No 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...

Alphaviruses are positive-sense RNA viruses in the family Togaviridae, whose members cause significant disease to livestock and humans (reviewed in reference 10). Cycling between mosquito vectors and vertebrate hosts, New World members of this genus are responsible for summertime epidemics of equine encephalitis. Human infection can also result in encephalitis for which there is currently no specific therapy. Similarly, summertime epidemics of polyarthritis, fever, and rash can occur upon human infection with Old World alphavirus members.The alphaviruses share a common replication strategy (reviewed in references 14 and 28) that has been extensively studied in, among other viruses, Sindbis virus (SINV). After virus entry via receptor-mediated endocytosis, fusion of the virion membrane with the endosomal membrane occurs, releasing the nucleocapsid into the cytoplasm. After uncoating of the RNA, the 5Ј two-thirds of the SINV genome of ϳ11,700 nucleotides is translated to generate a polyprotein that is coand posttranslationally processed to form the nonstructural proteins (nsPs), nsP1, nsP2, nsP3 as well as nsP4, the RNAdependent RNA polymerase. Together with host-derived factors, the nsPs form a replicase complex, which produces new genome RNA through a negative-strand intermediate. A subgenomic RNA, also produced from the negative-strand intermediate and representing the 3Ј one-third of the genome, is translated to generate the structural proteins, which include the capsid protein as well as two membrane glycoproteins. Progeny enveloped virions are generated after encapsidated genomic RNA buds through the plasma membrane.Viral infection of cells initiates a cascade of events (reviewed in references 18 and 29) resulting in the interferon (IFN) regulatory factor 3 (IRF3)-dependent production of IFN-␤ and IFN-␣4. Binding of these IFNs to type I IFN receptors on the cell surface initiates the JAK/STAT (Janus protein tyrosine kinase/signal transducers and activators of transcription) signaling cascade and results in the induction of IRF7 and a number of IFN-stimulated genes (ISGs) that confer an antiviral state upon the cell. IRF7 activation leads to expression of a family of related IFN-␣ species, which, when secreted, amplify the IFN response. Signaling through the type I IFN receptor and expression of the resulting ISGs confer an antiviral state upon neighboring cells.Previously we demonstrated that expression of the rat zinc finger antiviral protein (ZAP) results in dramatic inhibition of multiple Alphavirus genus members and established, using SINV, that rat ZAP prevents translation of the incoming genomic RNA (2). Our studies indicate that ZAP can bind to viral RNA sequences, that the CCCH-type zinc finger motifs are important for ZAP-mediated inhibition, and that the presence of specific viral sequences in a reporter RNA results in reduced steady-state levels of the RNA in cells expressing ZAP (11). Recently, it was reported that ZAP recruits the exosome to mediate mRNA degradation (12). Rat ZAP, or the amino

MicroRNAs have been predicted to regulate the stability and translation of many target mRNAs that are involved in modulating disease outcome. Thus, valuable strategies to enhance or to diminish the function of microRNAs are needed to manipulate microRNA-mediated target gene expression. Recently, it has become apparent that one class of antisense oligonucleotides, locked nucleic acids, can be used to sequester microRNAs in the liver of a variety of animals including humans, opening the possibility of applying locked nucleic acid-mediated gene therapy. This review summarizes the success of sequestration of liver-specific microRNA miR-122 by antisense locked nucleic acids and their use in combating hepatitis C virus in clinical trials.