Comparison of U2OS and Huh-7 cells for identifying host factors that affect hepatitis C virus RNA replication

Jones, Daniel M.; Domingues, Patricia; Targett-Adams, Paul; McLauchlan, John

doi:10.1099/vir.0.022210-0

Cited by 32 publications

(26 citation statements)

References 44 publications

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“…Previous efforts to investigate how Xrn1 influences HCV replication have produced conflicting results, with two studies showing no effects of Xrn1 depletion (26,27). Our results are consistent with those of Jones et al (28) and Ruggieri et al (29) and provide a mechanism for why Xrn1 depletion promotes HCV replication.…”

Section: Resultssupporting

confidence: 87%

Competing and noncompeting activities of miR-122 and the 5′ exonuclease Xrn1 in regulation of hepatitis C virus replication

Li¹,

Masaki²,

Yamane³

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

203

260

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Hepatitis C virus (HCV) replication is dependent on microRNA 122 (miR-122), a liver-specific microRNA that recruits Argonaute 2 to the 5′ end of the viral genome, stabilizing it and slowing its decay both in cell-free reactions and in infected cells. Here we describe the RNA degradation pathways against which miR-122 provides protection. Transfected HCV RNA is degraded by both the 5′ exonuclease Xrn1 and 3′ exonuclease exosome complex, whereas replicating RNA within infected cells is degraded primarily by Xrn1 with no contribution from the exosome. Consistent with this, sequencing of the 5′ and 3′ ends of RNA degradation intermediates in infected cells confirmed that 5′ decay is the primary pathway for HCV RNA degradation. Xrn1 knockdown enhances HCV replication, indicating that Xrn1 decay and the viral replicase compete to set RNA abundance within infected cells. Xrn1 knockdown and miR-122 supplementation have equal, redundant, and nonadditive effects on the rate of viral RNA decay, indicating that miR-122 protects HCV RNA from 5′ decay. Nevertheless, Xrn1 knockdown does not rescue replication of a viral mutant defective in miR-122 binding, indicating that miR-122 has additional yet uncharacterized function(s) in the viral life cycle.host factor | RNA decay | translation | viral replicase H epatitis C virus (HCV) is a positive-strand RNA virus classified in the family Flaviviridae. It is highly hepatotropic and an important cause of human liver disease (1). Its replication is uniquely dependent on miR-122, which is the most abundant microRNA (miRNA) in the liver and accounts for >50% of mature miRNAs in human hepatocytes (2, 3). There are two conserved miR-122 binding sites (S1 and S2) located near the 5′ end of the positive-sense viral RNA genome, immediately upstream of an internal ribosome entry site (IRES) that mediates translation of the viral polyprotein. Direct interactions between the miR-122 seed sequence (nts 2-8) and these sites in the 5′ UTR are essential for amplification of the HCV genome (4, 5). Additional supplemental base-pairing upstream of S1 and S2 has also been demonstrated and is important for viral replication (6, 7).Unlike typical interactions of miRNAs with mRNAs that involve binding within the 3′ UTR and promote translational repression and/or destabilization of the target RNA (8), binding of miR-122 to the 5′ UTR of HCV genomic RNA stimulates viral protein expression (4, 9) and, in association with Argonaute 2 (Ago2) protein, stabilizes HCV RNA (10). The rate of decay of either transfected synthetic genomic RNA or replicating viral RNA within infected cells is slowed when cells are transfected with synthetic duplex miR-122, thereby supplementing its endogenous abundance. Conversely, transfection of antisense RNA complementary to miR-122 enhances the rate with which HCV RNA decays in either context. HCV RNA is not thought to contain a 5′ cap structure, and the stabilizing action of miR-122 on synthetic RNA can be functionally substituted by addition of a 5′ cap analog (10). These obser...

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Section: Resultssupporting

confidence: 87%

Competing and noncompeting activities of miR-122 and the 5′ exonuclease Xrn1 in regulation of hepatitis C virus replication

Li¹,

Masaki²,

Yamane³

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

203

260

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“…More recently, researchers have been exploring whether the effect of miR-122 on viral translation is merely a stabilization of the viral RNA (8,11,18,32,43). In particular, Xrn1 has been implicated as the major host factor destabilizing and degrading the viral RNA (17,18,29,32), although not all screens involving Xrn1 knockdown show that it has a negative or any effect on HCV (17,(44)(45)(46). Our evidence (Fig.…”

