Virus-derived circular RNAs populate hepatitis C virus-infected cells.

It is known that pre-mRNAs in eukaryotic cells can be processed to circular RNAs by a backsplicing mechanism. Circular RNAs have great stability and can sequester proteins or small RNAs to exert functions on cellular pathways. Because viruses often exploit host pathways, we explored whether the RNA genome of the cytoplasmic hepatitis C virus is processed to yield virus-derived circRNAs (vcircRNAs). Computational analyses of RNA-seq experiments predicted that the viral RNA genome is fragmented to generate hundreds of vcircRNAs. More than a dozen of them were experimentally verified by rolling-circle amplification. VcircRNAs that contained the viral internal ribosome entry site were found to be translated into proteins that displayed proviral functions. Furthermore, two highly abundant, nontranslated vcircRNAs were shown to enhance viral RNA abundance. These findings argue that novel vcircRNA molecules modulate viral amplification in cells infected by a cytoplasmic RNA virus.

Loquacious modulates flaviviral RNA replication in mosquito cells.

Arthropod-borne viruses infect both mosquito and mammalian hosts. While much is known about virus-host interactions that modulate viral gene expression in their mammalian host, much less is known about the interactions that involve inhibition, subversion or avoidance strategies in the mosquito host. A novel RNA-Protein interaction detection assay was used to detect proteins that directly or indirectly bind to dengue viral genomes in infected mosquito cells. Membrane-associated mosquito proteins Sec61A1 and Loquacious (Loqs) were found to be in complex with the viral RNA. Depletion analysis demonstrated that both Sec61A1 and Loqs have pro-viral functions in the dengue viral infectious cycle. Co-localization and pull-down assays showed that Loqs interacts with viral protein NS3 and both full-length and subgenomic viral RNAs. While Loqs coats the entire positive-stranded viral RNA, it binds selectively to the 3' end of the negative-strand of the viral genome. In-depth analyses showed that the absence of Loqs did not affect translation or turnover of the viral RNA but modulated viral replication. Loqs also displayed pro-viral functions for several flaviviruses in infected mosquito cells, suggesting a conserved role for Loqs in flavivirus-infected mosquito cells.

Trans-kingdom mimicry underlies ribosome customization by a poxvirus kinase.

Ribosomes have the capacity to selectively control translation through changes in their composition that enable recognition of specific RNA elements. However, beyond differential subunit expression during development, evidence for regulated ribosome specification within individual cells has remained elusive. Here we report that a poxvirus kinase phosphorylates serine/threonine residues in the human small ribosomal subunit protein, receptor for activated C kinase (RACK1), that are not phosphorylated in uninfected cells or cells infected by other viruses. These modified residues cluster in an extended loop in RACK1, phosphorylation of which selects for translation of viral or reporter mRNAs with 5' untranslated regions that contain adenosine repeats, so-called polyA-leaders. Structural and phylogenetic analyses revealed that although RACK1 is highly conserved, this loop is variable and contains negatively charged amino acids in plants, in which these leaders act as translational enhancers. Phosphomimetics and inter-species chimaeras have shown that negative charge in the RACK1 loop dictates ribosome selectivity towards viral RNAs. By converting human RACK1 to a charged, plant-like state, poxviruses remodel host ribosomes so that adenosine repeats erroneously generated by slippage of the viral RNA polymerase confer a translational advantage. Our findings provide insight into ribosome customization through trans-kingdom mimicry and the mechanics of species-specific leader activity that underlie poxvirus polyA-leaders.

A transfer-RNA-derived small RNA regulates ribosome biogenesis.

Transfer-RNA-derived small RNAs (tsRNAs; also called tRNA-derived fragments) are an abundant class of small non-coding RNAs whose biological roles are not well understood. Here we show that inhibition of a specific tsRNA, LeuCAG3'tsRNA, induces apoptosis in rapidly dividing cells in vitro and in a patient-derived orthotopic hepatocellular carcinoma model in mice. This tsRNA binds at least two ribosomal protein mRNAs (RPS28 and RPS15) to enhance their translation. A decrease in translation of RPS28 mRNA blocks pre-18S ribosomal RNA processing, resulting in a reduction in the number of 40S ribosomal subunits. These data establish a post-transcriptional mechanism that can fine-tune gene expression during different physiological states and provide a potential new target for treating cancer.

An evolutionarily acquired microRNA shapes development of mammalian cortical projections.

