Picornavirus security proteins promote the release of extracellular vesicle enclosed viruses via the modulation of host kinases.

The discovery that extracellular vesicles (EVs) serve as carriers of virus particles calls for a reevaluation of the release strategies of non-enveloped viruses. Little is currently known about the molecular mechanisms that determine the release and composition of EVs produced by virus-infected cells, as well as conservation of these mechanisms among viruses. We previously described an important role for the Leader protein of the picornavirus encephalomyocarditis virus (EMCV) in the induction of virus-carrying EV subsets with distinct molecular and physical properties. EMCV L acts as a 'viral security protein' by suppressing host antiviral stress and type-I interferon (IFN) responses. Here, we tested the ability of functionally related picornavirus proteins of Theilers murine encephalitis virus (TMEV L), Saffold virus (SAFV L), and coxsackievirus B3 (CVB3 2Apro), to rescue EV and EV-enclosed virus release when introduced in Leader-deficient EMCV. We show that all viral security proteins tested were able to promote virus packaging in EVs, but that only the expression of EMCV L and CVB3 2Apro increased overall EV production. We provide evidence that one of the main antiviral pathways counteracted by this class of picornaviral proteins, i.e. the inhibition of PKR-mediated stress responses, affected EV and EV-enclosed virus release during infection. Moreover, we show that the enhanced capacity of the viral proteins EMCV L and CVB3 2Apro to promote EV-enclosed virus release is linked to their ability to simultaneously promote the activation of the stress kinase P38 MAPK. Taken together, we demonstrate that cellular stress pathways involving the kinases PKR and P38 are modulated by the activity of non-structural viral proteins to increase the release EV-enclosed viruses during picornavirus infections. These data shed new light on the molecular regulation of EV production in response to virus infection.

Genes for tRNA recycling are upregulated in response to infection with Theiler's mouse encephalitis virus.

The concept of tRNA recycling has recently emerged from the studies of ribosome-associated quality control. Therein tRNase ZS removes the 2', 3'>p from the ANKZF1-cleaved tRNA and the subsequent TRNT1 action re-generates the intact tRNA. To know the roles of the tRNA recycling in vivo, we investigated how viral infection affects the tRNA recycling system by analyzing the mRNA levels of tRNase ZS and TRNT1. We found that both genes in HeLa cells are upregulated in response to infection of Theiler's mouse encephalitis virus but not to that of an influenza A virus. Upregulation was also observed in cells infected with encephalomyocarditis virus with reduced efficiency. The levels of the IFN-ß mRNA appeared to positively correlate with those of the tRNase ZS and TRNT1 mRNAs. The tRNase ZS gene may be regulated post-transcriptionally in the cells infected with Theiler's mouse encephalitis virus.

Investigating molecular mechanisms of 2A-stimulated ribosomal pausing and frameshifting in Theilovirus.

The 2A protein of Theiler's murine encephalomyelitis virus (TMEV) acts as a switch to stimulate programmed -1 ribosomal frameshifting (PRF) during infection. Here, we present the X-ray crystal structure of TMEV 2A and define how it recognises the stimulatory RNA element. We demonstrate a critical role for bases upstream of the originally predicted stem-loop, providing evidence for a pseudoknot-like conformation and suggesting that the recognition of this pseudoknot by beta-shell proteins is a conserved feature in cardioviruses. Through examination of PRF in TMEV-infected cells by ribosome profiling, we identify a series of ribosomal pauses around the site of PRF induced by the 2A-pseudoknot complex. Careful normalisation of ribosomal profiling data with a 2A knockout virus facilitated the identification, through disome analysis, of ribosome stacking at the TMEV frameshifting signal. These experiments provide unparalleled detail of the molecular mechanisms underpinning Theilovirus protein-stimulated frameshifting.

The In Silico Prediction of Hotspot Residues that Contribute to the Structural Stability of Subunit Interfaces of a Picornavirus Capsid.

