The impact of small RNA binding on Hepatitis C virus replication via structural changes within the 5’ untranslated region

ABSTRACTHigh-mobility group box 1 (HMGB1) protein is a highly conserved nuclear protein involved in multiple human diseases, including infectious diseases, immune disorders, metabolic disorders, and cancer. HMGB1 is comprised of two tandem HMG boxes (the A box and the B box) containing DNA-binding domains and an acidic C-terminal peptide. It has been reported that HMGB1 enhances viral replication by binding to viral proteins. However, its role in hepatitis C virus (HCV) replication is unknown. Here, we show that HMGB1 promoted HCV replication but had no effect on HCV translation. RNA immunoprecipitation experiments indicated that the positive strand, not the negative strand, of HCV RNA interacted with HMGB1. HCV infection triggered HMGB1 protein translocation from the nucleus to the cytoplasm, in which it interacted with the HCV genome. Moreover, the A box of HMGB1 is the pivotal domain to interact with stem-loop 4 (SL4) of the HCV 5′ untranslated region. Deletion of the HMGB1 A box abrogated the enhancement of HCV replication by HMGB1. Our data suggested that HMGB1 serves as a proviral factor of HCV to facilitate viral replication in hepatocytes by interaction with the HCV genome.IMPORTANCEHepatitis C virus (HCV) is a major global health threat, affecting more than 170 million people infection worldwide. These patients are at high risk of developing severe liver diseases such as chronic hepatitis, cirrhosis, and hepatocellular carcinoma. Currently, no vaccine is available. Many host factors may be implicated in the pathogenesis of HCV-related diseases. In this study, we found a novel HCV RNA-binding protein, HMGB1, that promotes HCV RNA replication. Moreover, SL4 in the 5′ untranslated region of the HCV genome is the key region for HMGB1 binding, and the A box of HMGB1 protein is the functional domain to interact with HCV RNA and enhance viral replication. HMGB1 appears to play an important role in HCV-related diseases, and further investigation is warranted to elucidate the specific actions of HMGB1 in HCV pathogenesis.

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Location specific annealing of miR-122 and other small RNAs defines an Hepatitis C Virus 5′ UTR regulatory element with distinct impacts on virus translation and genome stability

Nucleic Acids Research ◽

10.1093/nar/gkaa664 ◽

2020 ◽

Vol 48 (16) ◽

pp. 9235-9249 ◽

Cited By ~ 2

Author(s):

Rasika D Kunden ◽

Sarah Ghezelbash ◽

Juveriya Q Khan ◽

Joyce A Wilson

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Virus Replication ◽

Small Rna ◽

Small Rnas ◽

Rna Structure ◽

Genome Stability ◽

Regulatory Element ◽

Entry Site ◽

Genome Stabilization

Abstract Hepatitis C virus (HCV) replication requires annealing of a liver specific small-RNA, miR-122 to 2 sites on 5′ untranslated region (UTR). Annealing has been reported to (a) stabilize the genome, (b) stimulate translation and (c) promote the formation of translationally active Internal Ribosome Entry Site (IRES) RNA structure. In this report, we map the RNA element to which small RNA annealing promotes HCV to nucleotides 1–44 and identify the relative impact of small RNA annealing on virus translation promotion and genome stabilization. We mapped the optimal region on the HCV genome to which small RNA annealing promotes virus replication to nucleotides 19–37 and found the efficiency of viral RNA accumulation decreased as annealing moved away from this region. Then, by using a panel of small RNAs that promote replication with varying efficiencies we link the efficiency of lifecycle promotion with translation stimulation. By contrast small RNA annealing stabilized the viral genome even if they did not promote virus replication. Thus, we propose that miR-122 annealing promotes HCV replication by annealing to an RNA element that activates the HCV IRES and stimulates translation, and that miR-122 induced HCV genome stabilization is insufficient alone but enhances virus replication.

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Location specific small RNA annealing to the HCV 5’ UTR promotes Hepatitis C Virus replication by favoring IRES formation and stimulating virus translation

10.1101/2020.03.25.008417 ◽

2020 ◽

Author(s):

Rasika D. Kunden ◽

Sarah Ghezelbash ◽

Juveriya Q. Khan ◽

Joyce A. Wilson

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Virus Replication ◽

Small Rna ◽

Small Rnas ◽

Rna Structure ◽

Mechanistic Model ◽

Genome Replication ◽

Entry Site ◽

Genome Stabilization

ABSTRACTHepatitis C virus (HCV) genome replication requires annealing of a liver specific small-RNA, miR-122 to 2 sites on 5’ untranslated region (UTR). Annealing has been reported to a) stabilize the genome, b) promote translation, and c) induce the canonical HCV 5’ UTR Internal Ribosome Entry Site (IRES) structure. In this report we identify the relative impact of small RNA annealing on the three functions ascribed to miR-122 and generate a mechanistic model for miR-122 promotion of HCV. First, we identified that perfectly complementary small RNAs that anneal to different locations on the HCV 5’ UTR stimulate replication with varying efficiencies and mapped the region on the HCV genome to which small RNA annealing promotes virus replication. Second, by using a panel of small RNAs that promote with varying efficiencies we link HCV replication induction with translation stimulation and 5’ UTR RNA structure modifications. However, replication promotion was not linked to genome stabilization since all small RNAs tested could stabilize the viral genome regardless of their ability to promote replication. Thus, we propose that miR-122 annealing promotes HCV replication primarily by activating the HCV IRES and stimulating translation, and that miR-122-induced HCV genome stabilization is insufficient alone but enhances virus replication.Graphical Abstract

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Seven nucleotide changes characteristic of the hepatitis C virus genotype 3 5′ untranslated region: correlation with reduced in vitro replication

Journal of General Virology ◽

10.1099/vir.0.83067-0 ◽

2008 ◽

Vol 89 (1) ◽

pp. 212-221 ◽

Cited By ~ 13

Author(s):

Cyril Masante ◽

Kathleen Mahias ◽

Sofia Lourenço ◽

Estelle Dumas ◽

Annie Cahour ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Untranslated Region ◽

Rna Synthesis ◽

Ex Vivo ◽

Minus Strand ◽

Genotype 1 ◽

Genotype 3 ◽

The Impact

Computer analysis of 158 hepatitis C virus (HCV) 5′ untranslated region (5′ UTR) sequences from the six genotypes showed that the 5′ UTR from genotype 3 displays seven specific non-contiguous nucleotide changes, at positions 8, 13, 14, 70, 97, 203 and 224. The purpose of this study was to investigate the impact of these changes on translation and replication activities. Indeed, these modifications could alter both the internal ribosome entry site (IRES) present in the 5′ UTR of the plus-strand RNA and the 3′ end of the minus strand involved in the initiation of plus-strand RNA synthesis. We found that the genotype 3-specific nucleotide changes do not modify the in vitro or ex vivo translation activity of the corresponding IRES, in comparison with that of genotype 1. In contrast, in vitro replication from the minus-strand RNA is eight times less efficient for genotype 3 than for genotype 1 RNA, suggesting the involvement of some nucleotide changes in the reduction of RNA synthesis. Nucleotides 13, 14 and 224 were found to be responsible for this effect. Moreover, a reduced replicative activity was confirmed ex vivo for genotype 3, but to a lesser extent than that observed in vitro, using an RNA minigenome.

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