The corticospinal tract is unique to mammals and the corpus callosum is unique to placental mammals (eutherians). The emergence of these structures is thought to underpin the evolutionary acquisition of complex motor and cognitive skills. Corticospinal motor neurons (CSMN) and callosal projection neurons (CPN) are the archetypal projection neurons of the corticospinal tract and corpus callosum, respectively. Although a number of conserved transcriptional regulators of CSMN and CPN development have been identified in vertebrates, none are unique to mammals and most are coexpressed across multiple projection neuron subtypes. Here, we discover 17 CSMN-enriched microRNAs (miRNAs), 15 of which map to a single genomic cluster that is exclusive to eutherians. One of these, miR-409-3p, promotes CSMN subtype identity in part via repression of LMO4, a key transcriptional regulator of CPN development. In vivo, miR-409-3p is sufficient to convert deep-layer CPN into CSMN. This is a demonstration of an evolutionarily acquired miRNA in eutherians that refines cortical projection neuron subtype development. Our findings implicate miRNAs in the eutherians' increase in neuronal subtype and projection diversity, the anatomic underpinnings of their complex behavior.

Host-derived circular RNAs display proviral activities in Hepatitis C virus-infected cells.

Viruses subvert macromolecular pathways in infected host cells to aid in viral gene amplification or to counteract innate immune responses. Roles for host-encoded, noncoding RNAs, including microRNAs, have been found to provide pro- and anti-viral functions. Recently, circular RNAs (circRNAs), that are generated by a nuclear back-splicing mechanism of pre-mRNAs, have been implicated to have roles in DNA virus-infected cells. This study examines the circular RNA landscape in uninfected and hepatitis C virus (HCV)-infected liver cells. Results showed that the abundances of distinct classes of circRNAs were up-regulated or down-regulated in infected cells. Identified circRNAs displayed pro-viral effects. One particular up-regulated circRNA, circPSD3, displayed a very pronounced effect on viral RNA abundances in both hepatitis C virus- and Dengue virus-infected cells. Though circPSD3 has been shown to bind factor eIF4A3 that modulates the cellular nonsense-mediated decay (NMD) pathway, circPSD3 regulates RNA amplification in a pro-viral manner at a post-translational step, while eIF4A3 exhibits the anti-viral property of the NMD pathway. Findings from the global analyses of the circular RNA landscape argue that pro-, and likely, anti-viral functions are executed by circRNAs that modulate viral gene expression as well as host pathways. Because of their long half-lives, circRNAs likely play hitherto unknown, important roles in viral pathogenesis.

Genetic dissection of Flaviviridae host factors through genome-scale CRISPR screens.

The Flaviviridae are a family of viruses that cause severe human diseases. For example, dengue virus (DENV) is a rapidly emerging pathogen causing an estimated 100 million symptomatic infections annually worldwide. No approved antivirals are available to date and clinical trials with a tetravalent dengue vaccine showed disappointingly low protection rates. Hepatitis C virus (HCV) also remains a major medical problem, with 160 million chronically infected patients worldwide and only expensive treatments available. Despite distinct differences in their pathogenesis and modes of transmission, the two viruses share common replication strategies. A detailed understanding of the host functions that determine viral infection is lacking. Here we use a pooled CRISPR genetic screening strategy to comprehensively dissect host factors required for these two highly important Flaviviridae members. For DENV, we identified endoplasmic-reticulum (ER)-associated multi-protein complexes involved in signal sequence recognition, N-linked glycosylation and ER-associated degradation. DENV replication was nearly completely abrogated in cells deficient in the oligosaccharyltransferase (OST) complex. Mechanistic studies pinpointed viral RNA replication and not entry or translation as the crucial step requiring the OST complex. Moreover, we show that viral non-structural proteins bind to the OST complex. The identified ER-associated protein complexes were also important for infection by other mosquito-borne flaviviruses including Zika virus, an emerging pathogen causing severe birth defects. By contrast, the most significant genes identified in the HCV screen were distinct and included viral receptors, RNA-binding proteins and enzymes involved in metabolism. We found an unexpected link between intracellular flavin adenine dinucleotide (FAD) levels and HCV replication. This study shows notable divergence in host-depenency factors between DENV and HCV, and illuminates new host targets for antiviral therapy.

Impact of a patient-derived hepatitis C viral RNA genome with a mutated microRNA binding site.