The assembly of picornavirus capsids proceeds through the stepwise oligomerization of capsid protein subunits and depends on interactions between critical residues known as hotspots. Few studies have described the identification of hotspot residues at the protein subunit interfaces of the picornavirus capsid, some of which could represent novel drug targets. Using a combination of accessible web servers for hotspot prediction, we performed a comprehensive bioinformatic analysis of the hotspot residues at the intraprotomer, interprotomer and interpentamer interfaces of the Theiler's murine encephalomyelitis virus (TMEV) capsid. Significantly, many of the predicted hotspot residues were found to be conserved in representative viruses from different genera, suggesting that the molecular determinants of capsid assembly are conserved across the family. The analysis presented here can be applied to any icosahedral structure and provides a platform for in vitro mutagenesis studies to further investigate the significance of these hotspots in critical stages of the virus life cycle with a view to identify potential targets for antiviral drug design.

Characterization of the stimulators of protein-directed ribosomal frameshifting in Theiler's murine encephalomyelitis virus.

Many viruses utilize programmed -1 ribosomal frameshifting (-1 PRF) to express additional proteins or to produce frameshift and non-frameshift protein products at a fixed stoichiometric ratio. PRF is also utilized in the expression of a small number of cellular genes. Frameshifting is typically stimulated by signals contained within the mRNA: a 'slippery' sequence and a 3'-adjacent RNA structure. Recently, we showed that -1 PRF in encephalomyocarditis virus (EMCV) is trans-activated by the viral 2A protein, leading to a temporal change in PRF efficiency from 0% to 70% during virus infection. Here we analyzed PRF in the related Theiler's murine encephalomyelitis virus (TMEV). We show that 2A is also required for PRF in TMEV and can stimulate PRF to levels as high as 58% in rabbit reticulocyte cell-free translations and 81% during virus infection. We also show that TMEV 2A trans-activates PRF on the EMCV signal but not vice versa. We present an extensive mutational analysis of the frameshift stimulators (mRNA signals and 2A protein) analysing activity in in vitro translation, electrophoretic mobility shift and in vitro ribosome pausing assays. We also investigate the PRF mRNA signal with RNA structure probing. Our results substantially extend previous characterization of protein-stimulated PRF.

TDP-43 proteinopathy in Theiler's murine encephalomyelitis virus infection.

TDP-43, an RNA-binding protein that is primarily nuclear and important in splicing and RNA metabolism, is mislocalized from the nucleus to the cytoplasm of neural cells in amyotrophic lateral sclerosis (ALS), and contributes to disease. We sought to investigate whether TDP-43 is mislocalized in infections with the acute neuronal GDVII strain and the persistent demyelinating DA strain of Theiler's virus murine encephalomyelitis virus (TMEV), a member of the Cardiovirus genus of Picornaviridae because: i) L protein of both strains is known to disrupt nucleocytoplasmic transport, including transport of polypyrimidine tract binding protein, an RNA-binding protein, ii) motor neurons and oligodendrocytes are targeted in both TMEV infection and ALS. TDP-43 phosphorylation, cleavage, and cytoplasmic mislocalization to an aggresome were observed in wild type TMEV-infected cultured cells, with predicted splicing abnormalities. In contrast, cells infected with DA and GDVII strains that have L deletion had rare TDP-43 mislocalization and no aggresome formation. TDP-43 mislocalization was also present in neural cells of TMEV acutely-infected mice. Of note, TDP-43 was mislocalized six weeks after DA infection to the cytoplasm of oligodendrocytes and other glial cells in demyelinating lesions of spinal white matter. A recent study showed that TDP-43 knock down in oligodendrocytes in mice led to demyelination and death of this neural cell [1], suggesting that TMEV infection mislocalization of TDP-43 and other RNA-binding proteins is predicted to disrupt key cellular processes and contribute to the pathogenesis of TMEV-induced diseases. Drugs that inhibit nuclear export may have a role in antiviral therapy.

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

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.

