The number of hepatitis C virus (HCV) RNA copies present within infected hepatocytes is determined by the rate of viral RNA synthesis in relation to the rates of intracellular HCV RNA decay and the packaging and export of newly replicated genomes in virions. Both viral RNA and viral proteins are typically present at low abundance, and this may contribute to cell survival, viral escape from the immune response and long-term viral persistence.
HCV RNA is synthesized by replicase complexes associated with the membranous web, which is a collection of cytoplasmic single- and double-membrane vesicles that are formed as protrusions from the endoplasmic reticulum. Formation of the membranous web is dependent on phosphatidylinositol 4-kinase IIIα and multiple nonstructural HCV proteins, including NS4B and NS5A.
Newly synthesized HCV positive-strand RNAs ((+)RNAs) are subject to four possible fates: cycling to ribosomes to direct translation of more viral protein; translocating to lipid droplets for packaging into new virions for export from the cell; remaining within the membranous web to act as a template for synthesis of negative-strand RNA; or being degraded by 5′-to-3′ exoribonuclease 1 (XRN1).
A network of variably conserved RNA structures extends across the entire HCV (+)RNA genome, but structure is especially prominent within 3′ and 5′ untranslated regions and an internal cis-acting replication element. RNA–RNA interactions within and between these structures, as well as RNA–protein interactions involving poly(rC)-binding protein 2 and possibly other host proteins, modulate the structure of the genome, including its circularization, and thereby probably regulate genome engagement in different stages of the viral life cycle.
HCV infection induces oxidative stress. This leads to cellular lipid peroxidation, which acts in an inhibitory manner on the replicase complex to reduce viral replication, thereby providing a negative feedback loop that helps to maintain viral RNA and protein abundance at low levels within the liver.
The microRNA miR-122 is an abundant, evolutionarily conserved liver-specific microRNA. It binds two sites near the 5′ end of the HCV genome in association with Argonaute 2, and it promotes replication of the virus by stabilizing and protecting the genome against XRN1-mediated decay and directly stimulating viral RNA synthesis by reducing the proportion of RNA genomes engaged in protein translation, thereby increasing those available to act as templates in RNA synthesis.
Hepatitis C virus (HCV) is an unusual RNA virus that has a striking capacity to persist for the remaining life of the host in the majority of infected individuals. In order to persist, HCV must balance viral RNA synthesis and decay in infected cells. In this Review, we focus on interactions between the positive-sense RNA genome of HCV and the host RNA-binding proteins and microRNAs, and describe how these interactions influence the competing processes of viral RNA synthesis and decay to achieve stable, long-term persistence of the viral genome. Furthermore, we discuss how these processes affect hepatitis C pathogenesis and therapeutic strategies against HCV.
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The authors apologize to the many colleagues whose work they have been unable to cite and thank K. McKnight and A. Perelson for helpful discussions. This work was supported in part by grants from the US National Institutes of Health (R01-AI095690 and R01-CA164029) and the University of North Carolina Cancer Research Fund.
S.M.L.'s laboratory has received research funding from industry (Merck, Scynexis and Johnson & Johnson) as well as from the US National Institutes of Health, and he has served as a consultant to Merck, AbbVie, Hoffman-LaRoche, Gilead, Santaris, Achillion and Janssen with respect to the clinical and preclinical development of direct-acting antivirals against hepatitis C virus. Y.L., D.Y. and T.M. declare no competing interests.
Also called a hairpin element. An element of RNA secondary structure formed when two segments of an RNA molecule have complementary nucleotide sequences and base pair to form a two-stranded helix with an intervening single-stranded loop segment.
- Internal ribosome entry site
(IRES). A highly structured RNA sequence that recruits ribosomes and initiates translation of the RNA independently of ribosome scanning from a 5′-cap. The 5′ untranslated region of the hepatitis C virus RNA genome contains an IRES. A variety of IRES elements with different RNA structures are found in other viruses, as well as in a subset of host mRNAs, where they permit translation when cap-dependent translation is inhibited during mitosis, apoptosis or hypoxia.
