Enhancement of replication of RNA viruses by ADAR1 via RNA editing and inhibition of RNA-activated protein kinase

Adenosine deaminase acting on RNA 1 (ADAR1) is a double-stranded RNA binding protein and RNA-editing enzyme that modifies cellular and viral RNAs, including coding and noncoding RNAs. This interferon (IFN)-induced protein was expected to have an antiviral role, but recent studies have demonstrated that it promotes the replication of many RNA viruses. The data from these experiments show that ADAR1 directly enhances replication of hepatitis delta virus, human immunodeficiency virus type 1, vesicular stomatitis virus, and measles virus. The proviral activity of ADAR1 occurs through two mechanisms: RNA editing and inhibition of RNA-activated protein kinase (PKR). While these pathways have been found independently, the two mechanisms can act in concert to increase viral replication and contribute to viral pathogenesis. This novel type of proviral regulation by an IFN-induced protein, combined with some antiviral effects of hyperediting, sheds new light on the importance of ADAR1 during viral infection and transforms our overall understanding of the innate immune response.     

Toll-like receptor 7 is required for effective adaptive immune responses that prevent persistent virus infection

TLR7 is an innate signaling receptor that recognizes single-stranded viral RNA and is activated by viruses that cause persistent infections. We show that TLR7 signaling dictates either clearance or establishment of life-long chronic infection by lymphocytic choriomeningitis virus (LCMV) Cl 13 but does not affect clearance of the acute LCMV Armstrong 53b strain. TLR7(-/-) mice infected with LCMV Cl 13 remained viremic throughout life from defects in the adaptive antiviral immune response-notably, diminished T?cell function, exacerbated T?cell exhaustion, decreased plasma cell maturation, and negligible antiviral antibody production. Adoptive transfer of TLR7(+/+) LCMV immune memory cells that enhanced clearance of persistent LCMV Cl 13 infection in TLR7(+/+) mice failed to purge LCMV Cl 13 infection in TLR7(-/-) mice, demonstrating that a TLR7-deficient environment renders antiviral responses ineffective. Therefore, methods that promote TLR7 signaling are promising treatment strategies for chronic viral infections.     

Early Virus-Host Interactions Dictate the Course of a Persistent Infection

Many persistent viral infections are characterized by a hypofunctional T cell response and the upregulation of negative immune regulators. These events occur days after the initiation of infection. However, the very early host-virus interactions that determine the establishment of viral persistence remain poorly uncharacterized. Here we show that to establish persistence, LCMV must counteract an innate anti-viral immune response within eight hours after infection. While the virus triggers cytoplasmic RNA sensing pathways soon after infection, LCMV counteracts this pathway through a rapid increase in viral titers leading to a dysfunctional immune response characterized by a high cytokine and chemokine expression profile. This altered immune environment allows for viral replication in the splenic white pulp as well as infection of immune cells essential to an effective anti-viral immune response. Our findings illustrate how early events during infection critically dictate the characteristics of the immune response to infection and facilitate either virus control and clearance or persistence.     

Double-stranded RNA deaminase ADAR1 increases host susceptibility to virus infection

The RNA-editing enzyme ADAR1 is a double-stranded RNA (dsRNA) binding protein that modifies cellular and viral RNA sequences by adenosine deamination. ADAR1 has been demonstrated to play important roles in embryonic erythropoiesis, viral response, and RNA interference. In human hepatitis virus infection, ADAR1 has been shown to target viral RNA and to suppress viral replication through dsRNA editing. It is not clear whether this antiviral effect of ADAR1 is a common mechanism in response to viral infection. Here, we report a proviral effect of ADAR1 that enhances replication of vesicular stomatitis virus (VSV) through a mechanism independent of dsRNA editing. We demonstrate that ADAR1 interacts with dsRNA-activated protein kinase PKR, inhibits its kinase activity, and suppresses the alpha subunit of eukaryotic initiation factor 2 (eIF-2alpha) phosphorylation. Consistent with the inhibitory effect on PKR activation, ADAR1 increases VSV infection in PKR+/+ mouse embryonic fibroblasts; however, no significant effect was found in PKR-/- cells. This proviral effect of ADAR1 requires the N-terminal domains but does not require the deaminase domain. These findings reveal a novel mechanism of ADAR1 that increases host susceptibility to viral infection by inhibiting PKR activation.     

