Frédéric Sorgeloos

Lambda Interferon Renders Epithelial Cells of the Respiratory and Gastrointestinal Tracts Resistant to Viral Infections

ABSTRACT Virus-infected cells secrete a broad range of interferons (IFN) which confer resistance to yet uninfected cells by triggering the synthesis of antiviral factors. The relative contributions of the various IFN subtypes to innate immunity against virus infections remain elusive. IFN-α, IFN-β, and other type I IFN molecules signal through a common, universally expressed cell surface receptor, whereas type III IFN (IFN-λ) uses a distinct cell-type-specific receptor complex for signaling. Using mice lacking functional receptors for type I IFN, type III IFN, or both, we found that IFN-λ plays an important role in the defense against several human pathogens that infect the respiratory tract, such as influenza A virus, influenza B virus, respiratory syncytial virus, human metapneumovirus, and severe acute respiratory syndrome (SARS) coronavirus. These viruses were more pathogenic and replicated to higher titers in the lungs of mice lacking both IFN receptors than in mice with single IFN receptor defects. In contrast, Lassa fever virus, which infects via the respiratory tract but primarily replicates in the liver, was not influenced by the IFN-λ receptor defect. Careful analysis revealed that expression of functional IFN-λ receptor complexes in the lung and intestinal tract is restricted to epithelial cells and a few other, undefined cell types. Interestingly, we found that SARS coronavirus was present in feces from infected mice lacking receptors for both type I and type III IFN but not in those from mice lacking single receptors, supporting the view that IFN-λ contributes to the control of viral infections in epithelial cells of both respiratory and gastrointestinal tracts.

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

Theilers 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 Theilers 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 Theilers 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 Theilers virus.

Murine Norovirus: Propagation, Quantification and Genetic Manipulation

Murine norovirus (MNV) is a positive‐sense, plus‐stranded RNA virus in the Caliciviridae family. It is the most common pathogen in biomedical research colonies. MNV is also related to the human noroviruses, which cause the majority of nonbacterial gastroenteritis worldwide. Like the human noroviruses, MNV is an enteric virus that replicates in the intestine and is transmitted by the fecal‐oral route. MNV replicates in murine macrophages and dendritic cells in cells in culture and in the murine host. This virus is often used to study mechanisms in norovirus biology, because human noroviruses are refractory to growth in cell culture. MNV combines the availability of a cell culture and reverse genetics system with the ability to study infection in the native host. Herein, we describe a panel of techniques that are commonly used to study MNV biology. Curr. Protoc. Microbiol 33:15K.2.1‐15K.2.61.

Both α2,3- and α2,6-Linked Sialic Acids on O-Linked Glycoproteins Act as Functional Receptors for Porcine Sapovirus

Sapovirus, a member of the Caliciviridae family, is an important cause of acute gastroenteritis in humans and pigs. Currently, the porcine sapovirus (PSaV) Cowden strain remains the only cultivable member of the Sapovirus genus. While some caliciviruses are known to utilize carbohydrate receptors for entry and infection, a functional receptor for sapovirus is unknown. To characterize the functional receptor of the Cowden strain of PSaV, we undertook a comprehensive series of protein-ligand biochemical assays in mock and PSaV-infected cell culture and/or piglet intestinal tissue sections. PSaV revealed neither hemagglutination activity with red blood cells from any species nor binding activity to synthetic histo-blood group antigens, indicating that PSaV does not use histo-blood group antigens as receptors. Attachment and infection of PSaV were markedly blocked by sialic acid and Vibrio cholerae neuraminidase (NA), suggesting a role for α2,3-linked, α2,6-linked or α2,8-linked sialic acid in virus attachment. However, viral attachment and infection were only partially inhibited by treatment of cells with sialidase S (SS) or Maackia amurensis lectin (MAL), both specific for α2,3-linked sialic acid, or Sambucus nigra lectin (SNL), specific for α2,6-linked sialic acid. These results indicated that PSaV recognizes both α2,3- and α2,6-linked sialic acids for viral attachment and infection. Treatment of cells with proteases or with benzyl 4-O-β-D-galactopyranosyl-β-D-glucopyranoside (benzylGalNAc), which inhibits O-linked glycosylation, also reduced virus binding and infection, whereas inhibition of glycolipd synthesis or N-linked glycosylation had no such effect on virus binding or infection. These data suggest PSaV binds to cellular receptors that consist of α2,3- and α2,6-linked sialic acids on glycoproteins attached via O-linked glycosylation.