The generation and characterisation of neutralising antibodies against the Theiler's murine encephalomyelitis virus (TMEV) GDVII capsid reveals the potential binding site of the host cell co-receptor, heparan sulfate.

The early stages of picornavirus capsid assembly and the host factors involved are poorly understood. Since the localisation of viral proteins in infected cells can provide information on their function, antibodies against purified Theiler's murine encephalomyelitis virus (TMEV) GDVII capsids were generated by immunisation of rabbits. The resultant anti-TMEV capsid antibodies recognised a C-terminal region of VP1 but not VP2 or VP3 by Western analysis. Examination of the sites of TMEV capsid assembly by indirect immunofluorescence and confocal microscopy showed that at 5h post infection, capsid signal was diffusely cytoplasmic with strong perinuclear staining and moved into large punctate structures from 6 to 8h post infection. A plaque reduction neutralisation assay showed that the anti-TMEV capsid antibodies but not anti-VP1 antibodies could neutralise viral infection in vitro. The VP1 C-terminal residues recognised by the anti-TMEV capsid antibodies were mapped to a loop on the capsid surface near to the putative receptor binding pocket. In silico docking experiments showed that the known TMEV co-receptor, heparan sulfate, interacts with residues of VP1 in the putative receptor binding pocket, residues of VP3 in the adjacent pit and residues of the adjoining VP1 C-terminal loop which is recognised by the anti-TMEV capsid antibodies. These findings suggest that the anti-TMEV capsid antibodies neutralise virus infection by preventing heparan sulfate from binding to the capsid. The antibodies produced in this study are an important tool for further investigating virus-host cell interactions essential to picornavirus assembly.

Intracellular localization of Saffold virus Leader (L) protein differs in Vero and HEp-2 cells.

The Saffold virus (SAFV) genome is translated as a single long polyprotein precursor and co-translationally cleaved to yield 12 separate viral proteins. Little is known about the activities of SAFV proteins although their homologs in other picornaviruses have already been described. To further support research on functions and activities of respective viral proteins, we investigated the spatio-temporal distribution of SAFV proteins in Vero and HEp-2 cells that had been either transfected with plasmids that express individual viral proteins or infected with live SAFV. Our results revealed that, with the exception of the Leader (L) protein, all viral proteins were localized in the cytoplasm at all the time points assayed. The L protein was found in the cytoplasm at an early time point but was subsequently translocated to the nucleus of HEp-2, but not Vero, cells. This was observed in both transfected and infected cells. Further mutational analysis of L protein revealed that Threonine 58 of the Ser/Thr-rich domain of L protein is crucial for protein trafficking between the cytoplasm and nucleus in HEp-2 cells. These findings contribute to a deeper understanding and stimulate investigation of the differetial cellular responses of HEp-2 cells in comparison to other mammalian cell lines during SAFV infection.

Modulatory effects of perforin gene dosage on pathogen-associated blood-brain barrier (BBB) disruption.

