Viral Determinants of miR-122-Independent Hepatitis C Virus Replication

Hopcraft, Sharon E.; Azarm, Kristopher D.; Israelow, Benjamin; Lévêque, Nicolas; Schwarz, Megan; Hsu, Tien-Huei; Chambers, Matthew T.; Sourisseau, Marion; Semler, Bert L.; Evans, Matthew J.

doi:10.1128/msphere.00009-15

Cited by 29 publications

(44 citation statements)

References 35 publications

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“…More recently, HCV was shown to replicate independent of miR-122 (Hopcraft et al, 2016; Thibault et al, 2013), and specific mutations in the HCV 5′ UTR not only facilitate HCV gene expression when miR-122 abundance is decreased but also in the absence of miR-122 (Hopcraft et al, 2016; Israelow et al, 2014). Interestingly depletion of DDX6 affects both miR-122 dependent and independent HCV gene expression.…”

Section: Discussionmentioning

confidence: 99%

Cellular DEAD-box RNA helicase DDX6 modulates interaction of miR-122 with the 5′ untranslated region of hepatitis C virus RNA

et al. 2017

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Hepatitis C virus (HCV) subverts the cellular DEAD-box RNA helicase DDX6 to promote virus infection. Using polysome gradient analysis and the subgenomic HCV Renilla reporter replicon genome, we determined that DDX6 does not affect HCV translation. Rather expression of the subgenomic HCV Renilla luciferase reporter at late times, as well as labeling of newly synthesized viral RNA with 4-thiouridine showed that DDX6 modulates replication. Because DDX6 is an effector protein of the microRNA pathway, we also investigated its role in miR-122-directed HCV gene expression. Similar to sequestering miR-122, depletion of DDX6 modulated HCV RNA stability. Interestingly, miR-122-HCV RNA interaction assays with mutant HCV genomes sites and compensatory exogenous miR-122 showed that DDX6 affects the function of miR-122 at one particular binding site. We propose that DDX6 facilitates the miR-122 interaction with HCV 5′ UTR, which is necessary for stabilizing the viral genome and the switch between translation and replication.

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Section: Discussionmentioning

confidence: 99%

Cellular DEAD-box RNA helicase DDX6 modulates interaction of miR-122 with the 5′ untranslated region of hepatitis C virus RNA

et al. 2017

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“…The identity of the Huh7 and Huh7.5 cell lines was verified using the Promega GenePrint STR kit (DNA Analysis Facility, Duke University), and cells were verified as mycoplasma free by the LookOut Mycoplasma PCR detection kit (Sigma). Huh7.5 CD81 KO cells were generated by CRISPR, as described before, with details given in the Supplemental Experimental Procedures (Hopcraft et al, 2016; Hopcraft and Evans, 2015). …”

Section: Methodsmentioning

confidence: 99%

N6 -Methyladenosine in Flaviviridae Viral RNA Genomes Regulates Infection

Gokhale

McIntyre

McFadden

et al. 2016

Cell Host & Microbe

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405

564

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SUMMARY The RNA modification N6-methyladenosine (m6A) post-transcriptionally regulates RNA function. The cellular machinery that controls m6A includes methyltransferases and demethylases that add or remove this modification as well as m6A-binding YTHDF proteins that promote the translation or degradation of m6A-modified mRNA. We demonstrate that m6A modulates infection by hepatitis C virus (HCV). Depletion of m6A-methyltransferases or an m6A-demethylase respectively increases and decreases infectious HCV particle production. During HCV infection, YTHDF proteins relocalize to lipid droplets, sites of viral assembly, and their depletion increases infectious viral particles. We further mapped m6A sites across the HCV genome and determine that inactivating m6A in one viral genomic region increases viral titer without affecting RNA replication. Additional mapping of m6A on the RNA genomes of other Flaviviridae, including dengue, Zika, yellow fever, and West Nile virus, identifies conserved regions modified by m6A. Together, this work identifies m6A as a conserved regulatory mark across Flaviviridae genomes.

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“…Approaching this point, a function of miR-122 in replication was proposed that could stimulate HCV RNA synthesis by altering the balance of viral RNAs engaged in replication versus translation ( Masaki et al, 2015 ). Moreover, it has been shown that revertants in the 5′ UTR miR-122 binding sites that do not support miR-122 binding can be stable ( Hopcraft et al, 2016 ), even suggesting a miR-122-independent role of the primary sequence of the respective HCV 5′ UTR region in the HCV life cycle. On the other hand, an decrease in cellular miR-122 concentration lead to the emergence of a mutation in the HCV sequence that slightly increased the affinity of miR-122 binding, indicating a certain need for miR-122 in efficient HCV replication ( Israelow et al, 2014 ).…”

Section: The Liver-specific Microrna-122mentioning

confidence: 99%

Signals Involved in Regulation of Hepatitis C Virus RNA Genome Translation and Replication

et al. 2018

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Hepatitis C virus (HCV) preferentially replicates in the human liver and frequently causes chronic infection, often leading to cirrhosis and liver cancer. HCV is an enveloped virus classified in the genus Hepacivirus in the family Flaviviridae and has a single-stranded RNA genome of positive orientation. The HCV RNA genome is translated and replicated in the cytoplasm. Translation is controlled by the Internal Ribosome Entry Site (IRES) in the 5′ untranslated region (5′ UTR), while also downstream elements like the cis-replication element (CRE) in the coding region and the 3′ UTR are involved in translation regulation. The cis-elements controlling replication of the viral RNA genome are located mainly in the 5′- and 3′-UTRs at the genome ends but also in the protein coding region, and in part these signals overlap with the signals controlling RNA translation. Many long-range RNA–RNA interactions (LRIs) are predicted between different regions of the HCV RNA genome, and several such LRIs are actually involved in HCV translation and replication regulation. A number of RNA cis-elements recruit cellular RNA-binding proteins that are involved in the regulation of HCV translation and replication. In addition, the liver-specific microRNA-122 (miR-122) binds to two target sites at the 5′ end of the viral RNA genome as well as to at least three additional target sites in the coding region and the 3′ UTR. It is involved in the regulation of HCV RNA stability, translation and replication, thereby largely contributing to the hepatotropism of HCV. However, we are still far from completely understanding all interactions that regulate HCV RNA genome translation, stability, replication and encapsidation. In particular, many conclusions on the function of cis-elements in HCV replication have been obtained using full-length HCV genomes or near-full-length replicon systems. These include both genome ends, making it difficult to decide if a cis-element in question acts on HCV replication when physically present in the plus strand genome or in the minus strand antigenome. Therefore, it may be required to use reduced systems that selectively focus on the analysis of HCV minus strand initiation and/or plus strand initiation.

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Viral Determinants of miR-122-Independent Hepatitis C Virus Replication

Cited by 29 publications

References 35 publications

Cellular DEAD-box RNA helicase DDX6 modulates interaction of miR-122 with the 5′ untranslated region of hepatitis C virus RNA

Cellular DEAD-box RNA helicase DDX6 modulates interaction of miR-122 with the 5′ untranslated region of hepatitis C virus RNA

N6 -Methyladenosine in Flaviviridae Viral RNA Genomes Regulates Infection

Signals Involved in Regulation of Hepatitis C Virus RNA Genome Translation and Replication

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