JFH1-based Core-NS2 genotype variants of HCV with genetic stability in vivo and in vitro: Important tools in evaluation of virus neutralization.

BACKGROUND AIMS: HCV infection continues to be a major global health burden, despite effective antiviral treatments. The urgent need for a protective vaccine is hindered by the scarcity of suitable HCV permissive animal models tractable in vaccination and challenge studies. Currently, only antibody neutralization studies in infectious cell culture systems or studies of protection by passive immunization of human-liver chimeric mice offer the possibility to evaluate the effect of vaccine-induced antibodies. However, differences between culture-permissive and in vivo-permissive viruses make it a challenge to compare analyses between platforms. To address this problem, we aimed at developing genotype-specific virus variants with genetic stability both in vitro and in vivo. APPROACH RESULTS: We demonstrated infection of human-liver chimeric mice with cell culture-adapted HCV JFH1-based Core-NS2 recombinants of genotype 1-6, with a panel of 10 virus strains used extensively in neutralization and receptor studies. Clonal re-engineering of mouse-selected mutations resulted in virus variants with robust replication both in Huh7.5 cells and human-liver chimeric mice, with genetic stability. Furthermore, we showed that overall, these virus variants have similar in vitro neutralization profiles as their parent strains and demonstrated their use for in vivo neutralization studies. CONCLUSIONS: These mouse-selected HCV recombinants enable triage of new vaccine-relevant antibodies in vitro and further allow characterization of protection from infection in vivo using identical viruses in human-liver chimeric mice. As such, these viruses will serve as important resources in testing novel antibodies and can thus guide strategies to develop an efficient protective vaccine against HCV infection.

Host genetic variation guides hepacivirus clearance, chronicity, and liver fibrosis in mice.

BACKGROUND AIMS: Human genetic variation is thought to guide the outcome of HCV infection, but model systems within which to dissect these host genetic mechanisms are limited. Norway rat hepacivirus, closely related to HCV, causes chronic liver infection in rats but causes acute self-limiting hepatitis in typical strains of laboratory mice, which resolves in 2 weeks. The Collaborative Cross (CC) is a robust mouse genetics resource comprised of a panel of recombinant inbred strains, which model the complexity of the human genome and provide a system within which to understand diseases driven by complex allelic variation. APPROACH RESULTS: We infected a panel of CC strains with Norway rat hepacivirus and identified several that failed to clear the virus after 4 weeks. Strains displayed an array of virologic phenotypes ranging from delayed clearance (CC046) to chronicity (CC071, CC080) with viremia for at least 10 months. Body weight loss, hepatocyte infection frequency, viral evolution, T-cell recruitment to the liver, liver inflammation, and the capacity to develop liver fibrosis varied among infected CC strains. CONCLUSIONS: These models recapitulate many aspects of HCV infection in humans and demonstrate that host genetic variation affects a multitude of viruses and host phenotypes. These models can be used to better understand the molecular mechanisms that drive hepacivirus clearance and chronicity, the virus and host interactions that promote chronic disease manifestations like liver fibrosis, therapeutic and vaccine performance, and how these factors are affected by host genetic variation.

Hepatitis C virus RNA is 5'-capped with flavin adenine dinucleotide.

RNA viruses have evolved elaborate strategies to protect their genomes, including 5' capping. However, until now no RNA 5' cap has been identified for hepatitis C virus1,2 (HCV), which causes chronic infection, liver cirrhosis and cancer3. Here we demonstrate that the cellular metabolite flavin adenine dinucleotide (FAD) is used as a non-canonical initiating nucleotide by the viral RNA-dependent RNA polymerase, resulting in a 5'-FAD cap on the HCV RNA. The HCV FAD-capping frequency is around 75%, which is the highest observed for any RNA metabolite cap across all kingdoms of life4-8. FAD capping is conserved among HCV isolates for the replication-intermediate negative strand and partially for the positive strand. It is also observed in vivo on HCV RNA isolated from patient samples and from the liver and serum of a human liver chimeric mouse model. Furthermore, we show that 5'-FAD capping protects RNA from RIG-I mediated innate immune recognition but does not stabilize the HCV RNA. These results establish capping with cellular metabolites as a novel viral RNA-capping strategy, which could be used by other viruses and affect anti-viral treatment outcomes and persistence of infection.