BACKGROUND: CD8 T cell-mediated blood-brain barrier (BBB) disruption is dependent on the effector molecule perforin. Human perforin has extensive single nucleotide variants (SNVs), the significance of which is not fully understood. These SNVs can result in reduced, but not ablated, perforin activity or expression. However, complete loss of perforin expression or activity results in the lethal disease familial hemophagocytic lymphohistiocytosis type 2 (FHL 2). In this study, we address the hypothesis that a single perforin allele can alter the severity of BBB disruption in vivo using a well-established model of CNS vascular permeability in C57Bl/6 mice. The results of this study provide insight into the significance of perforin SNVs in the human population. METHODS: We isolated the effect a single perforin allele has on CNS vascular permeability through the use of perforin-heterozygous (perforin+/-) C57BL/6 mice in the peptide-induced fatal syndrome (PIFS) model of immune-mediated BBB disruption. Seven days following Theiler's murine encephalomyelitis virus (TMEV) CNS infection, neuroinflammation and TMEV viral control were assessed through flow cytometric analysis and quantitative real-time PCR of the viral genome, respectively. Following immune-mediated BBB disruption, gadolinium-enhanced T1-weighted MRI, with 3D volumetric analysis, and confocal microscopy were used to define CNS vascular permeability. Finally, the open field behavior test was used to assess locomotor activity of mice following immune-mediated BBB disruption. RESULTS: Perforin-null mice had negligible CNS vascular permeability. Perforin-WT mice have extensive CNS vascular permeability. Interestingly, perforin-heterozygous mice had an intermediate level of CNS vascular permeability as measured by both gadolinium-enhanced T1-weighted MRI and fibrinogen leakage in the brain parenchyma. Differences in BBB disruption were not a result of increased CNS immune infiltrate. Additionally, TMEV was controlled in a perforin dose-dependent manner. Furthermore, a single perforin allele is sufficient to induce locomotor deficit during immune-mediated BBB disruption. CONCLUSIONS: Perforin modulates BBB disruption in a dose-dependent manner. This study demonstrates a potentially advantageous role for decreased perforin expression in reducing BBB disruption. This study also provides insight into the effect SNVs in a single perforin allele could have on functional deficit in neurological disease.

Neuroprotection mediated by inhibition of calpain during acute viral encephalitis.

Neurologic complications associated with viral encephalitis, including seizures and cognitive impairment, are a global health issue, especially in children. We previously showed that hippocampal injury during acute picornavirus infection in mice is associated with calpain activation and is the result of neuronal death triggered by brain-infiltrating inflammatory monocytes. We therefore hypothesized that treatment with a calpain inhibitor would protect neurons from immune-mediated bystander injury. C57BL/6J mice infected with the Daniel's strain of Theiler's murine encephalomyelitis virus were treated with the FDA-approved drug ritonavir using a dosing regimen that resulted in plasma concentrations within the therapeutic range for calpain inhibition. Ritonavir treatment significantly reduced calpain activity in the hippocampus, protected hippocampal neurons from death, preserved cognitive performance, and suppressed seizure escalation, even when therapy was initiated 36 hours after disease onset. Calpain inhibition by ritonavir may be a powerful tool for preserving neurons and cognitive function and preventing neural circuit dysregulation in humans with neuroinflammatory disorders.

Transgenic expression of non-structural genes of Theiler's virus suppresses initial viral replication and pathogenesis of demyelination.

BACKGROUND: Chronic infection with Theiler's murine encephalomyelitis virus (TMEV) in susceptible SJL/J mice induces an immune-mediated demyelinating disease and has extensively been used as a relevant infectious model for multiple sclerosis (MS). Infection of the host with many other viruses also leads to acute or chronic inflammatory diseases in the central nervous system (CNS). Levels of viral load in the host often play a critical role in the pathogenesis of virus-induced diseases. Thus, the inhibition of viral replication in the host against a broad spectrum of similar viruses is critically important for preventing the viral pathogenicity. METHODS: P2/P3-expressing transgenic (B6 X SJL)F1 founders were generated and bred onto the C57BL/6 and SJL/J backgrounds. Differences in the development of demyelinating disease were compared. Viral persistence, cytokine production, and immune responses in the CNS of infected control and P2/P3-Tg mice were analyzed after infection using quantitative PCR, ELISA, and flow cytometry. Various cell types from the control and P2/P3-Tg mice, as well as cells transfected in vitro with the P2 and/or P3 regions, were also analyzed for viral replication and innate cytokine production. RESULTS: P2/P3-transgenic (P2/P3-Tg) mice carrying the viral non-structural protein genes displayed significantly reduced virus-specific T cell responses in the CNS against both the structural and non-structural proteins. Consequently, viral loads in the CNS were greater in the Tg mice during the chronic infection. However, P2/P3-Tg SJL mice exhibited reduced disease incidence and less severe clinical symptoms than did their non-transgenic littermates. Interestingly, P2/P3-Tg mice showed low viral loads in the CNS at a very early period after infection (1-3 days) with TMEV and related EMCV but not unrelated VSV. Cells from P2/P3-Tg mice and cells transfected with the P2 and/or P3 regions in vitro yielded also lower viral replication but higher IFN-α/ß production. CONCLUSIONS: This study demonstrates that the expression of viral non-structural genes in mice inhibits initial viral replication and suppresses sustaining pathogenic anti-viral immune responses to broad viral determinants. It appears that the elevation of innate immune cytokines produced in the cells expressing the non-structural viral genes upon viral infection is responsible for the inhibitions. The inhibition is partially virus-specific as it is more efficient for a related virus compared to an unrelated virus, suggesting a role for the similarity in the viral genome structures. Therefore, the expression of viral non-structural genes may serve as a useful new method to prevent a broadly virus-specific pathogenesis in the hosts.

