A novel mechanism of RNase L inhibition: Theiler\u27s virus L* protein prevents 2-5A from binding to RNase L

<div><p>The OAS/RNase L pathway is one of the best-characterized effector pathways of the IFN antiviral response. It inhibits the replication of many viruses and ultimately promotes apoptosis of infected cells, contributing to the control of virus spread. However, viruses have evolved a range of escape strategies that act against different steps in the pathway. Here we unraveled a novel escape strategy involving Theiler’s murine encephalomyelitis virus (TMEV) L* protein. Previously we found that L* was the first viral protein binding directly RNase L. Our current data show that L* binds the ankyrin repeats R1 and R2 of RNase L and inhibits 2’-5’ oligoadenylates (2-5A) binding to RNase L. Thereby, L* prevents dimerization and oligomerization of RNase L in response to 2-5A. Using chimeric mouse hepatitis virus (MHV) expressing TMEV L*, we showed that L* efficiently inhibits RNase L <i>in vivo</i>. Interestingly, those data show that L* can functionally substitute for the MHV-encoded phosphodiesterase ns2, which acts upstream of L* in the OAS/RNase L pathway, by degrading 2-5A.</p></div

Antagonism of the antiviral OAS/RNase L pathway by Theiler's virus L* protein : molecular mechanisms and species-specificity

The DA strain of Theiler’s virus has a remarkable ability to evade the immune response of the host and to cause persistent infections of the central nervous system. During the chronic phase of infection, the virus is primarily detected in microglial cells, infiltrating macrophages and oligodendrocytes in the white matter of the spinal cord. The non-structural L* protein encoded by Theiler's virus was shown to enhance viral replication in macrophages in vitro and to be required for the establishment of persistent infections. Previous work in our lab revealed that L* directly interacts with RNase L and thereby inhibits the activity of this antiviral enzyme. Interestingly, RNase L antagonism by L* is a highly species-specific process; indeed, L* inhibits mouse RNase L but not its orthologues from other tested species. On the one hand, we characterized further the molecular mechanisms of RNase L antagonism by the Theiler’s virus L* protein. We identified a novel strategy of RNase L inhibition in which, upon binding to ankyrin repeats 1 and 2 of the mouse RNase L, L* competitively inhibits 2-5A binding to RNase L. Using chimeric MHV viruses expressing L*, we demonstrated that L* is able to counteract RNase L activity in vivo, in infected mice. On the other hand, we took advantage of the species-specificity of L* activity to identify the natural host of Vilyuisk human encephalitis virus (VHEV). VHEV was isolated from mice that were inoculated with CSF from a patient suffering from chronic encephalitis. It is therefore unclear whether the virus derives from the human sample or from the mice used to isolate the virus. We observed that L* of VHEV specifically binds to and inhibits mouse but not human RNase L, thereby showing that VHEV is of mouse origin and suggesting that it is a contaminant that originated from the mice that were used to inoculate the human sample.(BIFA - Sciences biomédicales et pharmaceutiques) -- UCL, 201

Inhibition of the OAS/RNase L pathway by viruses.

The OAS/RNase L system was one of the first characterized interferon effector pathways. It relies on the synthesis, by oligoadenylate synthetases (OAS), of short oligonucleotides that act as second messengers to activate the latent cellular RNase L. Viruses have developed diverse strategies to escape its antiviral effects. This underscores the importance of the OAS/RNase L pathway in antiviral defenses. Viral proteins such as the NS1 protein of Influenza virus A act upstream of the pathway while other viral proteins such as Theiler's virus L* protein act downstream. The diversity of escape strategies used by viruses likely stems from their relative susceptibility to OAS/RNase L and other antiviral pathways, which may depend on their host and cellular tropism

Nonstructural Protein L* Species Specificity Supports a Mouse Origin for Vilyuisk Human Encephalitis Virus

Vilyuisk human encephalitis virus (VHEV) is a picornavirus related to Theiler's murine encephalomyelitis virus (TMEV). VHEV was isolated from human material passaged in mice. Whether this VHEV is of human or mouse origin is therefore unclear. We took advantage of the species-specific activity of the nonstructural L* protein of theiloviruses to track the origin of TMEV isolates. TMEV L* inhibits RNase L, the effector enzyme of the interferon pathway. By using coimmunoprecipitation and functional RNase L assays, the species specificity of RNase L antagonism was tested for L* from mouse (DA) and rat (RTV-1) TMEV strains as well as for VHEV. Coimmunoprecipitation and functional assay data confirmed the species specificity of L* activity and showed that L* from rat strain RTV-1 inhibited rat but not mouse or human RNase L. Next, we showed that the VHEV L* protein was phylogenetically related to L* of mouse viruses and that it failed to inhibit human RNase L but readily antagonized mouse RNase L, unambiguously showing the mouse origin of VHEV.IMPORTANCE Defining the natural host of a virus can be a thorny issue, especially when the virus was isolated only once or when the isolation story is complex. The species Theilovirus includes Theiler's murine encephalomyelitis virus (TMEV), infecting mice and rats, and Saffold virus (SAFV), infecting humans. One TMEV strain, Vilyuisk human encephalitis virus (VHEV), however, was isolated from mice that were inoculated with cerebrospinal fluid of a patient presenting with chronic encephalitis. It is therefore unclear whether VHEV was derived from the human sample or from the inoculated mouse. The L* protein encoded by TMEV inhibits RNase L, a cellular enzyme involved in innate immunity, in a species-specific manner. Using binding and functional assays, we show that this species specificity even allows discrimination between TMEV strains of mouse and of rat origins. The VHEV L* protein clearly inhibited mouse but not human RNase L, indicating that this virus originates from mice

Innate Immune Detection of Cardioviruses and Viral Disruption of Interferon Signaling.