Characterising the RNA-binding protein atlas of the mammalian brain uncovers RBM5 misregulation in mouse models of Huntington's disease.

RNA-binding proteins (RBPs) are key players regulating RNA processing and are associated with disorders ranging from cancer to neurodegeneration. Here, we present a proteomics workflow for large-scale identification of RBPs and their RNA-binding regions in the mammalian brain identifying 526 RBPs. Analysing brain tissue from males of the Huntington's disease (HD) R6/2 mouse model uncovered differential RNA-binding of the alternative splicing regulator RBM5. Combining several omics workflows, we show that RBM5 binds differentially to transcripts enriched in pathways of neurodegeneration in R6/2 brain tissue. We further find these transcripts to undergo changes in splicing and demonstrate that RBM5 directly regulates these changes in human neurons derived from embryonic stem cells. Finally, we reveal that RBM5 interacts differently with several known huntingtin interactors and components of huntingtin aggregates. Collectively, we demonstrate the applicability of our method for capturing RNA interactor dynamics in the contexts of tissue and disease.

Differential activity of nucleotide analogs against tick-borne encephalitis and yellow fever viruses in human cell lines.

With no approved antiviral therapies, the continuous emergence and re-emergence of tick-borne encephalitis virus (TBEV) and yellow fever virus (YFV) is a rising concern. We performed head-to-head comparisons of the antiviral activity of available nucleos(t)ide analogs (nucs) using relevant human cell lines. Eight existing nucs inhibited TBEV and/or YFV with differential activity between cell lines and viruses. Remdesivir, uprifosbuvir and sofosbuvir were the most potent drugs against TBEV and YFV in liver cells, but they had reduced activity in neural cells, whereas galidesivir retained uniform activity across cell lines and viruses. Ribavirin, valopicitabine, molnupiravir and GS-6620 exhibited only moderate antiviral activity. We found antiviral activity for drugs previously reported as inactive, demonstrating the importance of using human cell lines and comparative experimental assays when screening the activity of nucs. The relatively high antiviral activity of remdesivir, sofosbuvir and uprifosbuvir against TBEV and YFV merits further investigation in clinical studies.

Molecular Determinants of Mouse Adaptation of Rat Hepacivirus.

The lack of robust immunocompetent animal models for hepatitis C virus (HCV) impedes vaccine development and studies of immune responses. Norway rat hepacivirus (NrHV) infection in rats shares HCV-defining characteristics, including hepatotropism, chronicity, immune responses, and aspects of liver pathology. To exploit genetic variants and research tools, we previously adapted NrHV to prolonged infection in laboratory mice. Through intrahepatic RNA inoculation of molecular clones of the identified variants, we here characterized four mutations in the envelope proteins responsible for mouse adaptation, including one disrupting a glycosylation site. These mutations led to high-titer viremia, similar to that observed in rats. In 4-week-old mice, infection was cleared after around 5 weeks compared to 2 to 3 weeks for nonadapted virus. In contrast, the mutations led to persistent but attenuated infection in rats, and they partially reverted, accompanied by an increase in viremia. Attenuated infection in rat but not mouse hepatoma cells demonstrated that the characterized mutations were indeed mouse adaptive rather than generally adaptive across species and that species determinants and not immune interactions were responsible for attenuation in rats. Unlike persistent NrHV infection in rats, acute resolving infection in mice was not associated with the development of neutralizing antibodies. Finally, infection of scavenger receptor B-I (SR-BI) knockout mice suggested that adaptation to mouse SR-BI was not a primary function of the identified mutations. Rather, the virus may have adapted to lower dependency on SR-BI, thereby potentially surpassing species-specific differences. In conclusion, we identified specific determinants of NrHV mouse adaptation, suggesting species-specific interactions during entry. IMPORTANCE A prophylactic vaccine is required to achieve the World Health Organization's objective for hepatitis C virus elimination as a serious public health threat. However, the lack of robust immunocompetent animal models supporting hepatitis C virus infection impedes vaccine development as well as studies of immune responses and viral evasion. Hepatitis C virus-related hepaciviruses were discovered in a number of animal species and provide useful surrogate infection models. Norway rat hepacivirus is of particular interest, as it enables studies in rats, an immunocompetent and widely used small laboratory animal model. Its adaptation to robust infection also in laboratory mice provides access to a broader set of mouse genetic lines and comprehensive research tools. The presented mouse-adapted infectious clones will be of utility for reverse genetic studies, and the Norway rat hepacivirus mouse model will facilitate studies of hepacivirus infection for in-depth characterization of virus-host interactions, immune responses, and liver pathology.