Section: Discussionsupporting

confidence: 58%

“…miR-122 has been observed to modestly increase translation of the viral RNA, but this has recently been attributed to simply increased stability of the viral RNA in the assays, allowing more viral RNA to be translated (10,11,14,15). In particular, miR-122 has been implicated in protecting the 5= end of the viral RNA from degradation by Xrn1, the major cytoplasmic 5=-to-3= exoribonuclease that normally functions to degrade decapped host mRNAs and other Flavivirus RNAs (16)(17)(18)(19). One model posits that the interaction of miR-122 with S1 generates a 3= overhang that shields the 5= triphosphate genome terminus, but the influence of each miR-122 binding interaction in protection from Xrn1 remains unknown.…”

mentioning

confidence: 99%

Regulation of Hepatitis C Virus Genome Replication by Xrn1 and MicroRNA-122 Binding to Individual Sites in the 5′ Untranslated Region

Thibault

Huys

Amador-Cañizares

et al. 2015

J Virol

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miR-122 is a liver-specific microRNA (miRNA) that binds to two sites (S1 and S2) on the 5= untranslated region (UTR) of the hepatitis C virus (HCV) genome and promotes the viral life cycle. It positively affects viral RNA stability, translation, and replication, but the mechanism is not well understood. To unravel the roles of miR-122 binding at each site alone or in combination, we employed miR-122 binding site mutant viral RNAs, Hep3B cells (which lack detectable miR-122), and complementation with wild-type miR-122, an miR-122 with the matching mutation, or both. We found that miR-122 binding at either site alone increased replication equally, while binding at both sites had a cooperative effect. Xrn1 depletion rescued miR-122-unbound fulllength RNA replication to detectable levels but not to miR-122-bound levels, confirming that miR-122 protects HCV RNA from Xrn1, a cytoplasmic 5=-to-3= exoribonuclease, but also has additional functions. In cells depleted of Xrn1, replication levels of S1-bound HCV RNA were slightly higher than S2-bound RNA levels, suggesting that both sites contribute, but their contributions may be unequal when the need for protection from Xrn1 is reduced. miR-122 binding at S1 or S2 also increased translation equally, but the effect was abolished by Xrn1 knockdown, suggesting that the influence of miR-122 on HCV translation reflects protection from Xrn1 degradation. Our results show that occupation of each miR-122 binding site contributes equally and cooperatively to HCV replication but suggest somewhat unequal contributions of each site to Xrn1 protection and additional functions of miR-122. Hepatitis C virus (HCV) is a hepatotropic virus that infects an estimated 150 million humans worldwide, a significant portion of whom do not know their status due to the largely asymptomatic nature of the infection (1). The virus is transmitted by blood-to-blood contact, and humans are the only known reservoir. Chronic infection occurs in approximately 70% of cases and can lead to sequelae such as metabolic disease, steatosis, hepatocellular carcinoma, and decompensated liver disease late in infection (2).One of the major determinants of the virus' hepatotropism is its requirement for the liver-specific, liver-abundant miR-122 microRNA (miRNA) (3, 4). miR-122 binds to two sites at the 5= end of the virus' positive-sense RNA genome and has been shown to directly enhance viral RNA accumulation, since mutation of the miR-122 binding sites abolishes RNA accumulation, and the provision of exogenous miR-122 sequences that have compensatory mutations to restore binding also reinstates RNA accumulation (4-10). Argonaute-2, one of the key effector proteins in the microRNA pathway and a component of the RNA-induced silencing complex (RISC), binds in association with miR-122 and is required to increase HCV replication, while several other proteins in the microRNA pathway and RISC have been implicated in either the biogenesis or activity of miR-122 (5, 11-14). Although miR-122 uses canonical microRNA se...

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“…Another protective role of miR-122's interaction with the 5′ terminus of the HCV genome could involve protection from 5′ exonucleases. Depletion of XRN-1 mRNA, encoding a 5′-3′ exonuclease that is involved in decay of cellular mRNAs (24,25), increases the expression of luciferase from tricistronic HCV replicons (26) and the abundance of electroporated HCV replicon RNA. Interestingly, Takahashi et al reported that the 5′ termini of HCV RNA obtained from HCV replicon cells contains a 5′ monophosphate (17), suggesting that the 5′ terminus of HCV RNA is sensitive to degradation by Xrn-1.…”

Section: Discussionmentioning

confidence: 99%

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

Machlin

Sarnow

Sagan

2011

Proc. Natl. Acad. Sci. U.S.A.

264

296

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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...

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Comparison of U2OS and Huh-7 cells for identifying host factors that affect hepatitis C virus RNA replication

Cited by 32 publications

References 44 publications

Competing and noncompeting activities of miR-122 and the 5′ exonuclease Xrn1 in regulation of hepatitis C virus replication

Competing and noncompeting activities of miR-122 and the 5′ exonuclease Xrn1 in regulation of hepatitis C virus replication

Regulation of Hepatitis C Virus Genome Replication by Xrn1 and MicroRNA-122 Binding to Individual Sites in the 5′ Untranslated Region

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

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