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1.
Proc Natl Acad Sci U S A ; 119(5)2022 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-35091472

RESUMO

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.


Assuntos
Interações entre Hospedeiro e Microrganismos/fisiologia , Proteínas Quinases S6 Ribossômicas 90-kDa/genética , Bactérias/patogenicidade , Infecções Bacterianas/genética , Infecções Bacterianas/metabolismo , Evolução Biológica , Linhagem Celular , Regulação Viral da Expressão Gênica/genética , Interações entre Hospedeiro e Microrganismos/genética , Humanos , Proteínas Imediatamente Precoces/genética , Sistema de Sinalização das MAP Quinases/fisiologia , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Proteínas Quinases S6 Ribossômicas 90-kDa/metabolismo , Viroses/genética , Viroses/metabolismo , Replicação Viral/fisiologia , Vírus/patogenicidade
2.
PLoS Pathog ; 18(12): e1011042, 2022 12.
Artigo em Inglês | MEDLINE | ID: mdl-36508477

RESUMO

Proteins from some unrelated pathogens, including small RNA viruses of the family Picornaviridae, large DNA viruses such as Kaposi sarcoma-associated herpesvirus and even bacteria of the genus Yersinia can recruit cellular p90-ribosomal protein S6 kinases (RSKs) through a common linear motif and maintain the kinases in an active state. On the one hand, pathogens' proteins might hijack RSKs to promote their own phosphorylation (direct target model). On the other hand, some data suggested that pathogens' proteins might dock the hijacked RSKs toward a third interacting partner, thus redirecting the kinase toward a specific substrate. We explored the second hypothesis using the Cardiovirus leader protein (L) as a paradigm. The L protein is known to trigger nucleocytoplasmic trafficking perturbation, which correlates with hyperphosphorylation of phenylalanine-glycine (FG)-nucleoporins (FG-NUPs) such as NUP98. Using a biotin ligase fused to either RSK or L, we identified FG-NUPs as primary partners of the L-RSK complex in infected cells. An L protein mutated in the central RSK-interaction motif was readily targeted to the nuclear envelope whereas an L protein mutated in the C-terminal domain still interacted with RSK but failed to interact with the nuclear envelope. Thus, L uses distinct motifs to recruit RSK and to dock the L-RSK complex toward the FG-NUPs. Using an analog-sensitive RSK2 mutant kinase, we show that, in infected cells, L can trigger RSK to use NUP98 and NUP214 as direct substrates. Our data therefore illustrate a novel virulence mechanism where pathogens' proteins hijack and retarget cellular protein kinases toward specific substrates, to promote their replication or to escape immunity.


Assuntos
Cardiovirus , Proteínas Quinases S6 Ribossômicas 90-kDa/genética , Proteínas Quinases S6 Ribossômicas 90-kDa/metabolismo , Proteínas Quinases/metabolismo , Fosforilação
3.
J Virol ; 95(20): e0113421, 2021 09 27.
Artigo em Inglês | MEDLINE | ID: mdl-34346771

RESUMO

Murine norovirus (MNV) infection results in a late translation shutoff that is proposed to contribute to the attenuated and delayed innate immune response observed both in vitro and in vivo. Recently, we further demonstrated the activation of the α subunit of eukaryotic initiation factor 2 (eIF2α) kinase GCN2 during MNV infection, which has been previously linked to immunomodulation and resistance to inflammatory signaling during metabolic stress. While viral infection is usually associated with activation of double-stranded RNA (dsRNA) binding pattern recognition receptor PKR, we hypothesized that the establishment of a metabolic stress in infected cells is a proviral event, exploited by MNV to promote replication through weakening the activation of the innate immune response. In this study, we used multi-omics approaches to characterize cellular responses during MNV replication. We demonstrate the activation of pathways related to the integrated stress response, a known driver of anti-inflammatory phenotypes in macrophages. In particular, MNV infection causes an amino acid imbalance that is associated with GCN2 and ATF2 signaling. Importantly, this reprogramming lacks the features of a typical innate immune response, with the ATF/CHOP target GDF15 contributing to the lack of antiviral responses. We propose that MNV-induced metabolic stress supports the establishment of host tolerance to viral replication and propagation. IMPORTANCE During viral infection, host defenses are typically characterized by the secretion of proinflammatory autocrine and paracrine cytokines, potentiation of the interferon (IFN) response, and induction of the antiviral response via activation of JAK and Stat signaling. To avoid these and propagate, viruses have evolved strategies to evade or counteract host sensing. In this study, we demonstrate that murine norovirus controls the antiviral response by activating a metabolic stress response that activates the amino acid response and impairs inflammatory signaling. This highlights novel tools in the viral countermeasures arsenal and demonstrates the importance of the currently poorly understood metabolic reprogramming occurring during viral infections.