Overexpression of caspase 1 in apoptosis-resistant astrocytes infected with the BeAn Theiler's virus.

In this study, we demonstrate the upregulation in the expression of caspases 1 and 11 by SJL/J mouse brain astrocytes infected with the BeAn strain of Theiler's murine encephalomyelitis virus (TMEV). The upregulation of both proteases hints at protection of astrocytic cells from apoptotic death. We therefore looked for the reason of the demonstrated absence of programmed cell death in BeAn-infected SJL/J astrocytes. Complementary RNA (cRNA) from mock- and TMEV-infected cells was hybridized to the whole murine genome U74v2 DNA microarray from Affymetrix. Those experiments demonstrated the upregulation of gene expression for caspases 1 and 11 in infected cells. We further confirmed and validated their messenger RNA (mRNA) increase by reverse transcriptase quantitative real-time PCR (qPCR). The presence of both enzymatically active caspases 1 and 11 was demonstrated in cell lysates using a colorimetric and fluorymetric assay, respectively. We also show that overexpressed caspase 11 activated caspase 1 after preincubation of cytosol in vitro following a time-dependent process. This induction was neutralized by an anti-caspase 11 polyclonal antibody. These results demonstrate the activation of the caspase 1 precursor by caspase 11 and suggest a new mechanism of protection of BeAn-infected astrocytes from apoptosis. The direct experimental evidence that the protection effect demonstrated in this article was mediated by caspase 1, is provided by the fact that its specific inhibitor Z-WEHD-FMK induced de novo apoptotic death.

Depletion of Olig2 in oligodendrocyte progenitor cells infected by Theiler's murine encephalomyelitis virus.

Theiler's murine encephalomyelitis virus (TMEV) infects the central nervous system of mice and causes a demyelinating disease that is a model for multiple sclerosis. During the chronic phase of the disease, TMEV persists in oligodendrocytes and macrophages. Lack of remyelination has been attributed to insufficient proliferation and differentiation of oligodendrocyte progenitor cells (OPCs), but the molecular mechanisms remain unknown. Here, we employed pluripotent stem cell technologies to generate pure populations of mouse OPCs to study the temporal and molecular effects of TMEV infection. Global transcriptome analysis of RNA sequencing data revealed that TMEV infection of OPCs caused significant up-regulation of 1926 genes, whereas 1853 genes were significantly down-regulated compared to uninfected cells. Pathway analysis revealed that TMEV disrupted many genes required for OPC growth and maturation. Down-regulation of Olig2, a transcription factor necessary for OPC proliferation, was confirmed by real-time PCR, immunofluorescence microscopy, and western blot analysis. Depletion of Olig2 was not found to be specific to viral strain and did not require expression of the leader (L) protein, which is a multifunctional protein important for persistence, modulation of gene expression, and cell death. These data suggest that direct infection of OPCs by TMEV may inhibit remyelination during the chronic phase of TMEV-induced demyelinating disease.

Cardiovirus Leader proteins bind exportins: Implications for virus replication and nucleocytoplasmic trafficking inhibition.