Hepatitis C virus (HCV) depends on liver-specific microRNA miR-122 for efficient viral RNA amplification in liver cells. This microRNA interacts with two different conserved sites at the very 5' end of the viral RNA, enhancing miR-122 stability and promoting replication of the viral RNA. Treatment of HCV patients with oligonucleotides that sequester miR-122 resulted in profound loss of viral RNA in phase II clinical trials. However, some patients accumulated in their sera a viral RNA genome that contained a single cytidine to uridine mutation at the third nucleotide from the 5' genomic end. It is shown here that this C3U variant indeed displayed higher rates of replication than that of wild-type HCV when miR-122 abundance is low in liver cells. However, when miR-122 abundance is high, binding of miR-122 to site 1, most proximal to the 5' end in the C3U variant RNA, is impaired without disrupting the binding of miR-122 to site 2. As a result, C3U RNA displays a much lower rate of replication than wild-type mRNA when miR-122 abundance is high in the liver. This phenotype was accompanied by binding of a different set of cellular proteins to the 5' end of the C3U RNA genome. In particular, binding of RNA helicase DDX6 was important for displaying the C3U RNA replication phenotype in liver cells. These findings suggest that sequestration of miR-122 leads to a resistance-associated mutation that has only been observed in treated patients so far, and raises the question about the function of the C3U variant in the peripheral blood.

Precursor microRNA-122 inhibits synthesis of Insig1 isoform mRNA by modulating polyadenylation site usage.

The insulin-induced gene 1 protein (Insig1) inhibits the cholesterol biosynthesis pathway by retaining transcription factor SREBP in the endoplasmic reticulum, and by causing the degradation of HMGCR, the rate-limiting enzyme in cholesterol biosynthesis. Liver-specific microRNA miR-122, on the other hand, enhances cholesterol biosynthesis by an unknown mechanism. We have found that Insig1 mRNAs are generated by alternative cleavage and polyadenylation, resulting in specific isoform mRNA species. During high cholesterol abundance, the short 1.4-kb Insig1 mRNA was found to be preferentially translated to yield Insig1 protein. Precursor molecules of miR-122 down-regulated the translation of the 1.4-kb Insig1 isoform mRNA by interfering with the usage of the promoter-proximal cleavage-polyadenylation site that gives rise to the 1.4-kb Insig1 mRNA. These findings argue that precursor miR-122 molecules modulate polyadenylation site usage in Insig1 mRNAs, resulting in down-regulation of Insig1 protein abundance. Thus, precursor microRNAs may have hitherto undetected novel functions in nuclear gene expression.

Kinetic pathway of 40S ribosomal subunit recruitment to hepatitis C virus internal ribosome entry site.

Translation initiation can occur by multiple pathways. To delineate these pathways by single-molecule methods, fluorescently labeled ribosomal subunits are required. Here, we labeled human 40S ribosomal subunits with a fluorescent SNAP-tag at ribosomal protein eS25 (RPS25). The resulting ribosomal subunits could be specifically labeled in living cells and in vitro. Using single-molecule Förster resonance energy transfer (FRET) between RPS25 and domain II of the hepatitis C virus (HCV) internal ribosome entry site (IRES), we measured the rates of 40S subunit arrival to the HCV IRES. Our data support a single-step model of HCV IRES recruitment to 40S subunits, irreversible on the initiation time scale. We furthermore demonstrated that after binding, the 40S:HCV IRES complex is conformationally dynamic, undergoing slow large-scale rearrangements. Addition of translation extracts suppresses these fluctuations, funneling the complex into a single conformation on the 80S assembly pathway. These findings show that 40S:HCV IRES complex formation is accompanied by dynamic conformational rearrangements that may be modulated by initiation factors.

Herpes Simplex Virus is Akt-ing in translational control.

All viruses depend on the cellular protein synthesis machinery for the production of viral proteins. Thus, viruses have evolved a variety of strategies to avoid innate host responses that inhibit protein synthesis. In this issue of Genes & Development, Chuluunbaatar and colleagues (pp. 2627-2639) demonstrate that Herpes Simplex Virus-1 counteracts this response through viral kinase Us3, which mimics cellular kinase Akt to phosphorylate and repress tuberous sclerosis complex 2 (TSC2), resulting in the activation of mammalian target of rapamycin complex 1 (mTORC1) and enhancement of mRNA translation.

Dissecting noncoding and pathogen RNA-protein interactomes.