Assuntos
Infecções por Caliciviridae/imunologia , Macrófagos/virologia , Fator 2 Ativador da Transcrição/metabolismo , Animais , Antivirais , Infecções por Caliciviridae/metabolismo , Linhagem Celular , Fator de Iniciação 2 em Eucariotos/metabolismo , Imunidade Inata/imunologia , Inflamação/imunologia , Interferons , Macrófagos/imunologia , Camundongos , Norovirus/patogenicidade , Proteínas Serina-Treonina Quinases/metabolismo , Células RAW 264.7 , RNA de Cadeia Dupla/genética , Transdução de Sinais/imunologia , Proteínas não Estruturais Virais/metabolismo , Replicação Viral/genética
4.
J Gen Virol ; 102(9)2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34491891

RESUMO

Murine norovirus (MNV) is widely used as a model for studying norovirus biology. While MNV isolates vary in their pathogenesis, infection of immunocompetent mice mostly results in persistent infection. The ability of a virus to establish a persistent infection is dependent on its ability to subvert or avoid the host immune response. Previously, we described the identification and characterization of virulence factor 1 (VF1) in MNV, and demonstrated its role as an innate immune antagonist. Here, we explore the role of VF1 during persistent MNV infection in an immunocompetent host. Using reverse genetics, we generated MNV-3 viruses carrying a single or a triple termination codon inserted in the VF1 ORF. VF1-deleted MNV-3 replicated to comparable levels to the wildtype virus in tissue culture. Comparative studies between MNV-3 and an acute MNV-1 strain show that MNV-3 VF1 exerts the same functions as MNV-1 VF1, but with reduced potency. C57BL/6 mice infected with VF1-deleted MNV-3 showed significantly reduced replication kinetics during the acute phase of the infection, but viral loads rapidly reached the levels seen in mice infected with wildtype virus after phenotypic restoration of VF1 expression. Infection with an MNV-3 mutant that had three termination codons inserted into VF1, in which reversion was suppressed, resulted in consistently lower replication throughout a 3 month persistent infection in mice, suggesting a role for VF1 in viral fitness in vivo. Our results indicate that VF1 expressed by a persistent strain of MNV also functions to antagonize the innate response to infection. We found that VF1 is not essential for viral persistence, but instead contributes to viral fitness in mice. These data fit with the hypothesis that noroviruses utilize multiple mechanisms to avoid and/or control the host response to infection and that VF1 is just one component of this.


Assuntos
Infecções por Caliciviridae/virologia , Norovirus/patogenicidade , Proteínas Virais/metabolismo , Fatores de Virulência/metabolismo , Animais , Apoptose , Infecções por Caliciviridae/imunologia , Linhagem Celular , Imunidade Inata , Interferon beta/biossíntese , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Norovirus/genética , Norovirus/fisiologia , Proteínas Virais/genética , Virulência , Fatores de Virulência/genética , Replicação Viral , Eliminação de Partículas Virais
5.
PLoS Pathog ; 15(4): e1007709, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-31017981