In vivo selection of a lymphocytic choriomeningitis virus variant that affects recognition of the GP33-43 epitope by H-2Db but not H-2Kb

CD8 T cells drive the protective immune response to lymphocytic choriomeningitis virus (LCMV) infection and are thus a determining force in the selection of viral variants. To examine how escape mutations affect the presentation and recognition of overlapping T-cell epitopes, we isolated an LCMV variant that is not recognized by T-cell receptor (TCR)-transgenic H-2Db-restricted LCMV GP33-41-specific cytotoxic T lymphocytes (CTL). The variant virus carried a single-amino-acid substitution (valine to alanine) at position 35 of the viral glycoprotein. This region of the LCMV glycoprotein encodes both the Db-restricted GP33-43 epitope and a second epitope (GP34-42) presented by the Kb molecule. We determined that the V-to-A CTL escape mutant failed to induce a Db GP33-43-specific CTL response and that Db-restricted GP33-43-specific CTL induced by the wild-type LCMV strain were unable to kill target cells infected with the variant LCMV strain. In contrast, the Kb-restricted response was much less affected. We found that the V-to-A substitution severely impaired peptide binding to Db but not to Kb molecules. Strikingly, the V-to-A mutation did not change any of the anchor residues, and the dramatic effect on binding was therefore unexpected. The strong decrease in Db binding explains why the variant virus escapes the Db GP33-43-specific response but still elicits the Kb-restricted response. These findings also illustrate that mutations within regions encoding overlapping T-cell epitopes can differentially affect the presentation and recognition of individual epitopes.     

Elevated activity of the large form of ADAR1 in vivo: very efficient RNA editing occurs in the cytoplasm

Mammalian cells express small and large forms of the RNA editing enzyme ADAR1, referred to as ADAR1-S and ADAR1-L, respectively. Here we observed that ADAR1-L was >70-fold more active than was ADAR1-S when assayed with a substrate that could be edited in either the nucleus or cytoplasm, and was also much more active when assayed with a substrate that was generated in the cytoplasm during viral replication. In contrast, when a substrate that could only be edited within the nucleus was assayed, the activity of ADAR1-S was found to be somewhat higher than that of ADAR1-L. We show here not only that editing could occur in the cytoplasm but also that the process was extremely efficient, occurred rapidly, and could occur in the absence of translation. Consistent with the observation that editing in the cytoplasm can be very efficient, deletion of the nuclear localization signal from ADAR2 resulted in a protein with 15-fold higher activity when tested with a substrate that contained an editing site in the mature message. In addition to its potential role in an antiviral response, we propose that ADAR1-L is the form primarily responsible for editing mRNAs in which the editing site is retained after processing.     

RNA sensing via the RIG‐I‐like receptor LGP2 is essential for the induction of a type I IFN response in ADAR1 deficiency

RNA editing by the adenosine deaminase ADAR1 prevents innate immune responses to endogenous RNAs. In ADAR1‐deficient cells, unedited self RNAs form base‐paired structures that resemble viral RNAs and inadvertently activate the cytosolic RIG‐I‐like receptor (RLR) MDA5, leading to an antiviral type I interferon (IFN) response. Mutations in ADAR1 cause Aicardi‐Goutières Syndrome (AGS), an autoinflammatory syndrome characterized by chronic type I IFN production. Conversely, ADAR1 loss and the consequent type I IFN production restricts tumor growth and potentiates the activity of some chemotherapeutics. Here, we show that another RIG‐I‐like receptor, LGP2, also has an essential role in the induction of a type I IFN response in ADAR1‐deficient human cells. This requires the canonical function of LGP2 as an RNA sensor and facilitator of MDA5‐dependent signaling. Furthermore, we show that the sensitivity of tumor cells to ADAR1 loss requires LGP2 expression. Finally, type I IFN induction in tumor cells depleted of ADAR1 and treated with some chemotherapeutics fully depends on LGP2 expression. These findings highlight a central role for LGP2 in self RNA sensing with important clinical implications.     

