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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.

International audience; Microbes have been coevolving with their host for millions of years, exploiting host resources to their own benefit. We show that viral and bacterial pathogens convergently evolved to hijack cellular mitogen-activated protein kinase (MAPK) p90-ribosomal S6-kinases (RSKs). Theiler’s virus leader (L) protein binds RSKs and prevents their dephosphorylation, thus maintaining the kinases active. Recruitment of RSKs enables L-protein-mediated inhibition of eukaryotic translation initiation factor 2 alpha kinase 2 (EIF2AK2 or PKR) and stress granule formation. Strikingly, ORF45 protein of Kaposi’s sarcoma-associated herpesvirus (KSHV) and YopM protein of Yersinia use the same peptide motif as L to recruit and activate RSKs. All three proteins interact with a conserved surface-located loop of RSKs, likely acting as an allosteric regulation site. Some unrelated viruses and bacteria thus evolved to harness RSKs in a common fashion, yet to target distinct aspects of innate immunity. As documented for Varicella zoster virus ORF11, additional pathogens likely evolved to hijack RSKs, using a similar short linear motif

RNase L is a well-known effector of the type I interferon pathway. This review focuses on the recent developments of RNase L activation and on the antagonism of the OAS-RNase L pathway by viral proteins. Recent structural data show that two 2'-5' oligoadenylate molecules can bridge ankyrin domains of two RNase L subunits bound in opposite orientations. The binding of nucleotides to the pseudokinase domain further strengthens the dimer and imparts an active conformation to the ribonuclease. The OAS/RNase L pathway is active against many viruses and viruses evolved in several ways to escape this pathway. Influenza virus A acts upstream of this pathway by hiding double stranded RNA through its NS1 protein. In this way, it also inhibits the PKR and TLR-3 activation by double stranded RNA. Theiler's virus acts downstream of the OAS/RNase L pathway, through the direct interaction between protein L* and RNase L. By acting on the effector enzyme, Theiler's virus ensures a strong RNase L inhibition, which seems to be particularly useful for the infection of macrophages. In conclusion, viruses have developed distinct strategies to escape RNase L activity, that are likely dependent on their tropism. The fact that viral proteins have evolved to specifically antagonize RNase L outlines the importance of this particular IFN effector in cells infected by those viruses.

Cardioviruses are members of the Picornaviridae 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 Picornaviridae, 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 in vivo. Additionally, we describe how cardiovirus proteins L, 3C and L∗ disrupt interferon production and antagonize the antiviral activity of interferon effector molecules.

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 in vivo. 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., Author summary Oligoadenylate synthetases (OAS) are a family of enzymes that produce 2’-5’ oligoadenylates (2-5A) from ATP. OAS accumulate as inactive enzymes in cells that have been exposed to interferon. When infection of these cells occurs, double-stranded RNA produced during virus replication activates OAS, which produce 2-5A. 2-5A act as second messengers. They bind a cellular endonuclease called RNase L, thereby triggering the dimerization and the activation of this RNase. Active RNase L degrades cellular and viral RNA molecules, ultimately killing the infected cell, thus preventing virus spread. Viruses have developed diverse mechanisms to escape RNase L activity. We report that Theiler’s murine encephalomyelitis virus, a virus that produces persistent infections of the central nervous system, evolved a novel mechanism to escape the OAS/RNase L pathway. The L* protein produced by this virus binds RNase L very close to the pocket known to sense 2-5A and thereby prevents 2-5A from activating RNase L. This mechanism contrasts with that used by Influenza (flu) virus, which sequesters double-stranded RNA and thereby also inhibits alternative antiviral pathways. We further showed that the RNase L antagonist activity of L* is effective in vivo and that L* can substitute for 2-5A degrading enzymes produced by coronaviruses.

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.

We observed previously that the L∗ protein of Theiler’s virus (picornavirus) inhibited RNase L, through direct protein–protein interaction. Interestingly, RNase L inhibition by L∗ exhibits species-specificity as L∗ inhibits mouse but not human RNase L [1] . We took advantage of this species specificity to map the L∗ binding site on RNase L, by testing L∗-mediated inhibition of a series of human-mouse RNase L chimeras. Our results suggest that L∗ interacts at two sites within RNase L ankyrin repeats 1 and 2. According to the recently solved structure of dimeric RNase L [2] , [3] , our results are compatible with RNase L inhibition through both dimerization and 2-5A-binding antagonism. We therefore established a crosslinking-based RNase L dimerization assay and show that L∗ indeed inhibits RNase L dimerization. Next, we analyzed whether L∗ could interfere with 2-5A binding. Competitive binding studies by surface plasmon resonance demonstrated that L∗ indeed competes with 2-5A binding to mouse but not human RNase L. In conclusion, protein L∗ of Theiler’s virus inhibits the OAS/RNase L pathway by binding to RNase L ankyrin repeats 1 and 2, thereby preventing 2-5A-mediated dimerization and activation of RNase L. L∗ is the first viral protein we know of that antagonizes the OAS/RNase L pathway by acting on the effector enzyme.