Identification of the viral and cellular microRNA interactomes during SARS-CoV-2 infection.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has had a tremendous impact worldwide. Mapping virus-host interactions is critical to understand disease progression. MicroRNAs (miRNAs) are important RNA regulators, but their interaction with SARS-CoV-2 RNA was not experimentally investigated. Here, using Argonaute (AGO) cross-linking immunoprecipitation combined with RNA proximity ligation (CLEAR-CLIP), we provide unbiased mapping of SARS-CoV-2/miRNA interactions. We identified six main regions on the viral RNA bound primarily by one specific miRNA. Targeted mutagenesis and AGO1-3 knockdown demonstrated that these interactions are not critical for virus production. Moreover, we identified perturbed regulation of cellular miRNA interactions during infection, including non-compensated viral sequestration of the miR-15 family. Transcriptome analysis further showed that mRNAs targeted by this miRNA family are derepressed. This work delineates the interphase between miRNA regulation and SARS-CoV-2 infection and further contributes to deciphering the full molecular interactome of this virus.

A Distinct Dexamethasone-Dependent Gene Expression Profile in the Lungs of COVID-19 Patients.

The effects of dexamethasone (DXM) treatment on pulmonary immunity in COVID-19-associated acute respiratory distress syndrome (CARDS) remain insufficiently understood. We performed transcriptomic RNA-seq analysis of bronchoalveolar lavage fluid from 20 mechanically ventilated patients: 12 with CARDS (with or without DXM) and 8 non-COVID-19 critically ill controls. CARDS with DXM was characterized by upregulation of genes related to B-cell and complement pathway activation, antigen presentation, phagocytosis, and FC-γ receptor signaling. Most interferon-stimulated genes were upregulated in CARDS, particularly in CARDS without DXM. In conclusion, DXM treatment was not associated with regulation of proinflammatory pathways in CARDS but with regulation of other local immune responses. Clinical Trials Registration. NCT04354584.

Neutralization and receptor use of infectious culture-derived rat hepacivirus as a model for HCV.

BACKGROUND AND AIMS: Lack of tractable immunocompetent animal models amenable to robust experimental challenge impedes vaccine efforts for HCV. Infection with rodent hepacivirus from Rattus norvegicus (RHV-rn1) in rats shares HCV-defining characteristics, including liver tropism, chronicity, and pathology. RHV in vitro cultivation would facilitate genetic studies on particle production, host factor interactions, and evaluation of antibody neutralization guiding HCV vaccine approaches. APPROACH AND RESULTS: We report an infectious reverse genetic cell culture system for RHV-rn1 using highly permissive rat hepatoma cells and adaptive mutations in the E2, NS4B, and NS5A viral proteins. Cell culture-derived RHV-rn1 particles (RHVcc) share hallmark biophysical characteristics of HCV and are infectious in mice and rats. Culture adaptive mutations attenuated RHVcc in immunocompetent rats, and the mutations reverted following prolonged infection, but not in severe combined immunodeficiency (SCID) mice, suggesting that adaptive immune pressure is a primary driver of reversion. Accordingly, sera from RHVcc-infected SCID mice or the early acute phase of immunocompetent mice and rats were infectious in culture. We further established an in vitro RHVcc neutralization assay, and observed neutralizing activity of rat sera specifically from the chronic phase of infection. Finally, we found that scavenger receptor class B type I promoted RHV-rn1 entry in vitro and in vivo. CONCLUSIONS: The RHV-rn1 infectious cell culture system enables studies of humoral immune responses against hepacivirus infection. Moreover, recapitulation of the entire RHV-rn1 infectious cycle in cell culture will facilitate reverse genetic studies and the exploration of tropism and virus-host interactions.