Cardioviruses are members of the family and infect a variety of mammals, from mice to humans. Replication of cardioviruses produces double stranded RNA that is detected by helicases in the RIG-I-like receptor family and leads to a signaling cascade to produce type I interferon. Like other viruses within , however, cardioviruses have evolved several mechanisms to inhibit interferon production. In this review, we summarize recent findings that have uncovered several proteins enabling efficient detection of cardiovirus dsRNA and discuss which cell types may be most important for interferon production . Additionally, we describe how cardiovirus proteins L, 3C and L disrupt interferon production and antagonize the antiviral activity of interferon effector molecules

A novel mechanism of RNase L inhibition: Theiler's virus L* protein prevents 2-5A from binding to RNase L

<div><p>The OAS/RNase L pathway is one of the best-characterized effector pathways of the IFN antiviral response. It inhibits the replication of many viruses and ultimately promotes apoptosis of infected cells, contributing to the control of virus spread. However, viruses have evolved a range of escape strategies that act against different steps in the pathway. Here we unraveled a novel escape strategy involving Theiler’s murine encephalomyelitis virus (TMEV) L* protein. Previously we found that L* was the first viral protein binding directly RNase L. Our current data show that L* binds the ankyrin repeats R1 and R2 of RNase L and inhibits 2’-5’ oligoadenylates (2-5A) binding to RNase L. Thereby, L* prevents dimerization and oligomerization of RNase L in response to 2-5A. Using chimeric mouse hepatitis virus (MHV) expressing TMEV L*, we showed that L* efficiently inhibits RNase L <i>in vivo</i>. Interestingly, those data show that L* can functionally substitute for the MHV-encoded phosphodiesterase ns2, which acts upstream of L* in the OAS/RNase L pathway, by degrading 2-5A.</p></div

L* compensates ns2 RNase L antagonist activity in bone marrow-derived macrophages (BMM) and in mice.

<p>A. Schematic diagram of recombinant MHV. B. L2 fibroblasts were infected (1 PFU/cell). At indicated time points post-infection, virus titers in the cell lysates combined with supernatants were determined by plaque assay (n = 3). C-D. BMM, derived from WT or RNase L−/− mice were infected (1 PFU/cell). At indicated time points post-infection, titers of viruses in the cell lysates combined with supernatants were determined by plaque assay (n = 3). E. Four-week-old WT or RNase L−/− B6 mice were inoculated intrahepatically with WT A59, mutant and chimeric viruses (2000 PFU/mouse). At 5 d.p.i., organs were harvested, homogenized and virus titers determined by plaque assay (n = 4 or 5). Statistics were done using the Mann-Whitney test. Error bars represent standard error of the means.</p

RNase L residues involved in 2-5A binding are not crucial for L* binding to mouse RNase L.

<p>A-B. Co-immunoprecipitation of indicated 2-5A binding-defective mouse RNase L mutants with HA-tagged L*DA. A. Immunoblots show Flag (RNase L) and HA (L*) detection after immunoprecipitation of HA (upper panels) and in cell lysates (Input, lower panels). B. Graphs showing the quantification of coimmunoprecipitated RNase L mutants relative to coimmunoprecipitated WT mouse RNase L (n = 3). Differences were non-significant according to one-way ANOVA followed by Tukey's test for multiple comparisons. C-D. Analysis of RNase L-mediated RNA degradation in HeLa-M cells overexpressing indicated Flag-RNase L and HA-L*_DA. RNA samples extracted 7 hours after polyI:C transfection were analyzed by RNA chips (C) and quantified (D). Graphs show the quantification of RNA degradation by RNase L mutants in the absence or in the presence of L*_DA. Data are normalized to those of WT RNase L in the absence of L*.</p

Inhibition of basal and glucagon-induced hepatic glucose production by 991 and other pharmacological AMPK activators.

Pharmacological AMPK activation represents an attractive approach for the treatment of type 2 diabetes (T2D). AMPK activation increases skeletal muscle glucose uptake, but there is controversy as to whether AMPK activation also inhibits hepatic glucose production (HGP) and pharmacological AMPK activators can have off-target effects that contribute to their anti-diabetic properties. The main aim was to investigate the effects of 991 and other direct AMPK activators on HGP and determine whether the observed effects were AMPK-dependent. In incubated hepatocytes, 991 substantially decreased gluconeogenesis from lactate, pyruvate and glycerol, but not from other substrates. Hepatocytes from AMPKβ1-/- mice had substantially reduced liver AMPK activity, yet the inhibition of glucose production by 991 persisted. Also, the glucose-lowering effect of 991 was still seen in AMPKβ1-/- mice subjected to an intraperitoneal pyruvate tolerance test. The AMPK-independent mechanism by which 991 treatment decreased gluconeogenesis could be explained by inhibition of mitochondrial pyruvate uptake and inhibition of mitochondrial sn-glycerol-3-phosphate dehydrogenase-2. However, 991 and new-generation direct small-molecule AMPK activators antagonized glucagon-induced gluconeogenesis in an AMPK-dependent manner. Our studies support the notion that direct pharmacological activation of hepatic AMPK as well as inhibition of pyruvate uptake could be an option for the treatment of T2D-linked hyperglycemia