Inducible microRNA-155 feedback promotes type I IFN signaling in antiviral innate immunity by targeting suppressor of cytokine signaling 1

Effective recognition of viral infection and subsequent triggering of antiviral innate immune responses are essential for the host antiviral defense, which is tightly regulated by multiple regulators, including microRNAs. Our previous study showed that a panel of microRNAs, including miR-155, was markedly upregulated in macrophages upon vesicular stomatitis virus infection; however, the biological function of miR-155 during viral infection remains unknown. In this paper, we show that RNA virus infection induces miR-155 expression in macrophages via TLR/MyD88-independent but retinoic acid-inducible gene I/JNK/NF-κB-dependent pathway. And the inducible miR-155 feedback promotes type I IFN signaling, thus suppressing viral replication. Furthermore, suppressor of cytokine signaling 1 (SOCS1), a canonical negative regulator of type I IFN signaling, is targeted by miR-155 in macrophages, and SOCS1 knockdown mediates the enhancing effect of miR-155 on type I IFN-mediated antiviral response. Therefore, we demonstrate that inducible miR-155 feedback positively regulates host antiviral innate immune response by promoting type I IFN signaling via targeting SOCS1.     

Virus-specific editing identification approach reveals the landscape of A-to-I editing and its impacts on SARS-CoV-2 characteristics and evolution

Upon SARS-CoV-2 infection, viral intermediates specifically activate the IFN response through MDA5-mediated sensing and accordingly induce ADAR1 p150 expression, which might lead to viral A-to-I RNA editing. Here, we developed an RNA virus-specific editing identification pipeline, surveyed 7622 RNA-seq data from diverse types of samples infected with SARS-CoV-2, and constructed an atlas of A-to-I RNA editing sites in SARS-CoV-2. We found that A-to-I editing was dynamically regulated, varied between tissue and cell types, and was correlated with the intensity of innate immune response. On average, 91 editing events were deposited at viral dsRNA intermediates per sample. Moreover, editing hotspots were observed, including recoding sites in the spike gene that affect viral infectivity and antigenicity. Finally, we provided evidence that RNA editing accelerated SARS-CoV-2 evolution in humans during the epidemic. Our study highlights the ability of SARS-CoV-2 to hijack components of the host antiviral machinery to edit its genome and fuel its evolution, and also provides a framework and resource for studying viral RNA editing.     

Functional and therapeutic analysis of hepatitis C virus NS3.4A protease control of antiviral immune defense

Chronic hepatitis C virus (HCV) infection is a major global public health problem. HCV infection is supported by viral strategies to evade the innate antiviral response wherein the viral NS3.4A protease complex targets and cleaves the interferon promoter stimulator-1 (IPS-1) adaptor protein to ablate signaling of interferon alpha/beta immune defenses. Here we examined the structural requirements of NS3.4A and the therapeutic potential of NS3.4A inhibitors to control the innate immune response against virus infection. The structural composition of NS3 includes an amino-terminal serine protease domain and a carboxyl-terminal RNA helicase domain. NS3 mutants lacking the helicase domain retained the ability to control virus signaling initiated by retinoic acid-inducible gene-I (RIG-I) or melanoma differentiation antigen 5 and suppressed the downstream activation of interferon regulatory factor-3 (IRF-3) and nuclear factor kappaB (NF-kappaB) through the targeted proteolysis of IPS-1. This regulation was abrogated by truncation of the NS3 protease domain or by point mutations that ablated protease activity. NS3.4A protease control of antiviral immune signaling was due to targeted proteolysis of IPS-1 by the NS3 protease domain and minimal NS4A cofactor. Treatment of HCV-infected cells with an NS3 protease inhibitor prevented IPS-1 proteolysis by the HCV protease and restored RIG-I immune defense signaling during infection. Thus, the NS3.4A protease domain can target IPS-1 for cleavage and is essential for blocking RIG-I signaling to IRF-3 and NF-kappaB, whereas the helicase domain is dispensable for this action. Our results indicate that NS3.4A protease inhibitors have immunomodulatory potential to restore innate immune defenses to HCV infection.     

Molecular basis of viral persistence: a single amino acid change in the glycoprotein of lymphocytic choriomeningitis virus is associated with suppression of the antiviral cytotoxic T-lymphocyte response and establishment of persistence.