Versatile SARS-CoV-2 Reverse-Genetics Systems for the Study of Antiviral Resistance and Replication.

The COVID-19 pandemic continues to threaten healthcare systems worldwide due to the limited access to vaccines, suboptimal treatment options, and the continuous emergence of new and more transmissible SARS-CoV-2 variants. Reverse-genetics studies of viral genes and mutations have proven highly valuable in advancing basic virus research, leading to the development of therapeutics. We developed a functional and highly versatile full-length SARS-CoV-2 infectious system by cloning the sequence of a COVID-19 associated virus isolate (DK-AHH1) into a bacterial artificial chromosome (BAC). Viruses recovered after RNA-transfection of in vitro transcripts into Vero E6 cells showed growth kinetics and remdesivir susceptibility similar to the DK-AHH1 virus isolate. Insertion of reporter genes, green fluorescent protein, and nanoluciferase into the ORF7 genomic region led to high levels of reporter activity, which facilitated high throughput treatment experiments. We found that putative coronavirus remdesivir resistance-associated substitutions F480L and V570L-and naturally found polymorphisms A97V, P323L, and N491S, all in nsp12-did not decrease SARS-CoV-2 susceptibility to remdesivir. A nanoluciferase reporter clone with deletion of spike (S), envelope (E), and membrane (M) proteins exhibited high levels of transient replication, was inhibited by remdesivir, and therefore could function as an efficient non-infectious subgenomic replicon system. The developed SARS-CoV-2 reverse-genetics systems, including recombinants to modify infectious viruses and non-infectious subgenomic replicons with autonomous genomic RNA replication, will permit high-throughput cell culture studies-providing fundamental understanding of basic biology of this coronavirus. We have proven the utility of the systems in rapidly introducing mutations in nsp12 and studying their effect on the efficacy of remdesivir, which is used worldwide for the treatment of COVID-19. Our system provides a platform to effectively test the antiviral activity of drugs and the phenotype of SARS-CoV-2 mutants.

Hepatitis C Virus Protease Inhibitors Show Differential Efficacy and Interactions with Remdesivir for Treatment of SARS-CoV-2 In Vitro.

Antivirals targeting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) could improve treatment of COVID-19. We evaluated the efficacy of clinically relevant hepatitis C virus (HCV) NS3 protease inhibitors (PIs) against SARS-CoV-2 and their interactions with remdesivir, the only direct-acting antiviral approved for COVID-19 treatment. HCV PIs showed differential potency in short-term treatment assays based on the detection of SARS-CoV-2 spike protein in Vero E6 cells. Linear PIs boceprevir, telaprevir, and narlaprevir had 50% effective concentrations (EC50) of ∼40 µM. Among the macrocyclic PIs, simeprevir had the highest (EC50, 15 µM) and glecaprevir the lowest (EC50, >178 µM) potency, with paritaprevir, grazoprevir, voxilaprevir, vaniprevir, danoprevir, and deldeprevir in between. Acyclic PIs asunaprevir and faldaprevir had EC50s of 72 and 23 µM, respectively. ACH-806, inhibiting the HCV NS4A protease cofactor, had an EC50 of 46 µM. Similar and slightly increased PI potencies were found in human hepatoma Huh7.5 cells and human lung carcinoma A549-hACE2 cells, respectively. Selectivity indexes based on antiviral and cell viability assays were highest for linear PIs. In short-term treatments, combination of macrocyclic but not linear PIs with remdesivir showed synergism in Vero E6 and A549-hACE2 cells. Longer-term treatment of infected Vero E6 and A549-hACE2 cells with 1-fold EC50 PI revealed minor differences in the barrier to SARS-CoV-2 escape. Viral suppression was achieved with 3- to 8-fold EC50 boceprevir or 1-fold EC50 simeprevir or grazoprevir, but not boceprevir, in combination with 0.4- to 0.8-fold EC50 remdesivir; these concentrations did not lead to viral suppression in single treatments. This study could inform the development and application of protease inhibitors for optimized antiviral treatments of COVID-19.