RESUMO

Norovirus infection is the leading cause of food-borne gastroenteritis worldwide, being responsible for over 200,000 deaths annually. Studies with murine norovirus (MNV) showed that protective STAT1 signaling controls viral replication and pathogenesis, but the immune mechanisms that noroviruses exploit to induce pathology are elusive. Here, we show that gastrointestinal MNV infection leads to widespread IL-1ß maturation in MNV-susceptible STAT1-deficient mice. MNV activates the canonical Nlrp3 inflammasome in macrophages, leading to maturation of IL-1ß and to Gasdermin D (GSDMD)-dependent pyroptosis. STAT1-deficient macrophages displayed increased MAVS-mediated expression of pro-IL-1ß, facilitating elevated Nlrp3-dependent release of mature IL-1ß upon MNV infection. Accordingly, MNV-infected Stat1-/- mice showed Nlrp3-dependent maturation of IL-1ß as well as Nlrp3-dependent pyroptosis as assessed by in vivo cleavage of GSDMD to its active N-terminal fragment. While MNV-induced diarrheic responses were not affected, Stat1-/- mice additionally lacking either Nlrp3 or GSDMD displayed lower levels of the fecal inflammatory marker Lipocalin-2 as well as delayed lethality after gastrointestinal MNV infection. Together, these results uncover new insights into the mechanisms of norovirus-induced inflammation and cell death, thereby revealing Nlrp3 inflammasome activation and ensuing GSDMD-driven pyroptosis as contributors to MNV-induced immunopathology in susceptible STAT1-deficient mice.


Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Infecções por Caliciviridae/patologia , Trato Gastrointestinal/patologia , Inflamassomos/imunologia , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Piroptose/fisiologia , Fator de Transcrição STAT1/fisiologia , Animais , Proteínas Reguladoras de Apoptose/genética , Infecções por Caliciviridae/imunologia , Infecções por Caliciviridae/metabolismo , Infecções por Caliciviridae/virologia , Células Cultivadas , Trato Gastrointestinal/imunologia , Trato Gastrointestinal/metabolismo , Trato Gastrointestinal/virologia , Inflamassomos/metabolismo , Interleucina-1beta/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular , Macrófagos/imunologia , Macrófagos/metabolismo , Macrófagos/patologia , Macrófagos/virologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Norovirus/imunologia , Norovirus/patogenicidade , Proteínas de Ligação a Fosfato
6.
J Virol ; 93(19)2019 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-31292248

RESUMO

Leader (L) proteins encoded by cardioviruses are multifunctional proteins that contribute to innate immunity evasion. L proteins of Theiler's murine encephalomyelitis virus (TMEV), Saffold virus (SAFV), and encephalomyocarditis virus (EMCV) were reported to inhibit stress granule assembly in infected cells. Here, we show that TMEV L can act at two levels in the stress granule formation pathway: on the one hand, it can inhibit sodium arsenite-induced stress granule assembly without preventing eIF2α phosphorylation and, thus, acts downstream of eIF2α; on the other hand, it can inhibit eucaryotic translation initiation factor 2 alpha kinase 2 (PKR) activation and the consequent PKR-mediated eIF2α phosphorylation. Interestingly, coimmunostaining experiments revealed that PKR colocalizes with viral double-stranded RNA (dsRNA) in cells infected with L-mutant viruses but not in cells infected with the wild-type virus. Furthermore, PKR coprecipitated with dsRNA from cells infected with L-mutant viruses significantly more than from cells infected with the wild-type virus. These data strongly suggest that L blocks PKR activation by preventing the interaction between PKR and viral dsRNA. In infected cells, L also rendered PKR refractory to subsequent activation by poly(I·C). However, no interaction was observed between L and either dsRNA or PKR. Taken together, our results suggest that, unlike other viral proteins, L indirectly acts on PKR to negatively regulate its responsiveness to dsRNA.IMPORTANCE The leader (L) protein encoded by cardioviruses is a very short multifunctional protein that contributes to evasion of the host innate immune response. This protein notably prevents the formation of stress granules in infected cells. Using Theiler's virus as a model, we show that L proteins can act at two levels in the stress response pathway leading to stress granule formation, the most striking one being the inhibition of eucaryotic translation initiation factor 2 alpha kinase 2 (PKR) activation. Interestingly, the leader protein appears to inhibit PKR via a novel mechanism by rendering this kinase unable to detect double-stranded RNA, its typical activator. Unlike other viral proteins, such as influenza virus NS1, the leader protein appears to interact with neither PKR nor double-stranded RNA, suggesting that it acts indirectly to trigger the inhibition of the kinase.


