Masking the 5′ terminal nucleotides of the hepatitis C virus genome by an unconventional microRNA-target RNA complex

Abstract

Hepatitis C virus subverts liver-specific microRNA, miR-122, to upregulate viral RNA abundance in both infected cultured cells and in the liver of infected chimpanzees. These findings have identified miR-122 as an attractive antiviral target. Thus, it is imperative to know whether a distinct functional complex exists between miR-122 and the viral RNA versus its normal cellular target mRNAs. Toward this goal, effects on viral RNA abundance of mutated miR-122 duplex molecules, bound at each of the two target sites in the viral genome, were compared to effects on microRNA-or siRNA-mediated regulation of reporter target mRNAs. It was found that miR-122 formed an unusual microRNA complex with the viral RNA that is distinct from miR-122 complexes with reporter mRNAs. Notably, miR-122 forms an oligomeric complex in which one miR-122 molecule binds to the 5′ terminus of the hepatitis C virus (HCV) RNA with 3′ overhanging nucleotides, masking the 5′ terminal sequences of the HCV genome. Furthermore, specific internal nucleotides as well as the 3′ terminal nucleotides in miR-122 were absolutely required for maintaining HCV RNA abundance but not for microRNA function. Both miR-122 molecules utilize similar internal nucleotides to interact with the viral genome, creating a bulge and tail in the miR-122 molecules, revealing tandemly oriented oligomeric RNA complexes. These findings suggest that miR-122 protects the 5′ terminal viral sequences from nucleolytic degradation or from inducing innate immune responses to the RNA terminus. Finally, this remarkable microRNA-mRNA complex could be targeted with compounds that inactivate miR-122 or interfere with this unique RNA structure. H epatitis C virus (HCV) infection is a global health problem with an estimated 170 million people infected worldwide (reviewed in ref. 1). HCV-infected individuals typically develop persistent infections that can lead to chronic hepatitis, cirrhosis, and hepatocellular carcinoma (2). Currently, there are no vaccines available, and the clinical efficacies of modern therapeutics are limited. Thus, understanding fundamental aspects of hostvirus interactions in HCV infection is important for the discovery of unique anti-HCV treatments. HCV is a hepatotrophic, positive-sense RNA virus that belongs to the family Flaviviridae. The HCV genome contains a single open reading frame encoding the viral polyprotein, which is subsequently cleaved into at least 10 viral proteins by host and viral proteases. The HCV open reading frame is flanked by 5′ and 3′ noncoding regions (NCRs) that contain RNA elements that are important for viral replication and translation (reviewed in ref.3). Recently, interactions between the liver-specific microRNA, miR-122, with two sites in the HCV 5′NCR (Fig. 1A) have been shown to be essential to maintain HCV RNA abundance during virus infection in cultured cells (4, 5) and in infected chimpanzees (6).MicroRNAs (miRNAs) are small, noncoding RNAs that are predicted to regulate at least one-third of all human mRNAs (7,8). As a general ru...

Functional analysis of microRNA-122 binding sequences of hepatitis C virus and identification of variants with high resistance against a specific antagomir

“…Until recently, efficient HCV infectious culture systems were based on the genotype 2a isolate JFH1 in systems permitting studies in vitro and in vivo (Bukh, 2012;(nt 1-42; numbering according to genotype 2a isolate HC-J6, GenBank accession no. NC_009823) contains one stemloop structure (stem-loop I, SLI) (Honda et al, 1999), and two microRNA 122 (miR-122) binding sites (S1 and S2) (Henke et al, 2008;Jopling, 2008;Jopling et al, 2005;Machlin et al, 2011). It has been shown that the SLI is important for viral RNA replication (Friebe et al, 2001;Luo et al, 2003), and we demonstrated that it is essential for the viability of infectious HCV genotype 1-6 recombinants (Li et al, 2011a).…”

“…1a) (Machlin et al, 2011). We previously found that deletion of the first nucleotide, A, of the J6 5¢UTR-NS2 /JFH1 recombinant could be repaired by addition of A or AC at the 5¢ terminus during virus replication in Huh7.5 cells, thus producing viruses with a variable length of 5¢E upstream of SLI (Li et al, 2011a, b).…”

“…ml À1 , comparable to that of the control J6 5¢UTR-NS2 /JFH1. Taken together, HCV was able to repair the mutated 5¢E by acquiring (or deleting) additional nucleotides to maintain a specific length, optimally 4 nt (ACCC in the genotype 2a Machlin et al (2011). (b) Permissive mutations identified in second-passage viruses in this study are shown in red and aligned with the nucleotides of the original HCV sequence; single mutations are shown individually, combined mutations in S1 or S2 are underlined, combined mutations in S1 and S2 are indicated by a connecting dotted line, a dash indicates a deletion of the nucleotide corresponding to the aligned original sequence, and the arrow indicates a nucleotide insertion.…”

“…Thus, we also tested a mutant with S1_A8G plus S2_432CGA, designated S1_A8G +S2_432CGA. In addition, we constructed S1_A8G plus S1S2_432CGA and deletion of the GCCAU stretch between S1 and S2 (DGCCAU, the 'CC' of 'GCCAU' between S1 and S2 is involved in miR-122 binding; Machlin et al, 2011), designated S1_A8G+S1S2m (Fig. 1e).…”

“…miR-122 is a liver-abundant miRNA (Chang et al, 2004), and it stimulates viral RNA replication and translation by directly binding to S1 and S2, as well as to upstream nucleotides, in the HCV 5¢UTR (Fig. 1a) (Henke et al, 2008;Jopling, 2008;Jopling et al, 2005;Machlin et al, 2011). miR-122 binds HCV RNA in association with Ago2, thus stabilizing the viral RNA (Shimakami et al, 2012) and protecting the 5¢ end of the viral RNA from degradation by Xrn1 (Li et al, 2013).…”