Argonaute-CLIP delineates versatile, functional RNAi networks in Aedes aegypti, a major vector of human viruses.

Argonaute (AGO) proteins bind small RNAs to silence complementary RNA transcripts, and they are central to RNA interference (RNAi). RNAi is critical for regulation of gene expression and antiviral defense in Aedes aegypti mosquitoes, which transmit Zika, chikungunya, dengue, and yellow fever viruses. In mosquitoes, AGO1 mediates miRNA interactions, while AGO2 mediates siRNA interactions. We applied AGO-crosslinking immunoprecipitation (AGO-CLIP) for both AGO1 and AGO2, and we developed a universal software package for CLIP analysis (CLIPflexR), identifying 230 small RNAs and 5,447 small RNA targets that comprise a comprehensive RNAi network map in mosquitoes. RNAi network maps predicted expression levels of small RNA targets in specific tissues. Additionally, this resource identified unexpected, context-dependent AGO2 target preferences, including endogenous viral elements and 3'UTRs. Finally, contrary to current thinking, mosquito AGO2 repressed imperfect targets. These findings expand our understanding of small RNA networks and have broad implications for the study of antiviral RNAi.

Pathogenesis, MicroRNA-122 Gene-Regulation, and Protective Immune Responses After Acute Equine Hepacivirus Infection.

BACKGROUND AND AIMS: Equine hepacivirus (EqHV) is phylogenetically the closest relative of HCV and shares genome organization, hepatotropism, transient or persistent infection outcome, and the ability to cause hepatitis. Thus, EqHV studies are important to understand equine liver disease and further as an outbred surrogate animal model for HCV pathogenesis and protective immune responses. Here, we aimed to characterize the course of EqHV infection and associated protective immune responses. APPROACH AND RESULTS: Seven horses were experimentally inoculated with EqHV, monitored for 6 months, and rechallenged with the same and, subsequently, a heterologous EqHV. Clearance was the primary outcome (6 of 7) and was associated with subclinical hepatitis characterized by lymphocytic infiltrate and individual hepatocyte necrosis. Seroconversion was delayed and antibody titers waned slowly. Clearance of primary infection conferred nonsterilizing immunity, resulting in shortened duration of viremia after rechallenge. Peripheral blood mononuclear cell responses in horses were minimal, although EqHV-specific T cells were identified. Additionally, an interferon-stimulated gene signature was detected in the liver during EqHV infection, similar to acute HCV in humans. EqHV, as HCV, is stimulated by direct binding of the liver-specific microRNA (miR), miR-122. Interestingly, we found that EqHV infection sequesters enough miR-122 to functionally affect gene regulation in the liver. This RNA-based mechanism thus could have consequences for pathology. CONCLUSIONS: EqHV infection in horses typically has an acute resolving course, and the protective immune response lasts for at least a year and broadly attenuates subsequent infections. This could have important implications to achieve the primary goal of an HCV vaccine; to prevent chronicity while accepting acute resolving infection after virus exposure.

Equine pegiviruses cause persistent infection of bone marrow and are not associated with hepatitis.