Cardiovirus Leader proteins (LX) inhibit cellular nucleocytoplasmic trafficking by directing host kinases to phosphorylate Phe/Gly-containing nuclear pore proteins (Nups). Resolution of the Mengovirus LM structure bound to Ran GTPase, suggested this complex would further recruit specific exportins (karyopherins), which in turn mediate kinase selection. Pull-down experiments and recombinant complex reconstitution now confirm that Crm1 and CAS exportins form stable dimeric complexes with encephalomyocarditis virus LE, and also larger complexes with LE:Ran. shRNA knockdown studies support this idea. Similar activities could be demonstrated for recombinant LS and LT from Theiloviruses. When mutations were introduced to alter the LE zinc finger domain, acidic domain, or dual phosphorylation sites, there was reduced exportin selection. These regions are not involved in Ran interactions, so the Ran and Crm1 binding sites on LE must be non-overlapping. The involvement of exportins in this mechanism is important to viral replication and the observation of trafficking inhibition by LE.

IRF3 deficiency impacts granzyme B expression and maintenance of memory T cell function in response to viral infection.

The role of interferon regulatory factor 3 (IRF3) in the innate immune response to infection has been well studied. However, less is known about IRF3 signaling in shaping the adaptive T cell response. To determine the role of IRF3 in the generation and maintenance of effective anti-viral T cell responses, mice deficient in IRF3 were infected with a potentially persistent virus, Theiler's murine encephalomyelitis virus (TMEV) or with a model acute infection, influenza A virus (IAV). IRF3 was required to prevent TMEV persistence and induce robust TMEV specific effector T cell responses at the site of infection. This defect was more pronounced in the memory phase with an apparent lack of TMEV-specific memory T cells expressing granzyme B (GrB) in IRF3 deficient mice. In contrast, IRF3 had no effect on antigen specific T cell responses at the effector stage during IAV infection. However, memory T cell responses to IAV were also impaired in IRF3 deficient mice. Furthermore, addition of cytokines during peptide restimulation could not restore GrB expression in IRF3 deficient memory T cells. Taken together, IRF3 plays an important role in the maintenance of effective anti-viral T cell memory responses.

A CD8 T-cell epitope variant enhances immune targeting to a recombinant picornavirus vaccine antigen.

Recombinant virus vaccines are often less effective due to immunodominant responses against endogenous vector antigens. However, the use of small RNA virus vectors provides an opportunity to limit host exposure to endogenous virus antigens and focus immune responses on the desired vaccine antigen. Using the Daniel's strain of Theiler's murine encephalomyelitis virus, we have identified strategies to modulate responses to endogenous viral proteins by manipulating the host CD8+ T-cell repertoire prior to infection or through the use of mutations introduced into the virus genome. Both of these approaches enhance responses to vaccine antigens introduced into the picornavirus. However, the use of mutant immunodominant epitopes provides an opportunity for enhancing vaccine responses without further manipulation of the host. Using this strategy, we demonstrate that modification of the consensus MHC class I anchor residue within the virus genome can promote enhanced immunity to foreign antigens and self-antigens embedded in the virus genome.

Mutation of the Theiler's virus leader protein zinc-finger domain impairs apoptotic activity in murine macrophages.

The Theiler's murine encephalomyelitis virus (TMEV) leader (L) protein zinc-finger domain was mutated to study its role in cell death in infection of the murine macrophage cell line M1-D, revealing that an intact zinc-finger domain is required for full apoptotic activity. A functional L zinc-finger domain was also required for activation of p38 MAPK that results in phosphorylation and activation of p53, and in turn, alteration of the conformation of the anti-apoptotic proteins Puma and Mcl-1, leading to the release of pro-apoptotic Bax and apoptosis through the intrinsic pathway. TMEV infection also inhibits host protein synthesis, a stress shown by others to induce apoptosis. Since inhibition of host protein synthesis follows rather than precedes activation of MKK3/6 and p38, it seems less likely that it triggers apoptosis in infected cells. Finally, we showed that the levels of reactive oxygen species following infection were consistent with apoptotic rather than necrotic cell death. Thus, these experiments support an important role for the TMEV L protein zinc-finger domain in apoptosis in an infected murine macrophage line.