Isolates of lymphocytic choriomeningitis virus (LCMV) that elicit a cytotoxic T-lymphocyte response (CTL+) have been compared with isolates that suppress the CTL response (CTL-) in an effort to map this phenotype. A single amino acid change in the glycoprotein of the LCMV Armstrong (ARM) strain is consistently associated with the CTL- trait and the ability of the virus to persist (P+). The CTL+ P- parental strain spontaneously gives rise to CTL- P+ variants within lymphoid tissues of mice persistently infected from birth. To map the structural basis of the phenotype, the complete RNA sequence of LCMV ARM 53b (CTL+) was compared with that of its variant ARM clone 13 (CTL-). Differences in 5 of 10,600 nucleotides were found. Three changes are noted in the large L RNA segment, and two are noted in the small S RNA segment. Only two of the changes distinguishing CTL+ from CTL- isolates affect amino acid coding: lysine to glutamine at amino acid 1079 of the polymerase protein, and phenylalanine to leucine at amino acid 260 of the envelope glycoprotein (GP). We also analyzed two additional CTL- variants and four spontaneous CTL+ revertants. All three CTL- variants differ from the original CTL+ parental strain at GP amino acid 260, indicating that this amino acid change is consistently associated with the CTL- phenotype. By contrast the other four mutations in LCMV are not associated with the CTL- phenotype. Sequence analysis of the coding regions of four CTL+ revertants of ARM clone 13 did not reveal back mutations at the GP 260 locus. This finding indicates that the GP 260 mutation is necessary but not sufficient for a CTL- P+ phenotype and that the reversion to CTL+ P- is likely either due to secondary mutations in other regions of the viral genome or to quasispecies within the revertant population that make significant contributions to the phenotype.     

Disruption of Type I Interferon Induction by HIV Infection of T Cells

Our main objective of this study was to determine how Human Immunodeficiency Virus (HIV) avoids induction of the antiviral Type I Interferon (IFN) system. To limit viral infection, the innate immune system produces important antiviral cytokines such as the IFN. IFN set up a critical roadblock to virus infection by limiting further replication of a virus. Usually, IFN production is induced by the recognition of viral nucleic acids by innate immune receptors and subsequent downstream signaling. However, the importance of IFN in the defense against viruses has lead most pathogenic viruses to evolve strategies to inhibit host IFN induction or responses allowing for increased pathogenicity and persistence of the virus. While the adaptive immune responses to HIV infection have been extensively studied, less is known about the balance between induction and inhibition of innate immune defenses, including the antiviral IFN response, by HIV infection. Here we show that HIV infection of T cells does not induce significant IFN production even IFN I Interferon production. To explain this paradox, we screened HIV proteins and found that two HIV encoded proteins, Vpu and Nef, strongly antagonize IFN induction, with expression of these proteins leading to loss of expression of the innate immune viral RNA sensing adaptor protein, IPS-1 (IFN-β promoter stimulator-1). We hypothesize that with lower levels of IPS-1 present, infected cells are defective in mounting antiviral responses allowing HIV to replicate without the normal antiviral actions of the host IFN response. Using cell lines as well as primary human derived cells, we show that HIV targeting of IPS-1 is key to limiting IFN induction. These findings describe how HIV infection modulates IFN induction providing insight into the mechanisms by which HIV establishes infection and persistence in a host.     

Modeling within-host dynamics of influenza virus infection including immune responses

Influenza virus infection remains a public health problem worldwide. The mechanisms underlying viral control during an uncomplicated influenza virus infection are not fully understood. Here, we developed a mathematical model including both innate and adaptive immune responses to study the within-host dynamics of equine influenza virus infection in horses. By comparing modeling predictions with both interferon and viral kinetic data, we examined the relative roles of target cell availability, and innate and adaptive immune responses in controlling the virus. Our results show that the rapid and substantial viral decline (about 2 to 4 logs within 1 day) after the peak can be explained by the killing of infected cells mediated by interferon activated cells, such as natural killer cells, during the innate immune response. After the viral load declines to a lower level, the loss of interferon-induced antiviral effect and an increased availability of target cells due to loss of the antiviral state can explain the observed short phase of viral plateau in which the viral level remains unchanged or even experiences a minor second peak in some animals. An adaptive immune response is needed in our model to explain the eventual viral clearance. This study provides a quantitative understanding of the biological factors that can explain the viral and interferon kinetics during a typical influenza virus infection.     