Pegiviruses frequently cause persistent infection (as defined by >6 months), but unlike most other Flaviviridae members, no apparent clinical disease. Human pegivirus (HPgV, previously GBV-C) is detectable in 1-4% of healthy individuals and another 5-13% are seropositive. Some evidence for infection of bone marrow and spleen exists. Equine pegivirus 1 (EPgV-1) is not linked to disease, whereas another pegivirus, Theiler's disease-associated virus (TDAV), was identified in an outbreak of acute serum hepatitis (Theiler's disease) in horses. Although no subsequent reports link TDAV to disease, any association with hepatitis has not been formally examined. Here, we characterized EPgV-1 and TDAV tropism, sequence diversity, persistence and association with liver disease in horses. Among more than 20 tissue types, we consistently detected high viral loads only in serum, bone marrow and spleen, and viral RNA replication was consistently identified in bone marrow. PBMCs and lymph nodes, but not liver, were sporadically positive. To exclude potential effects of co-infecting agents in experimental infections, we constructed full-length consensus cDNA clones; this was enabled by determination of the complete viral genomes, including a novel TDAV 3' terminus. Clone derived RNA transcripts were used for direct intrasplenic inoculation of healthy horses. This led to productive infection detectable from week 2-3 and persisting beyond the 28 weeks of study. We did not observe any clinical signs of illness or elevation of circulating liver enzymes. The polyprotein consensus sequences did not change, suggesting that both clones were fully functional. To our knowledge, this is the first successful extrahepatic viral RNA launch and the first robust reverse genetics system for a pegivirus. In conclusion, equine pegiviruses are bone marrow tropic, cause persistent infection in horses, and are not associated with hepatitis. Based on these findings, it may be appropriate to rename the group of TDAV and related viruses as EPgV-2.

Evolutionary selection of pestivirus variants with altered or no microRNA dependency.

Host microRNA (miRNA) dependency is a hallmark of the human pathogen hepatitis C virus (HCV) and was also described for the related pestiviruses, which are important livestock pathogens. The liver-specific miR-122 binds within the HCV 5' untranslated region (UTR), whereas the broadly expressed let-7 and miR-17 families bind two sites (S1 and S2, respectively) in the pestiviral 3' UTR. Here, we dissected the mechanism of miRNA dependency of the pestivirus bovine viral diarrhea virus (BVDV). Argonaute 2 (AGO2) and miR-17 binding were essential for viral replication, whereas let-7 binding was mainly required for full translational efficiency. Furthermore, using seed site randomized genomes and evolutionary selection experiments, we found that tropism could be redirected to different miRNAs. AGO cross-linking and immunoprecipitation (CLIP) experiments and miRNA antagonism demonstrated that these alternative variants bound and depended on the corresponding miRNAs. Interestingly, we also identified miRNA-independent variants that were obtained through acquisition of compensatory mutations near the genomic 3' terminus. Rescue experiments demonstrated that miRNA binding and 3' mutagenesis contribute to replication through mutually exclusive mechanisms. Altogether, our findings suggest that pestiviruses, although capable of miRNA-independent replication, took advantage of miRNAs as essential host factors, suggesting a favorable path during evolutionary adaptation.

Replicons of a Rodent Hepatitis C Model Virus Permit Selection of Highly Permissive Cells.

Animal hepaciviruses represent promising surrogate models for hepatitis C virus (HCV), for which there are no efficient immunocompetent animal models. Experimental infection of laboratory rats with rodent hepacivirus isolated from feral Rattus norvegicus (RHV-rn1) mirrors key aspects of HCV infection in humans, including chronicity, hepatitis, and steatosis. Moreover, RHV has been adapted to infect immunocompetent laboratory mice. RHV in vitro systems have not been developed but would enable detailed studies of the virus life cycle crucial for designing animal experiments to model HCV infection. Here, we established efficient RHV-rn1 selectable subgenomic replicons with and without reporter genes. Rat and mouse liver-derived cells did not readily support the complete RHV life cycle, but replicon-containing cell clones could be selected with and without acquired mutations. Replication was significantly enhanced by mutations in NS4B and NS5A and in cell clones cured of replicon RNA. These mutations increased RHV replication of both mono- and bicistronic constructs, and CpG/UpA-dinucleotide optimization of reporter genes allowed replication. Using the replicon system, we show that the RHV-rn1 NS3-4A protease cleaves a human mitochondrial antiviral signaling protein reporter, providing a sensitive readout for virus replication. RHV-rn1 replication was inhibited by the HCV polymerase inhibitor sofosbuvir and high concentrations of HCV NS5A antivirals but not by NS3 protease inhibitors. The microRNA-122 antagonist miravirsen inhibited RHV-rn1 replication, demonstrating the importance of this HCV host factor for RHV. These novel RHV in vitro systems will be useful for studies of tropism, molecular virology, and characterization of virus-host interactions, thereby providing important complements to in vivo systems.IMPORTANCE A vaccine against hepatitis C virus (HCV) is crucial for global control of this important pathogen, which induces fatal human liver diseases. Vaccine development has been hampered by the lack of immunocompetent animal models. Discovery of rodent hepacivirus (RHV) enabled establishment of novel surrogate animal models. These allow robust infection and reverse genetic and immunization studies of laboratory animals, which develop HCV-like chronicity. Currently, there are no RHV in vitro systems available to study tropism and molecular virology. Here, we established the first culture systems for RHV, recapitulating the intracellular phase of the virus life cycle in vitro These replicon systems enabled identification of replication-enhancing mutations and selection of cells highly permissive to RHV replication, which allow study of virus-host interactions. HCV antivirals targeting NS5A, NS5B, and microRNA-122 efficiently inhibited RHV replication. Hence, several important aspects of HCV replication are shared by the rodent virus system, reinforcing its utility as an HCV model.

