MicroRNAs are important regulators of gene expression that control both physiological and pathological processes such as development and cancer. Although their mode of action has attracted great attention, the principles governing their expression and activity are only beginning to emerge. Recent studies have introduced a paradigm shift in our understanding of the microRNA biogenesis pathway, which was previously believed to be universal to all microRNAs. Maturation steps specific to individual microRNAs have been uncovered, and these offer a plethora of regulatory options after transcription with multiple proteins affecting microRNA processing efficiency. Here we review the recent advances in knowledge of the microRNA biosynthesis pathways and discuss their impact on post-transcriptional microRNA regulation during tumour development.

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Many roads to maturity: microRNA biogenesis pathways and their regulation

Antiviral immunity against a pathogen is mounted upon recognition by the host of virally associated structures. One of these viral 'signatures', double-stranded (ds) RNA, is a replication product of most viruses within infected cells and is sensed by Toll-like receptor 3 (TLR3) and the recently identified cytosolic RNA helicases RIG-I (retinoic acid inducible gene I, also known as Ddx58) and Mda5 (melanoma differentiation-associated gene 5, also known as Ifih1 or Helicard). Both helicases detect dsRNA, and through their protein-interacting CARD domains, relay an undefined signal resulting in the activation of the transcription factors interferon regulatory factor 3 (IRF3) and NF-kappaB. Here we describe Cardif, a new CARD-containing adaptor protein that interacts with RIG-I and recruits IKKalpha, IKKbeta and IKKvarepsilon kinases by means of its C-terminal region, leading to the activation of NF-kappaB and IRF3. Overexpression of Cardif results in interferon-beta and NF-kappaB promoter activation, and knockdown of Cardif by short interfering RNA inhibits RIG-I-dependent antiviral responses. Cardif is targeted and inactivated by NS3-4A, a serine protease from hepatitis C virus known to block interferon-beta production. Cardif thus functions as an adaptor, linking the cytoplasmic dsRNA receptor RIG-I to the initiation of antiviral programmes.

Cardif is an adaptor protein in the RIG-I antiviral pathway and is targeted by hepatitis C virus

Many aspects of the hepatitis C virus (HCV) life cycle have not been reproduced in cell culture, which has slowed research progress on this important human pathogen. Here, we describe a full-length HCV genome that replicates and produces virus particles that are infectious in cell culture (HCVcc). Replication of HCVcc was robust, producing nearly 10(5) infectious units per milliliter within 48 hours. Virus particles were filterable and neutralized with a monoclonal antibody against the viral glycoprotein E2. Viral entry was dependent on cellular expression of a putative HCV receptor, CD81. HCVcc replication was inhibited by interferon-alpha and by several HCV-specific antiviral compounds, suggesting that this in vitro system will aid in the search for improved antivirals.

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Complete Replication of Hepatitis C Virus in Cell Culture

Review of recent research on hepatitis C therapy for 54th annual meeting of the American association for the study of liver diseases

Since the discovery of interferons (IFNs), considerable progress has been made in describing the nature of the cytokines themselves, the signalling components that direct the cell response and their antiviral activities. Gene targeting studies have distinguished four main effector pathways of the IFN-mediated antiviral response: the Mx GTPase pathway, the 2',5'-oligoadenylate-synthetase-directed ribonuclease L pathway, the protein kinase R pathway and the ISG15 ubiquitin-like pathway. As discussed in this Review, these effector pathways individually block viral transcription, degrade viral RNA, inhibit translation and modify protein function to control all steps of viral replication. Ongoing research continues to expose additional activities for these effector proteins and has revealed unanticipated functions of the antiviral response.

Interferon-inducible antiviral effectors

The absence of a robust cell culture model of hepatitis C virus (HCV) infection has severely limited analysis of the HCV life cycle and the development of effective antivirals and vaccines. Here we report the establishment of a simple yet robust HCV cell culture infection system based on the HCV JFH-1 molecular clone and Huh-7-derived cell lines that allows the production of virus that can be efficiently propagated in tissue culture. This system provides a powerful tool for the analysis of host-virus interactions that should facilitate the discovery of antiviral drugs and vaccines for this important human pathogen.