RNA-protein interactions are central to biological regulation. Cross-linking immunoprecipitation (CLIP)-seq is a powerful tool for genome-wide interrogation of RNA-protein interactomes, but current CLIP methods are limited by challenging biochemical steps and fail to detect many classes of noncoding and nonhuman RNAs. Here we present FAST-iCLIP, an integrated pipeline with improved CLIP biochemistry and an automated informatic pipeline for comprehensive analysis across protein coding, noncoding, repetitive, retroviral, and nonhuman transcriptomes. FAST-iCLIP of Poly-C binding protein 2 (PCBP2) showed that PCBP2-bound CU-rich motifs in different topologies to recognize mRNAs and noncoding RNAs with distinct biological functions. FAST-iCLIP of PCBP2 in hepatitis C virus-infected cells enabled a joint analysis of the PCBP2 interactome with host and viral RNAs and their interplay. These results show that FAST-iCLIP can be used to rapidly discover and decipher mechanisms of RNA-protein recognition across the diversity of human and pathogen RNAs.

Supporting Role for GTPase Rab27a in Hepatitis C Virus RNA Replication through a Novel miR-122-Mediated Effect.

The small GTPase Rab27a has been shown to control membrane trafficking and microvesicle transport pathways, in particular the secretion of exosomes. In the liver, high expression of Rab27a correlates with the development of hepatocellular carcinoma. We discovered that low abundance of Rab27a resulted in decreased hepatitis C virus (HCV) RNA and protein abundances in virus-infected cells. Curiously, both cell-associated and extracellular virus yield decreased in Rab27a depleted cells, suggesting that reduced exosome secretion did not cause the observed effect. Instead, Rab27a enhanced viral RNA replication by a mechanism that involves the liver-specific microRNA miR-122. Rab27a surrounded lipid droplets and was enriched in membrane fractions that harbor viral replication proteins, suggesting a supporting role for Rab27a in viral gene expression. Curiously, Rab27a depletion decreased the abundance of miR-122, whereas overexpression of miR-122 in Rab27a-depleted cells rescued HCV RNA abundance. Because intracellular HCV RNA abundance is enhanced by the binding of two miR-122 molecules to the extreme 5' end of the HCV RNA genome, the diminished amounts of miR-122 in Rab27a-depleted cells could have caused destabilization of HCV RNA. However, the abundance of HCV RNA carrying mutations on both miR-122-binding sites and whose stability was supported by ectopically expressed miR-122 mimetics with compensatory mutations also decreased in Rab27a-depleted cells. This result indicates that the effect of Rab27a depletion on HCV RNA abundance does not depend on the formation of 5' terminal HCV/miR-122 RNA complexes, but that miR-122 has a Rab27a-dependent function in the HCV lifecycle, likely the downregulation of a cellular inhibitor of HCV gene expression. These findings suggest that the absence of miR-122 results in a vulnerability not only to exoribonucleases that attack the viral genome, but also to upregulation of one more cellular factor that inhibit viral gene expression.

Interaction of host cell microRNAs with the HCV RNA genome during infection of liver cells.

It has remained an enigma how hepatitis C viral (HCV) RNA can persist in the liver of infected patients for many decades. With the recent discovery of roles for microRNAs in gene expression, it was reported that the HCV RNA genome subverts liver-specific microRNA miR-122 to protect its 5' end from degradation by host cell exoribonucleases. Sequestration of miR-122 in cultured liver cells and in the liver of chimpanzees by small, modified antisense RNAs resulted in dramatic loss of HCV RNA and viral yield. This finding led to the first successful human trial in which subcutaneous administration of antisense molecules against miR-122 lowered viral yield in HCV patients, without the emergence of resistant virus. In this review, the authors summarize the molecular mechanism by which miR-122 protects the HCV RNA genome from degradation by exoribonucleases Xrn1 and Xrn2 and discuss the application of miR-122 antisense molecules in the clinic.

Enhancement of hepatitis C viral RNA abundance by precursor miR-122 molecules.

The hepatitis C viral RNA genome forms a complex with liver-specific microRNA (miR-122) at the extreme 5' end of the viral RNA. This complex is essential to stabilize the viral RNA in infected cultured cells and in the liver of humans. The abundances of primary and precursor forms of miR-122, but not the abundance of mature miR-122, are regulated in a circadian rhythm in the liver of animals, suggesting a possible independent function of precursor molecules of miR-122 in regulating viral gene expression. Modified precursor molecules of miR-122 were synthesized that were refractory to cleavage by Dicer. These molecules were found to enhance the abundance of HCV RNA. Furthermore, they diminished the expression of mRNAs that contained binding sites for miR-122 in their 3' noncoding regions. By use of duplex and precursor miR-122 mimetic molecules that carried mutations in the passenger strand of miR-122, the effects on viral and reporter gene expression could be pinpointed to the action of precursor miR-122 molecules. Targeting the circadian expression of precursor miR-122 by specific compounds likely provides novel therapeutic strategies.