Assuntos
Ativação Enzimática , Interações Hospedeiro-Patógeno , Evasão da Resposta Imune , Theilovirus/fisiologia , Proteínas Virais/metabolismo , eIF-2 Quinase/antagonistas & inibidores , Animais , Linhagem Celular , Humanos , Ligação Proteica , RNA de Cadeia Dupla/metabolismo , RNA Viral/metabolismo
7.
Mol Cell Proteomics ; 16(4 suppl 1): S215-S229, 2017 04.
Artigo em Inglês | MEDLINE | ID: mdl-28087593

RESUMO

Noroviruses produce viral RNAs lacking a 5' cap structure and instead use a virus-encoded viral protein genome-linked (VPg) protein covalently linked to viral RNA to interact with translation initiation factors and drive viral protein synthesis. Norovirus infection results in the induction of the innate response leading to interferon stimulated gene (ISG) transcription. However, the translation of the induced ISG mRNAs is suppressed. A SILAC-based mass spectrometry approach was employed to analyze changes to protein abundance in both whole cell and m7GTP-enriched samples to demonstrate that diminished host mRNA translation correlates with changes to the composition of the eukaryotic initiation factor complex. The suppression of host ISG translation correlates with the activity of the viral protease (NS6) and the activation of cellular caspases leading to the establishment of an apoptotic environment. These results indicate that noroviruses exploit the differences between viral VPg-dependent and cellular cap-dependent translation in order to diminish the host response to infection.


Assuntos
Infecções por Caliciviridae/genética , Norovirus/metabolismo , Proteômica/métodos , Capuzes de RNA/metabolismo , RNA Mensageiro/metabolismo , Proteínas Virais/metabolismo , Apoptose , Infecções por Caliciviridae/imunologia , Infecções por Caliciviridae/virologia , Interações Hospedeiro-Patógeno , Humanos , Imunidade Inata , Marcação por Isótopo/métodos , Espectrometria de Massas/métodos , Norovirus/genética , RNA Viral/metabolismo
8.
PLoS Pathog ; 12(1): e1005379, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26734730

RESUMO

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.


Assuntos
Fator de Iniciação Eucariótico 4G/metabolismo , Norovirus/fisiologia , Iniciação Traducional da Cadeia Peptídica/fisiologia , Proteínas Virais/metabolismo , Motivos de Aminoácidos , Animais , Calorimetria , Linhagem Celular , Cromatografia em Gel , Imunoprecipitação , Espectroscopia de Ressonância Magnética , Camundongos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Estrutura Secundária de Proteína
9.
J Virol ; 90(14): 6489-6501, 2016 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-27147742

RESUMO

UNLABELLED: In response to stress such as virus infection, cells can stall translation by storing mRNAs away in cellular compartments called stress granules (SGs). This defense mechanism favors cell survival by limiting the use of energy and nutrients until the stress is resolved. In some cases it may also block viral propagation as viruses are dependent on the host cell resources to produce viral proteins. Human norovirus is a member of the Caliciviridae family responsible for gastroenteritis outbreaks worldwide. Previous studies on caliciviruses have identified mechanisms by which they can usurp the host translational machinery, using the viral protein genome-linked VPg, or regulate host protein synthesis through the mitogen-activated protein kinase (MAPK) pathway. Here, we examined the effect of feline calicivirus (FCV) infection on SG accumulation. We show that FCV infection impairs the assembly of SGs despite an increased phosphorylation of eukaryotic initiation factor eIF2α, a hallmark of stress pathway activation. Furthermore, SGs did not accumulate in FCV-infected cells that were stressed with arsenite or hydrogen peroxide. FCV infection resulted in the cleavage of the SG-nucleating protein Ras-GTPase activating SH3 domain-binding protein (G3BP1), which is mediated by the viral 3C-like proteinase NS6(Pro) Using mutational analysis, we identified the FCV-induced cleavage site within G3BP1, which differs from the poliovirus 3C proteinase cleavage site previously identified. Finally, we showed that NS6(Pro)-mediated G3BP1 cleavage impairs SG assembly. In contrast, murine norovirus (MNV) infection did not impact arsenite-induced SG assembly or G3BP1 integrity, suggesting that related caliciviruses have distinct effects on the stress response pathway. IMPORTANCE: Human noroviruses are a major cause of viral gastroenteritis, and it is important to understand how they interact with the infected host cell. Feline calicivirus (FCV) and murine norovirus (MNV) are used as models to understand norovirus biology. Recent studies have suggested that the assembly of stress granules is central in orchestrating stress and antiviral responses to restrict viral replication. Overall, our study provides the first insight on how caliciviruses impair stress granule assembly by targeting the nucleating factor G3BP1 via the viral proteinase NS6(Pro) This work provides new insights into host-pathogen interactions that regulate stress pathways during FCV infection.