http://www.pnas.org/content/pnas/102/26/9294.full.pdf

Robust hepatitis C virus infection in vitro

Plants and invertebrates can use RNA silencing as a protective mechanism in viral infection. Now cellular microRNAs (miRNAs) have been found to have an antiviral function in mammalian cells too. Interferon-β is involved in the regulation of a number of cellular miRNAs in human cells, and eight of these are active against sequences on the hepatitis C virus. In addition, modulation of cellular miRNA levels are found to contribute significantly towards the antiviral effects of interferon-β, suggesting that they are a functioning component of the mammalian innate immune response. Plants and invertebrates can use RNA silencing as a protective mechanism in viral infection. Cellular microRNAs can have anti-viral activity also in mammalian cells, in this case by contributing to the antiviral effects of interferon beta against hepatitis C virus. RNA interference through non-coding microRNAs (miRNAs) represents a vital component of the innate antiviral immune response in plants and invertebrate animals; however, a role for cellular miRNAs in the defence against viral infection in mammalian organisms has thus far remained elusive1. Here we show that interferon beta (IFNβ) rapidly modulates the expression of numerous cellular miRNAs, and that eight of these IFNβ-induced miRNAs have sequence-predicted targets within the hepatitis C virus (HCV) genomic RNA. The introduction of synthetic miRNA-mimics corresponding to these IFNβ-induced miRNAs reproduces the antiviral effects of IFNβ on HCV replication and infection, whereas neutralization of these antiviral miRNAs with anti-miRNAs reduces the antiviral effects of IFNβ against HCV. In addition, we demonstrate that IFNβ treatment leads to a significant reduction in the expression of the liver-specific miR-122, an miRNA that has been previously shown to be essential for HCV replication2. Therefore, our findings strongly support the notion that mammalian organisms too, through the interferon system, use cellular miRNAs to combat viral infections.

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Interferon modulation of cellular microRNAs as an antiviral mechanism.

Intracellular infectious hepatitis C virus (HCV) particles display a distinctly higher buoyant density than do secreted virus particles, suggesting that the characteristic low density of extracellular HCV particles is acquired during viral egress. We took advantage of this difference to examine the determinants of assembly, maturation, degradation, and egress of infectious HCV particles. The results demonstrate that HCV assembly and maturation occur in the endoplasmic reticulum (ER) and post-ER compartments, respectively, and that both depend on microsomal transfer protein and apolipoprotein B, in a manner that parallels the formation of very-low-density lipoproteins (VLDL). In addition, they illustrate that only low-density particles are efficiently secreted and that immature particles are actively degraded, in a proteasome-independent manner, in a post-ER compartment of the cell. These results suggest that by coopting the VLDL assembly, maturation, degradation, and secretory machinery of the cell, HCV acquires its hepatocyte tropism and, by mimicry, its tendency to persist.

Cellular Determinants of Hepatitis C Virus Assembly, Maturation, Degradation, and Secretion

The virological and cellular consequences of persistent hepatitis C virus (HCV) infection have been elusive due to the absence of the requisite experimental systems. Here, we report the establishment and the characteristics of persistent in vitro infection of human hepatoma-derived cells by a recently described HCV genotype 2a infectious molecular clone. Persistent in vitro infection was characterized by the selection of viral variants that displayed accelerated expansion kinetics, higher peak titers, and increased buoyant densities. Sequencing analysis revealed the selection of a single adaptive mutation in the HCV E2 envelope protein that was largely responsible for the variant phenotype. In parallel, as the virus became more aggressive, cells that were resistant to infection emerged, displaying escape mechanisms operative at the level of viral entry, HCV RNA replication, or both. Collectively, these results reveal the existence of coevolutionary events during persistent HCV infection that favor survival of both virus and host.

Persistent hepatitis C virus infection in vitro: coevolution of virus and host.

Virus infection triggers IFN immune defenses in infected cells in part through viral nucleic acid interactions, but the pathways by which dsDNA and DNA viruses trigger innate defenses are only partially understood. Here we present evidence that both retinoic acid-induced gene I (RIG-I) and mitochondrial antiviral signaling protein (MAVS) are required for dsDNA-induced IFN-β promoter activation in a human hepatoma cell line (Huh-7), and that activation is efficiently blocked by the hepatitis C virus NS3/4A protease, which is known to block dsRNA signaling by cleaving MAVS. These findings suggest that dsDNA and dsRNA share a common pathway to trigger the innate antiviral defense response in human cells, although dsDNA appears to trigger that pathway upstream of the dsRNA-interacting protein RIG-I.

https://www.pnas.org/content/pnas/104/21/9035.full.pdf

Guofeng Cheng

Papers

Robust hepatitis C virus infection in vitro

Interferon modulation of cellular microRNAs as an antiviral mechanism.

Cellular Determinants of Hepatitis C Virus Assembly, Maturation, Degradation, and Secretion

Persistent hepatitis C virus infection in vitro: coevolution of virus and host.

Double-stranded DNA and double-stranded RNA induce a common antiviral signaling pathway in human cells