Functional Interplay between RNA Viruses and Non-Coding RNA in Mammals.

Exploring virus⁻host interactions is key to understand mechanisms regulating the viral replicative cycle and any pathological outcomes associated with infection. Whereas interactions at the protein level are well explored, RNA interactions are less so. Novel sequencing methodologies have helped uncover the importance of RNA⁻protein and RNA⁻RNA interactions during infection. In addition to messenger RNAs (mRNAs), mammalian cells express a great number of regulatory non-coding RNAs, some of which are crucial for regulation of the immune system whereas others are utilized by viruses. It is thus becoming increasingly clear that RNA interactions play important roles for both sides in the arms race between virus and host. With the emerging field of RNA therapeutics, such interactions are promising antiviral targets. In this review, we discuss direct and indirect RNA interactions occurring between RNA viruses or retroviruses and host non-coding transcripts upon infection. In addition, we review RNA virus derived non-coding RNAs affecting immunological and metabolic pathways of the host cell typically to provide an advantage to the virus. The relatively few known examples of virus⁻host RNA interactions suggest that many more await discovery.

Correction: miRNA independent hepacivirus variants suggest a strong evolutionary pressure to maintain miR-122 dependence.

[This corrects the article DOI: 10.1371/journal.ppat.1006694.].

Viral persistence, liver disease, and host response in a hepatitis C-like virus rat model.

The lack of a relevant, tractable, and immunocompetent animal model for hepatitis C virus (HCV) has severely impeded investigations of viral persistence, immunity, and pathogenesis. In the absence of immunocompetent models with robust HCV infection, homolog hepaciviruses in their natural host could potentially provide useful surrogate models. We isolated a rodent hepacivirus from wild rats (Rattus norvegicus), RHV-rn1; acquired the complete viral genome sequence; and developed an infectious reverse genetics system. RHV-rn1 resembles HCV in genomic features including the pattern of polyprotein cleavage sites and secondary structures in the viral 5' and 3' untranslated regions. We used site-directed and random mutagenesis to determine that only the first of the two microRNA-122 seed sites in the viral 5' untranslated region is required for viral replication and persistence in rats. Next, we used the clone-derived virus progeny to infect several inbred and outbred rat strains. Our results determined that RHV-rn1 possesses several HCV-defining hallmarks: hepatotropism, propensity to persist, and the ability to induce gradual liver damage. Histological examination of liver samples revealed the presence of lymphoid aggregates, parenchymal inflammation, and macrovesicular and microvesicular steatosis in chronically infected rats. Gene expression analysis demonstrated that the intrahepatic response during RHV-rn1 infection in rats mirrors that of HCV infection, including persistent activation of interferon signaling pathways. Finally, we determined that the backbone drug of HCV direct-acting antiviral therapy, sofosbuvir, effectively suppresses chronic RHV-rn1 infection in rats. CONCLUSION: We developed RHV-rn1-infected rats as a fully immunocompetent and informative surrogate model to delineate the mechanisms of HCV-related viral persistence, immunity, and pathogenesis. (Hepatology 2018).