Assuntos
Infecções por Caliciviridae/virologia , Calicivirus Felino/patogenicidade , Proteínas de Transporte/metabolismo , Grânulos Citoplasmáticos/metabolismo , Interações Hospedeiro-Patógeno , Replicação Viral , Proteases Virais 3C , Animais , Infecções por Caliciviridae/metabolismo , Infecções por Caliciviridae/patologia , Proteínas de Transporte/genética , Gatos , Cisteína Endopeptidases/metabolismo , Grânulos Citoplasmáticos/virologia , DNA Helicases , Fator de Iniciação 2 em Eucariotos/metabolismo , Células HeLa , Humanos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fosforilação , Proteínas de Ligação a Poli-ADP-Ribose , RNA Helicases , Proteínas com Motivo de Reconhecimento de RNA , Proteínas Virais/metabolismo
10.
J Gen Virol ; 97(6): 1350-1355, 2016 06.
Artigo em Inglês | MEDLINE | ID: mdl-26959376

RESUMO

Saffold virus (SAFV) is a highly seroprevalent human Cardiovirus discovered recently. No clear association between SAFV infection and human disease has been established. Rare infection cases, however, correlated with neurological symptoms. To gain insight into the pathogenesis potential of the virus, we performed experimental mouse infection with SAFV strains of genotypes 2 and 3 (SAFV-2 and SAFV-3). After intraperitoneal infection, both strains exhibited a typical Cardiovirus tropism. Viral load was most prominent in the pancreas. Heart, spleen, brain and spinal cord were also infected. In IFN-receptor 1 deficient (IFNAR-KO) mice, SAFV-3 caused a severe encephalitis. The virus was detected by immunohistochemistry in many parts of the brain and spinal cord, both in neurons and astrocytes, but astrocyte infection was more extensive. In vitro, SAFV-3 also infected astrocytes better than neurons in mixed primary cultures. Astrocytes were, however, very efficiently protected by IFN-α/ß treatment.


Assuntos
Infecções por Cardiovirus/patologia , Infecções por Cardiovirus/virologia , Sistema Nervoso/patologia , Sistema Nervoso/virologia , Theilovirus/fisiologia , Tropismo Viral , Estruturas Animais/virologia , Animais , Modelos Animais de Doenças , Imuno-Histoquímica , Camundongos , Microscopia , Carga Viral
11.
PLoS Pathog ; 10(6): e1004172, 2014 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-24901849

RESUMO

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-ß-D-galactopyranosyl-ß-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.


Assuntos
Interações Hospedeiro-Patógeno , Mucosa Intestinal/virologia , Glicoproteínas de Membrana/metabolismo , Modelos Moleculares , Receptores Virais/metabolismo , Sapovirus/fisiologia , Ácidos Siálicos/metabolismo , Animais , Infecções por Caliciviridae/patologia , Infecções por Caliciviridae/veterinária , Infecções por Caliciviridae/virologia , Linhagem Celular , Inibidores Enzimáticos/farmacologia , Gastroenterite/patologia , Gastroenterite/veterinária , Gastroenterite/virologia , Glicosilação/efeitos dos fármacos , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Humanos , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patologia , Ligantes , Glicoproteínas de Membrana/antagonistas & inibidores , Glicoproteínas de Membrana/química , Estabilidade Proteica , Receptores Virais/antagonistas & inibidores , Receptores Virais/química , Sapovirus/efeitos dos fármacos , Sapovirus/patogenicidade , Ácidos Siálicos/antagonistas & inibidores , Ácidos Siálicos/química , Estereoisomerismo , Sus scrofa , Suínos , Doenças dos Suínos/patologia , Doenças dos Suínos/virologia
12.
Cancer Sci ; 106(11): 1568-75, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26334652

RESUMO

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).


