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MicroRNA let-7b expression is induced by infection of hepatitis C virus (HCV) and is involved in the regulation of HCV replication by directly targeting the HCV genome. The current study demonstrated that let-7b directly targets negative regulators of type I interferon (IFN) signaling thereby limiting HCV replication in the early stage of HCV infection. Let-7b-regulated genes which are involved in host cellular responses to HCV infection were unveiled by microarray profiling and bioinformatic analyses, followed by various molecular and cellular assays using Huh7 cells expressing wild-type (WT) or the seed region-mutated let-7b. Let-7b targeted the cytokine signaling 1 (SOCS1) protein, a negative regulator of JAK/STAT signaling, which then enhanced STAT1-Y701 phosphorylation leading to increased expression of the downstream interferon-stimulated genes (ISGs). Let-7b augmented retinoic acid-inducible gene I (RIG-I) signaling, but not MDA5, to phosphorylate and nuclear translocate IRF3 leading to increased expression of IFN-β. Let-7b directly targeted the ATG12 and IκB kinase alpha (IKKα) transcripts and reduced the interaction of the ATG5-ATG12 conjugate and RIG-I leading to increased expression of IFN, which may further stimulate JAK/STAT signaling. Let-7b induced by HCV infection elicits dual effects on IFN expression and signaling, along with targeting the coding sequences of NS5B and 5′ UTR of the HCV genome, and limits HCV RNA accumulation in the early stage of HCV infection. Controlling let-7b expression is thereby crucial in the intervention of HCV infection. IMPORTANCE HCV is a leading cause of liver disease, with an estimated 71 million people infected worldwide. During HCV infection, type I interferon (IFN) signaling displays potent antiviral and immunomodulatory effects. Host factors, including microRNAs (miRNAs), play a role in upregulating IFN signaling to limit HCV replication. Let-7b is a liver-abundant miRNA that is induced by HCV infection and targets the HCV genome to suppress HCV RNA accumulation. In this study, we demonstrated that let-7b, as a positive regulator of type I IFN signaling, plays dual roles against HCV replication by increasing the expression of IFN and interferon-sensitive response element (ISRE)-driven interferon-stimulated genes (ISGs) in the early stage of HCV infection. This study sheds new insight into understanding the role of let-7b in combatting HCV infection. Clarifying IFN signaling regulated by miRNA during the early phase of HCV infection may help researchers understand the initial defense mechanisms to other RNA viruses.

Hepatitis C virus (HCV) nonstructural protein 5B (NS5B) is an RNA-dependent RNA polymerase (RdRp) that acts as a key player in the HCV replication complex. Understanding the interplay between the viral and cellular components of the HCV replication complex could provide new insight for prevention of the progression of HCV-associated hepatocellular carcinoma (HCC). In this study, the NS5B protein was used as the bait in a pulldown assay to screen for NS5B-interacting proteins that are present in Huh7 hepatoma cell lysates. After mass spectrophotometric analysis, fatty acid synthase (FASN) was found to interact with NS5B. Coimmunoprecipitation and double staining assays further confirmed the direct binding between NS5B and FASN. The domain of NS5B that interacts with FASN was also determined. Moreover, FASN was associated with detergent-resistant lipid rafts and colocalized with NS5B in active HCV replication complexes. In addition, overexpression of FASN enhanced HCV expression in Huh7/Rep-Feo cells, while transfection of FASN small interfering RNA (siRNA) or treatment with FASN-specific inhibitors decreased HCV replication and viral production. Notably, FASN directly increased HCV NS5B RdRp activity in vitro . These results together indicate that FASN interacts with NS5B and modulates HCV replication through a direct increase of NS5B RdRp activity. FASN may thereby serve as a target for the treatment of HCV infection and the prevention of HCV-associated HCC progression.