Modulation of GB virus B RNA abundance by microRNA-122: dependence on and escape from microRNA-122 restriction.

Hepatitis C virus (HCV) RNA forms an unusual interaction with human microRNA-122 (miR-122) that promotes viral RNA accumulation in cultured human liver cells and in the livers of infected chimpanzees. GB virus B (GBV-B) is a hepatotropic virus and close relative of HCV. Thus, GBV-B has been used as a surrogate system to study HCV amplification in cultured cells and in infected tamarins. It was discovered that the 5'-terminal sequences of GBV-B RNA, like HCV RNA, forms an Argonaute 2-mediated complex with two miR-122 molecules that are essential for accumulation of GBV-B subgenomic replicon RNA. However, sequences in miR-122 that anneal to each viral RNA genome were different, suggesting distinct overall structural features in HCV:miR-122 and GBV-B:miR-122 complexes. Surprisingly, a deletion that removed both miR-122 binding sites from the subgenomic GBV-B RNAs rendered viral RNA amplification independent from miR-122 and Argonaute 2. This finding suggests that structural features at the end of the viral genome dictate whether miR-122 is required to aid in maintaining viral RNA abundance.

LNA-mediated microRNA silencing in non-human primates.

microRNAs (miRNAs) are small regulatory RNAs that are important in development and disease and therefore represent a potential new class of targets for therapeutic intervention. Despite recent progress in silencing of miRNAs in rodents, the development of effective and safe approaches for sequence-specific antagonism of miRNAs in vivo remains a significant scientific and therapeutic challenge. Moreover, there are no reports of miRNA antagonism in primates. Here we show that the simple systemic delivery of a unconjugated, PBS-formulated locked-nucleic-acid-modified oligonucleotide (LNA-antimiR) effectively antagonizes the liver-expressed miR-122 in non-human primates. Acute administration by intravenous injections of 3 or 10 mg kg(-1) LNA-antimiR to African green monkeys resulted in uptake of the LNA-antimiR in the cytoplasm of primate hepatocytes and formation of stable heteroduplexes between the LNA-antimiR and miR-122. This was accompanied by depletion of mature miR-122 and dose-dependent lowering of plasma cholesterol. Efficient silencing of miR-122 was achieved in primates by three doses of 10 mg kg(-1) LNA-antimiR, leading to a long-lasting and reversible decrease in total plasma cholesterol without any evidence for LNA-associated toxicities or histopathological changes in the study animals. Our findings demonstrate the utility of systemically administered LNA-antimiRs in exploring miRNA function in rodents and primates, and support the potential of these compounds as a new class of therapeutics for disease-associated miRNAs.

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

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.

CRISPR Screen Reveals PACT as a Pro-Viral Factor for Dengue Viral Replication.

The dengue virus is a single-stranded, positive-sense RNA virus that infects ~400 million people worldwide. Currently, there are no approved antivirals available. CRISPR-based screening methods have greatly accelerated the discovery of host factors that are essential for DENV infection and that can be targeted in host-directed antiviral interventions. In the present study, we performed a focused CRISPR (Clustered Regularly Interspaced Palindromic Repeats) library screen to discover the key host factors that are essential for DENV infection in human Huh7 cells and identified the Protein Activator of Interferon-Induced Protein Kinase (PACT) as a novel pro-viral factor for DENV. PACT is a double-stranded RNA-binding protein generally known to activate antiviral responses in virus-infected cells and block viral replication. However, in our studies, we observed that PACT plays a pro-viral role in DENV infection and specifically promotes viral RNA replication. Knockout of PACT resulted in a significant decrease in DENV RNA and protein abundances in infected cells, which was rescued upon ectopic expression of full-length PACT. An analysis of global gene expression changes indicated that several ER-associated pro-viral genes such as ERN1, DDIT3, HERPUD1, and EIF2AK3 are not upregulated in DENV-infected PACT knockout cells as compared to infected wildtype cells. Thus, our study demonstrates a novel role for PACT in promoting DENV replication, possibly through modulating the expression of ER-associated pro-viral genes.