Evasion of antiviral innate immunity by Theiler's virus L* protein through direct inhibition of RNase L.

Theiler's virus is a neurotropic picornavirus responsible for chronic infections of the central nervous system. The establishment of a persistent infection and the subsequent demyelinating disease triggered by the virus depend on the expression of L*, a viral accessory protein encoded by an alternative open reading frame of the virus. We discovered that L* potently inhibits the interferon-inducible OAS/RNase L pathway. The antagonism of RNase L by L* was particularly prominent in macrophages where baseline oligoadenylate synthetase (OAS) and RNase L expression levels are elevated, but was detectable in fibroblasts after IFN pretreatment. L* mutations significantly affected Theiler's virus replication in primary macrophages derived from wild-type but not from RNase L-deficient mice. L* counteracted the OAS/RNase L pathway through direct interaction with the ankyrin domain of RNase L, resulting in the inhibition of this enzyme. Interestingly, RNase L inhibition was species-specific as Theiler's virus L* protein blocked murine RNase L but not human RNase L or RNase L of other mammals or birds. Direct RNase L inhibition by L* and species specificity were confirmed in an in vitro assay performed with purified proteins. These results demonstrate a novel viral mechanism to elude the antiviral OAS/RNase L pathway. By targeting the effector enzyme of this antiviral pathway, L* potently inhibits RNase L, underscoring the importance of this enzyme in innate immunity against Theiler's virus.

Anandamide inhibits Theiler's virus induced VCAM-1 in brain endothelial cells and reduces leukocyte transmigration in a model of blood brain barrier by activation of CB(1) receptors.

BACKGROUND: VCAM-1 represents one of the most important adhesion molecule involved in the transmigration of blood leukocytes across the blood-brain barrier (BBB) that is an essential step in the pathogenesis of MS. Several evidences have suggested the potential therapeutic value of cannabinoids (CBs) in the treatment of MS and their experimental models. However, the effects of endocannabinoids on VCAM-1 regulation are poorly understood. In the present study we investigated the effects of anandamide (AEA) in the regulation of VCAM-1 expression induced by Theiler's virus (TMEV) infection of brain endothelial cells using in vitro and in vivo approaches. METHODS: i) in vitro: VCAM-1 was measured by ELISA in supernatants of brain endothelial cells infected with TMEV and subjected to AEA and/or cannabinoid receptors antagonist treatment. To evaluate the functional effect of VCAM-1 modulation we developed a blood brain barrier model based on a system of astrocytes and brain endothelial cells co-culture. ii) in vivo: CB(1) receptor deficient mice (Cnr1(-/-)) infected with TMEV were treated with the AEA uptake inhibitor UCM-707 for three days. VCAM-1 expression and microglial reactivity were evaluated by immunohistochemistry. RESULTS: Anandamide-induced inhibition of VCAM-1 expression in brain endothelial cell cultures was mediated by activation of CB(1) receptors. The study of leukocyte transmigration confirmed the functional relevance of VCAM-1 inhibition by AEA. In vivo approaches also showed that the inhibition of AEA uptake reduced the expression of brain VCAM-1 in response to TMEV infection. Although a decreased expression of VCAM-1 by UCM-707 was observed in both, wild type and CB(1) receptor deficient mice (Cnr1(-/-)), the magnitude of VCAM-1 inhibition was significantly higher in the wild type mice. Interestingly, Cnr1(-/-) mice showed enhanced microglial reactivity and VCAM-1 expression following TMEV infection, indicating that the lack of CB(1) receptor exacerbated neuroinflammation. CONCLUSIONS: Our results suggest that CB(1) receptor dependent VCAM-1 inhibition is a novel mechanism for AEA-reduced leukocyte transmigration and contribute to a better understanding of the mechanisms underlying the beneficial role of endocannabinoid system in the Theiler's virus model of MS.