Antiviral Type I and Type III Interferon Responses in the Central Nervous System

The central nervous system (CNS) harbors highly differentiated cells, such as neurons that are essential to coordinate the functions of complex organisms. This organ is partly protected by the blood-brain barrier (BBB) from toxic substances and pathogens carried in the bloodstream. Yet, neurotropic viruses can reach the CNS either by crossing the BBB after viremia, or by exploiting motile infected cells as Trojan horses, or by using axonal transport. Type I and type III interferons (IFNs) are cytokines that are critical to control early steps of viral infections. Deficiencies in the IFN pathway have been associated with fatal viral encephalitis both in humans and mice. Therefore, the IFN system provides an essential protection of the CNS against viral infections. Yet, basal activity of the IFN system appears to be low within the CNS, likely owing to the toxicity of IFN to this organ. Moreover, after viral infection, neurons and oligodendrocytes were reported to be relatively poor IFN producers and appear to keep some susceptibility to neurotropic viruses, even in the presence of IFN. This review addresses some trends and recent developments concerning the role of type I and type III IFNs in: i) preventing neuroinvasion and infection of CNS cells; ii) the identity of IFN-producing cells in the CNS; iii) the antiviral activity of ISGs; and iv) the activity of viral proteins of neurotropic viruses that target the IFN pathway.

Random Mutagenesis Defines a Domain of Theiler's Virus Leader Protein That Is Essential for Antagonism of Nucleocytoplasmic Trafficking and Cytokine Gene Expression

ABSTRACT The leader protein of cardioviruses, Theilers murine encephalomyelitis virus (TMEV) and encephalomyocarditis virus (EMCV), is a multifunctional protein known to antagonize type I interferon expression and to interfere with nucleocytoplasmic trafficking of host proteins and mRNA. This protein plays an important role in the capacity of TMEV to establish persistent infection of the central nervous system. Mutant forms of the TMEV leader protein were generated by random mutagenesis and selected after retroviral transduction on the basis of the loss of the highly toxic nature of this protein. Selected mutations define a short C-terminal domain of the leader conserved in TMEV and Saffold virus but lacking in the EMCV leader and thus called the Theilo domain. Mutations in this domain had a dramatic impact on TMEV L protein activity. Like the zinc finger mutation, Theilo domain mutations affected all of the activities of the L protein tested: interferon gene transcription and IRF-3 dimerization antagonism, alteration of nucleocytoplasmic trafficking, nucleoporin 98 hyperphosphorylation, and viral persistence in vivo. This suggests that the Zn finger and the Theilo domain of the protein cooperate for function. Moreover, the fact that all of the activities tested were affected by these mutations suggests that the various leader protein functions are somehow coupled.

Deregulation of SYCP2 predicts early stage human papillomavirus-positive oropharyngeal carcinoma: A prospective whole transcriptome analysis.

This study was designed to identify significant differences in gene expression profiles of human papillomavirus (HPV)‐positive and HPV‐negative oropharyngeal squamous cell carcinomas (OPSCC) and to better understand the functional and biological effects of HPV infection in the premalignant pathway. Twenty‐four consecutive patients with locally advanced primary OPSCC were included in a prospective clinical trial. Fresh tissue samples (tumor vs. matched normal epithelium) were subjected to whole transcriptome analysis and the results validated on the same cohort with RT–quantitative real‐time PCR. In a separate retrospective cohort of 27 OPSCC patients, laser capture microdissection of formalin‐fixed, paraffin‐embedded tissue allowed RNA extraction from adjacent regions of normal epithelium, carcinoma in situ (premalignant) and invasive SCC tissue. The majority of patients showed evidence of high‐risk HPV16 positivity (80.4%). Predictable fold changes of RNA expression in HPV‐associated disease included multiple transcripts within the p53 oncogenic pathway (e.g. CDKN2A/CCND1). Other candidate transcripts found to have altered levels of expression in this study have not previously been established (SFRP1, CRCT1, DLG2, SYCP2, and CRNN). Of these, SYCP2 showed the most consistent fold change from baseline in premalignant tissue; aberrant expression of this protein may contribute to genetic instability during HPV‐associated cancer development. If further corroborated, this data may contribute to the development of a non‐invasive screening tool. This study is registered with the UK Clinical Research Network (ref.: 11945).

Inefficient Type I Interferon-Mediated Antiviral Protection of Primary Mouse Neurons Is Associated with the Lack of Apolipoprotein L9 Expression

