial ribos- (CC BY). witch (18), resulting in the formation of SLII and allowing the viral 1To whom correspondence may be addressed. Email: [email protected]. IRES (SLII-IV) to form (15–17). Secondly, this change in con- This article contains supporting information online at https://www.pnas.org/lookup/suppl/ formation allows the recruitment of Ago:miR-122 to site 1, which doi:10.1073/pnas.2103671118/-/DCSupplemental. protects the 5′ terminus from pyrophosphatase activity and Published August 12, 2021. PNAS 2021 Vol. 118 No. 33 e2103671118 https://doi.org/10.1073/pnas.2103671118 | 1of11 Downloaded by guest on September 30, 2021 alt SLII Ago:miR-122 Ago:miR-122 3 Translation Site 2 SLII SLII Site 1 5' 3'5' 3'5'5' 3' SLI SLI SLI 12Riboswitch Stability Fig. 1. Model of miR-122 interactions with the HCV genome. The HCV genomic RNA is thought to enter the cell in an energetically stable conformation termed SLIIalt. The recruitment of the first Ago:miR-122 molecule to the accessible (unpaired) site 2 serves as an RNA chaperone, akin to a bacterial riboswitch, which refolds the RNA into the functional SLII conformation and allows the viral IRES (SLII-IV) to form (1). Subsequent recruitment of a second Ago:miR-122 molecule to site 1 promotes genome stability by protecting the 5′ terminus from cellular pyrophosphatases and exoribonuclease-mediated decay (2). In order to accommodate the Ago:miR-122 complex at site 1, the Ago:miR-122 complex at site 2 releases its auxiliary interactions but is likely stabilized by interactions between the Ago protein and SLII of the HCV IRES (3). Collectively, these interactions promote HCV IRES-mediated translation. miR-122 seed and auxiliary binding sequences are indicated (red). identified in cell culture, including studies performed with genotype present in genotype 2 (save for A4C, although U4C was deemed an 1 (A4C) and genotype 2 (U4C, G28A, and C37U), which were equivalent RAV observed in this genotype), herein, we explored the identified alone (A4C and G28A) or in combination with other mechanism of action of these collective RAVs (C2GC3U, C3U, RAVs (i.e., G28A+C37U, U4C+G28A+C37U) (24–26). As the U4C, G28A, and C37U) using genotype2a(J6/JFH-1)reporter cell culture–based studies identifying RAVs were performed with RNAs (Fig. 2A)(22–26). Previous work suggests that the G28A genotype 2 and the majority of the RAV nucleotide identities are mutation is “riboswitched” and promotes the formation of the A BCWT Huh-7.5 cells miR-122 KO cells C2GC3U 108 108 C3U U4C 107 107 G28A 106 106 C37U 105 GNN 105 104 104 103 103 102 102 101 101 2h1234567 Luciferase expression (RLU) 2h1234567 Luciferase expression (RLU) Time (Days) Time (Days) D E WT C2GC3U 108 Site 1 complemented Site 2 complemented 108 C3U 107 U4C 7 10 G28A 106 106 C37U 105 GNN 105 104 104 103 103 102 102 1 10 101 2h1234567 Luciferase expression (RLU) 2h1234567 Luciferase expression (RLU) Time (Days) Time (Days) Fig. 2. RAV accumulation in cell culture. (A) The positions of the RAVs on the 5′ UTR of the HCV RNA.
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