The nonstructural protein 3 (NS3) of hepatitis C virus (HCV) possesses protease, nucleoside triphosphatase, and helicase activities. Although the enzymatic activities have been extensively studied, the ATP- and RNA-binding domains of the NS3 helicase are not well-characterized. In this study, NS3 proteins with point mutations in the conserved helicase motifs were expressed in Escherichia coli , purified, and analyzed for their effects on ATP binding, RNA binding, ATP hydrolysis, and RNA unwinding. UV cross-linking experiments indicate that the lysine residue in the AX 4 GKS motif is directly involved in ATP binding, whereas the NS3(GR1490DT) mutant in which the arginine-rich motif (1486-QRRGRTGR-1493) was changed to QRRDTTGR bound ATP as well as the wild type. The binding activity of HCV NS3 helicase to the viral RNA was drastically reduced with the mutation at Arg1488 (R1488A) and was also affected by the K1236E substitution in the AX 4 GKS motif and the R1490A and GR1490DT mutations in the arginine-rich motif. Previously, Arg1490 was suggested, based on the crystal structure of an NS3-deoxyuridine octamer complex, to directly interact with the γ-phosphate group of ATP. Nevertheless, our functional analysis demonstrated the critical roles of Arg1490 in binding to the viral RNA, ATP hydrolysis, and RNA unwinding, but not in ATP binding.

Hepatitis C virus (HCV) NS5B protein is the viral RNA-dependent RNA polymerase capable of directing RNA synthesis. In this study, an electrophoretic mobility shift assay demonstrated the interaction between a partially purified recombinant NS5B protein and a 3* viral genomic RNA with or without the conserved 98-nucleotide tail. The NS5B-RNA complexes were specifically competed away by the unlabeled homologous RNA but not by the viral 5* noncoding region and very poorly by the 3* conserved 98-nucleotide tail. A 3* coding region with conserved stem-loop structures rather than the 3* noncoding region of the HCV genome is critical for the specific binding of NS5B. Nevertheless, no direct interaction between the 3* coding region and the HCV NS5A protein was detected. Furthermore, two independent RNA-binding domains (RBDs) of NS5B were identified, RBD1, from amino acid residues 83 to 194, and RBD2, from residues 196 to 298. Interestingly, the conserved motifs of RNA-dependent RNA polymerase for putative RNA binding (220-DxxxxD-225) and template/primer position (282-S/TGxxxTxxxNS/T-292) are present in the RBD2. Nevertheless, the RNA-binding activity of RBD2 was abolished when it was linked to the carboxy-terminal half of the NS5B. These results provide some clues to understanding the initiation of HCV replication. Hepatitis C virus (HCV) is an enveloped virus that possesses a single-stranded positive-sense RNA genome encoding a polyprotein of approximately 3,000 amino acid residues (7, 20, 29). The conserved 59 noncoding region (59NCR) of the HCV genome (4, 13) is highly structured and contributes to the internal ribosome entry site important for the translation initiation of HCV RNA (14, 15, 25, 34, 35, 39). The 39 noncoding region (39NCR) consists of a short genotype-specific region and a poly(U)-C(U)n repeat stretch of variable length followed by a highly conserved tail of 98 nucleotides (nt) (12, 21, 30, 37). Studies to understand the molecular mechanism of HCV replication have been restricted by the lack of a well-established cell culture system, but studies from other positive-sense RNA viruses may provide some clues. Upon flavivirus infection, translation of incoming viral genomic RNA occurs, and replication of the viral RNA begins with the synthesis of minus-strand RNA which then serves as the template for the synthesis of progeny genomic RNA. The replication appears to take place at the perinuclear endoplasmic reticulum and requires virus-encoded proteins NS3 (proteinase/helicase) and NS5 (polymerase) as components of the presumed replicative complex (36). In addition, the 39 terminus of the flavivirus genomic RNA forms a conserved pseudoknot structure (26). It is generally believed that conserved sequences and structures at the 39 terminus of viral genomic RNA function as cis-acting signals that interact with viral and cellular proteins to initiate the synthesis of minus-strand RNA during viral replication. The NS5B protein of HCV is a membrane-associated phosphoprotein (16) that possesses the conserved GDD motif of RNA-dependent RNA polymerase (RdRp) (19). RdRp activity of the HCV NS5B has been demonstrated in vitro, and