ABSTRACT We examined the antiviral response promoted by type I interferons (IFN) in primary mouse neurons. IFN treatment of neuron cultures strongly upregulated the transcription of IFN-stimulated genes but conferred a surprisingly low resistance to infection by neurotropic viruses such as Theilers murine encephalomyelitis virus (TMEV) or vesicular stomatitis virus (VSV). Response of primary mouse neurons to IFN treatment was heterogeneous, as many neurons failed to express the typical IFN response marker Mx1 after IFN treatment. This heterogeneous response of primary neurons correlated with a low level of basal expression of IFN-stimulated genes, such as Stat1, that are involved in signal transduction of the IFN response. In addition, transcriptomic analysis identified 15 IFN-responsive genes whose expression was low in IFN-treated primary neurons compared to that of primary fibroblasts derived from the same mice (Dhx58, Gvin1, Sp100, Ifi203 isoforms 1 and 2, Irgm2, Lgals3bp, Ifi205, Apol9b, Ifi204, Ifi202b, Tor3a, Slfn2, Ifi35, Lgals9). Among these genes, the gene coding for apolipoprotein L9b (Apol9b) displayed antiviral activity against Theilers virus when overexpressed in L929 cells or in primary neurons. Accordingly, knocking down Apol9b expression in L929 cells increased viral replication. Therefore, we identified a new antiviral protein induced by interferon, ApoL9b, whose lack of expression in primary neurons likely contributes to the high sensitivity of these cells to viral infection. IMPORTANCE The type I interferon (IFN) response is an innate immune defense mechanism that is critical to contain viral infection in the host until an adaptive immune response can be mounted. Neurons are a paradigm for postmitotic, highly differentiated cells. Our data show that primary mouse neurons that are exposed to type I interferon remain surprisingly susceptible to viral infection. On one hand, the low level of basal expression of some factors in neurons might prevent a rapid response of these cells. On the other hand, some genes that are typically activated by type I interferon in other cell types are expressed at much lower levels in neurons. Among these genes is the gene encoding apolipoprotein L9, a protein that proved to have antiviral activity against the neurotropic Theilers murine encephalomyelitis virus. Our data suggest important functional differences in the IFN response mounted by specific cell populations.

Differential IFN-alpha/beta production suppressing capacities of the leader proteins of mengovirus and foot-and-mouth disease virus.

Picornaviruses encompass a large family of RNA viruses. Some picornaviruses possess a leader (L) protein at the N‐terminus of their polyprotein. The L proteins of encephalomyocarditis virus, a cardiovirus, and foot‐and‐mouth disease virus (FMDV), an aphthovirus, are both dispensable for replication and their major function seems to be the suppression of antiviral host cell responses. Previously, we showed that the L protein of mengovirus, a strain of encephalomyocarditis virus, inhibits antiviral responses by inhibiting type I interferon (IFN‐α/β) gene transcription. The L protein of the FMDV is a protease (Lpro) that cleaves cellular factors to reduce cytokine and chemokine mRNA production and to inhibit cap‐dependent cellular host mRNA translation, thereby limiting the production of proteins with antiviral activity. In this study, we constructed a viable chimeric mengovirus that expresses FMDV Lpro in place of the authentic L protein in order to compare the efficiency of the immune evasion mechanisms mediated by L and Lpro respectively. We show that in this mengovirus background the L protein is more potent than FMDV Lpro in suppressing IFN‐α/β responses. Yet, FMDV Lpro is important to antagonize infection‐limiting responses both in vitro and in vivo.

A Conserved Interaction between a C-Terminal Motif in Norovirus VPg and the HEAT-1 Domain of eIF4G Is Essential for Translation Initiation

Translation initiation is a critical early step in the replication cycle of the positive-sense, single-stranded RNA genome of noroviruses, a major cause of gastroenteritis in humans. Norovirus RNA, which has neither a 5´ m7G cap nor an internal ribosome entry site (IRES), adopts an unusual mechanism to initiate protein synthesis that relies on interactions between the VPg protein covalently attached to the 5´-end of the viral RNA and eukaryotic initiation factors (eIFs) in the host cell. For murine norovirus (MNV) we previously showed that VPg binds to the middle fragment of eIF4G (4GM; residues 652–1132). Here we have used pull-down assays, fluorescence anisotropy, and isothermal titration calorimetry (ITC) to demonstrate that a stretch of ~20 amino acids at the C terminus of MNV VPg mediates direct and specific binding to the HEAT-1 domain within the 4GM fragment of eIF4G. Our analysis further reveals that the MNV C terminus binds to eIF4G HEAT-1 via a motif that is conserved in all known noroviruses. Fine mutagenic mapping suggests that the MNV VPg C terminus may interact with eIF4G in a helical conformation. NMR spectroscopy was used to define the VPg binding site on eIF4G HEAT-1, which was confirmed by mutagenesis and binding assays. We have found that this site is non-overlapping with the binding site for eIF4A on eIF4G HEAT-1 by demonstrating that norovirus VPg can form ternary VPg-eIF4G-eIF4A complexes. The functional significance of the VPg-eIF4G interaction was shown by the ability of fusion proteins containing the C-terminal peptide of MNV VPg to inhibit in vitro translation of norovirus RNA but not cap- or IRES-dependent translation. These observations define important structural details of a functional interaction between norovirus VPg and eIF4G and reveal a binding interface that might be exploited as a target for antiviral therapy.