A Novel System for the Launch of Alphavirus RNA Synthesis Reveals a Role for the Imd Pathway in Arthropod Antiviral Response

Alphaviruses are RNA viruses transmitted between vertebrate hosts by arthropod vectors, primarily mosquitoes. How arthropods counteract alphaviruses or viruses per se is not very well understood. Drosophila melanogaster is a powerful model system for studying innate immunity against bacterial and fungal infections. In this study we report the use of a novel system to analyze replication of Sindbis virus (type species of the alphavirus genus) RNA following expression of a Sindbis virus replicon RNA from the fly genome. We demonstrate deficits in the immune deficiency (Imd) pathway enhance viral replication while mutations in the Toll pathway fail to affect replication. Similar results were observed with intrathoracic injections of whole virus and confirmed in cultured mosquito cells. These findings show that the Imd pathway mediates an antiviral response to Sindbis virus replication. To our knowledge, this is the first demonstration of an antiviral role for the Imd pathway in insects.     

Interaction of the innate immune system with positive-strand RNA virus replication organelles

The potential health risks associated with (re-)emerging positive-strand RNA (+RNA) viruses emphasizes the need for understanding host-pathogen interactions for these viruses. The innate immune system forms the first line of defense against pathogenic organisms like these and is responsible for detecting pathogen-associated molecular patterns (PAMPs). Viral RNA is a potent inducer of antiviral innate immune signaling, provoking an antiviral state by directing expression of interferons (IFNs) and pro-inflammatory cytokines. However, +RNA viruses developed various methods to avoid detection and downstream signaling, including isolation of viral RNA replication in membranous viral replication organelles (ROs). These structures therefore play a central role in infection, and consequently, loss of RO integrity might simultaneously result in impaired viral replication and enhanced antiviral signaling. This review summarizes the first indications that the innate immune system indeed has tools to disrupt viral ROs and other non- or aberrant-self membrane structures, and may do this by marking these membranes with proteins such as microtubule-associated protein 1A/1B-light chain 3 (LC3) and ubiquitin, resulting in the recruitment of IFN-inducible GTPases. Further studies should evaluate whether this process forms a general effector mechanism in +RNA virus infection, thereby creating the opportunity for development of novel antiviral therapies.     

Effects of promyelocytic leukemia protein on virus-host balance

The cellular promyelocytic leukemia protein (PML) associates with the proteins of several viruses and in some cases reduces viral propagation in cell culture. To examine the role of PML in vivo, we compared immune responses and virus loads of PML-deficient and control mice infected with lymphocytic choriomeningitis virus (LCMV) and vesicular stomatitis virus (VSV). PML(-/-) mice exhibited accelerated primary footpad swelling reactions to very-low-dose LCMV, higher swelling peaks upon high-dose inoculation, and higher viral loads in the early phase of systemic LCMV infection. T-cell-mediated hepatitis and consequent mortality upon infection with a hepatotropic LCMV strain required 10- to 100-times-lower inocula despite normal cytotoxic T-lymphocyte reactivity in PML(-/-) mice. Furthermore, PML deficiency rendered mice 10 times more susceptible to lethal immunopathology upon intracerebral LCMV inoculation. Accordingly, 10-times-lower VSV inocula elicited specific neutralizing-antibody responses, a replication-based effect not observed with inactivated virus or after immunization with recombinant VSV glycoprotein. These in vivo observations corroborated our results showing more virus production in PML(-/-) fibroblasts. Thus, PML is a contributor to innate immunity, defining host susceptibility to viral infections and to immunopathology.     

ADAR1 Facilitates HIV-1 Replication in Primary CD4+ T Cells

Unlike resting CD4⁺ T cells, activated CD4⁺T cells are highly susceptible to infection of human immunodeficiency virus 1 (HIV-1). HIV-1 infects T cells and macrophages without activating the nucleic acid sensors and the anti-viral type I interferon response. Adenosine deaminase acting on RNA 1 (ADAR1) is an RNA editing enzyme that displays antiviral activity against several RNA viruses. Mutations in ADAR1 cause the autoimmune disorder Aicardi-Goutieères syndrome (AGS). This disease is characterized by an inappropriate activation of the interferon-stimulated gene response. Here we show that HIV-1 replication, in ADAR1-deficient CD4⁺T lymphocytes from AGS patients, is blocked at the level of protein translation. Furthermore, viral protein synthesis block is accompanied by an activation of interferon-stimulated genes. RNA silencing of ADAR1 in Jurkat cells also inhibited HIV-1 protein synthesis. Our data support that HIV-1 requires ADAR1 for efficient replication in human CD4⁺T cells.