Assuntos
Biomarcadores Tumorais/análise , Proteínas de Ligação a DNA/biossíntese , Neoplasias Orofaríngeas/virologia , Infecções por Papillomavirus/complicações , Adulto , Área Sob a Curva , Proteínas de Ciclo Celular , Feminino , Perfilação da Expressão Gênica , Humanos , Imuno-Histoquímica , Hibridização In Situ , Estimativa de Kaplan-Meier , Microdissecção e Captura a Laser , Masculino , Dados de Sequência Molecular , Neoplasias Orofaríngeas/metabolismo , Neoplasias Orofaríngeas/mortalidade , Modelos de Riscos Proporcionais , Estudos Prospectivos , Curva ROC , Reação em Cadeia da Polimerase em Tempo Real , Estudos Retrospectivos , Transcriptoma
13.
J Gen Virol ; 96(Pt 1): 74-84, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25304652

RESUMO

Porcine sapovirus (PSaV) of the family Caliciviridae, is the only member of the genus Sapovirus with cell culture and reverse genetics systems. When combined with the piglet model, these approaches provide a system to understand the molecular basis of sapovirus pathogenesis. The replication of PSaV in cell culture is, however, restricted, displaying an absolute requirement for bile acids and producing lower levels of infectious virus than other caliciviruses. The effect of bile acids has previously been linked to a reduction in the signal transducer and activator of transcription (STAT1)-mediated signalling pathway. In the current study, we observed that even in the presence of bile acids, PSaV replication in cell culture was restricted by soluble factors produced from infected cells. This effect was at least partially due to secreted IFN because treatment of cells with recombinant porcine IFN-ß resulted in significantly reduced viral replication. Moreover, IFN-mediated signalling pathways (IFN, STAT1 and the 2',5'-oligoadenylate synthetase) were activated during PSaV infection. Characterization of PSaV growth in cell lines deficient in their ability to induce or respond to IFN showed a 100-150-fold increase in infectious virus production, indicating that the primary role of bile acids was not the inactivation of the innate immune response. Furthermore, the use of IFN-deficient cell lines enabled more efficient recovery of PSaV from cDNA constructs. Overall, the highly efficient cell culture and reverse genetics system established here for PSaV highlighted the key role of the innate immune response in the restriction of PSaV infection and should greatly facilitate further molecular studies on sapovirus host-cell interactions.


Assuntos
Infecções por Caliciviridae/imunologia , Replicação do DNA/genética , Interferon beta/imunologia , Sapovirus/genética , Sapovirus/imunologia , Replicação Viral/genética , Animais , Infecções por Caliciviridae/virologia , Linhagem Celular , Replicação do DNA/imunologia , DNA Complementar/genética , Imunidade Inata/imunologia , RNA Viral/genética , RNA Viral/imunologia , Suínos , Replicação Viral/imunologia
14.
J Virol ; 88(7): 3874-84, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24453359

RESUMO

UNLABELLED: 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 Theiler's 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 Theiler's 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 Theiler's murine encephalomyelitis virus. Our data suggest important functional differences in the IFN response mounted by specific cell populations.


Assuntos
Apolipoproteínas/biossíntese , Expressão Gênica , Interferon Tipo I/imunologia , Neurônios/imunologia , Neurônios/virologia , Theilovirus/imunologia , Vesiculovirus/imunologia , Animais , Células Cultivadas , Fibroblastos/imunologia , Fibroblastos/virologia , Perfilação da Expressão Gênica , Técnicas de Silenciamento de Genes , Camundongos
15.
PLoS Pathog ; 9(6): e1003474, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23825954

RESUMO

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.