- Lipid droplets
Ubiquitous, dynamic cellular organelles that are rich in lipids and mainly composed of cholesteryl esters and triglycerides. They function in energy storage by sequestering fatty acids in triglycerides and as a platform for the metabolism and transport of lipids, as well as playing a part in cellular signalling through the generation of bioactive lipids.
- Retinoic acid-inducible gene I
(RIG-I). A cytoplasmic RNA helicase that senses viral RNAs and initiates signalling that leads to host interferon production. RIG-I preferentially binds short double-stranded, AU-rich RNAs with 5′ di- or triphosphates and subsequently activates its downstream adaptor, mitochondrial antiviral signalling protein (MAVS), through interactions between shared caspase activation and recruitment domains (CARDs). RIG-I and a second RIG-I-like receptor, MDA5 (also known as IFIH1), contribute to innate immune sensing of distinct types of viruses.
- Protein kinase R
(PKR). An interferon-induced protein kinase that is activated in response to double-stranded RNA and is central to cellular responses to a variety of stresses, such as viral infection, cytokine exposure, nutrient depletion, irradiation and endoplasmic reticulum stress. PKR-mediated phosphorylation of eukaryotic translation initiation factor 2A (EIF2A) results in global translational repression.
A higher-order RNA structure that contains at least two stem–loops and in which the bases in one half of one stem pair with those in the loop of a second stem–loop.
- Ribosome scanning
The process by which a 40S ribosome subunit with associated initiation factors moves in a 3′ direction along an mRNA molecule in search of a start codon (typically AUG) following its initial interactions with and binding to the 5′ terminal 7-methylguanylate cap structure. This is an essential component of eukaryotic cap-dependent translation initiation. Assembly of the mature 80S ribosome and the initiation of polypeptide synthesis commence at the start codon.
- Selective 2′-hydroxyl acylation and primer extension
(SHAPE). An experimental method that assesses the backbone flexibility of an RNA molecule at a single-nucleotide resolution based on the reactivity of 2′-hydroxyl groups towards an electrophile. Nucleotides that are flexible (unpaired) are preferentially chemically modified and are recognized as stops in a subsequent primer extension reaction, allowing unpaired bases to be distinguished from those for which flexibility is constrained by base-pairing.
A mechanism that was initially discovered in plants and involves small non-coding RNAs suppressing gene expression by targeting specific mRNA transcripts in association with an RNA-induced silencing complex. Synthetic short interfering RNAs (siRNAs) may be transfected into cells to deplete a specific protein.
- RNA-induced silencing complex
(RISC). A ribonucleoprotein complex with a core comprising a small non-coding RNA (typically a microRNA or short interfering RNA) guide strand loaded into an Argonaute protein (AGO1–AGO4). The RISC acts to suppress translation and, in some cases, directly cleave an mRNA target.
A synthetic, typically nuclease-resistant, chemically modified oligonucleotide that is complementary to the sequence of a microRNA and antagonizes its activity by binding to and sequestering the microRNA from its mRNA target, as well as possibly blocking microRNA biogenesis.
- Small nucleolar RNA
(snoRNA). A class of small non-coding RNAs that act to guide specific post-transcriptional modifications, such as 2′-O-methylation or uridine isomerization, and occasionally cleavage, of other RNAs (primarily rRNAs and tRNAs).
A large complex formed by multiple mature 80S ribosomes bound to an mRNA that is actively undergoing translation.
A family of enveloped positive-strand RNA viruses comprising four genera: Flavivirus (dengue virus, yellow fever virus and others), Hepacivirus (hepatitis C virus), Pestivirus and Pegivirus.
(Processing bodies). Cytoplasmic aggregates of proteins engaged in the transport, storage and decay of host mRNA. These bodies represent foci of accumulation of 5′-to-3′ exonuclease 1 (XRN1).
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Li, Y., Yamane, D., Masaki, T. et al. The yin and yang of hepatitis C: synthesis and decay of hepatitis C virus RNA. Nat Rev Microbiol 13, 544–558 (2015). https://doi.org/10.1038/nrmicro3506
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