Assuntos
Infecções por Cardiovirus/metabolismo , Endorribonucleases/antagonistas & inibidores , Evasão da Resposta Imune/fisiologia , Imunidade Inata , Theilovirus/metabolismo , Proteínas Virais/metabolismo , Animais , Infecções por Cardiovirus/genética , Infecções por Cardiovirus/imunologia , Infecções por Cardiovirus/patologia , Linhagem Celular , Cricetinae , Endorribonucleases/genética , Endorribonucleases/imunologia , Endorribonucleases/metabolismo , Humanos , Camundongos , Camundongos Mutantes , Estrutura Terciária de Proteína , Especificidade da Espécie , Theilovirus/genética , Theilovirus/imunologia , Proteínas Virais/genética , Proteínas Virais/imunologia
16.
Virologie (Montrouge) ; 18(5): 264-277, 2014 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-33065920

RESUMO

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.

17.
STAR Protoc ; 5(1): 102913, 2024 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-38393950

RESUMO

Leakage of mitochondrial or nuclear DNA into the cytosol can occur following viral infections, radiation damage, and some cancers. Here, we present an optimized protocol for isolating and quantifying cytosolic DNA from mammalian cells. We describe steps for collecting cytosolic fractions from cells, extracting DNA using columns, and quantifying extracted DNA using qPCR. This straightforward protocol can be completed in as little as 5 hours, and allows for the identification of the source of DNA. For complete details on the use and execution of this protocol, please refer to Jahun et al.1.


Assuntos
DNA , Mitocôndrias , Animais , Citosol , Mamíferos/genética
18.
Viruses ; 16(2)2024 02 03.
Artigo em Inglês | MEDLINE | ID: mdl-38400023

RESUMO

Human sapoviruses (HuSaVs) and noroviruses are considered the leading cause of acute gastroenteritis worldwide. While extensive research has focused on noroviruses, our understanding of sapoviruses (SaVs) and their interactions with the host's immune response remains limited. HuSaVs have been challenging to propagate in vitro, making the porcine sapovirus (PSaV) Cowden strain a valuable model for studying SaV pathogenesis. In this study we show, for the first time, that PSaV Cowden strain has mechanisms to evade the host's innate immune response. The virus 3C-like protease (NS6) inhibits type I IFN production by targeting TBK1. Catalytically active NS6, both during ectopic expression and during PSaV infection, targets TBK1 which is then led for rapid degradation by the proteasome. Moreover, deletion of TBK1 from porcine cells led to an increase in PSaV titres, emphasizing its role in regulating PSaV infection. Additionally, we successfully established PSaV infection in IPEC-J2 cells, an enterocytic cell line originating from the jejunum of a neonatal piglet. Overall, this study provides novel insights into PSaV evasion strategies, opening the way for future investigations into SaV-host interactions, and enabling the use of a new cell line model for PSaV research.


Assuntos
Infecções por Caliciviridae , Sapovirus , Animais , Linhagem Celular , Expressão Gênica , Imunidade Inata , Peptídeo Hidrolases , Proteínas Serina-Treonina Quinases , Sapovirus/genética , Suínos
20.
Cell Rep ; 42(3): 112179, 2023 03 28.
Artigo em Inglês | MEDLINE | ID: mdl-36943868

RESUMO

The cGAS-STING pathway is central to the interferon response against DNA viruses. However, recent studies are increasingly demonstrating its role in the restriction of some RNA viruses. Here, we show that the cGAS-STING pathway also contributes to the interferon response against noroviruses, currently the commonest causes of infectious gastroenteritis worldwide. We show a significant reduction in interferon-ß induction and a corresponding increase in viral replication in norovirus-infected cells after deletion of STING, cGAS, or IFI16. Further, we find that immunostimulatory host genome-derived DNA and mitochondrial DNA accumulate in the cytosol of norovirus-infected cells. Lastly, overexpression of the viral NS4 protein is sufficient to drive the accumulation of cytosolic DNA. Together, our data find a role for cGAS, IFI16, and STING in the restriction of noroviruses and show the utility of host genomic DNA as a damage-associated molecular pattern in cells infected with an RNA virus.


Assuntos
DNA Mitocondrial , Transdução de Sinais , DNA Mitocondrial/genética , Genômica , Imunidade Inata/genética , Interferons , Nucleotidiltransferases/metabolismo , Transdução de Sinais/genética , Proteínas de Membrana/metabolismo
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