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1.
J Virol ; 2020 Nov 11.
Artigo em Inglês | MEDLINE | ID: mdl-33177207

RESUMO

Noroviruses are a leading cause of gastrointestinal infection in humans and mice. Understanding human norovirus (HuNoV) cell tropism has important implications for our understanding of viral pathogenesis. Murine norovirus (MNoV) is extensively used as a surrogate model for HuNoV. We previously identified CD300lf as the receptor for MNoV. Here, we generated a Cd300lf conditional knockout (CD300lfF/F ) mouse to elucidate the cell tropism of persistent and non-persistent strains of murine norovirus. Using this mouse model, we demonstrate that CD300lf expression on intestinal epithelial cells (IECs), and on tuft cells in particular, is essential for transmission of the persistent MNoV strain CR6 (MNoVCR6) in vivo In contrast, the non-persistent MNoV strain CW3 (MNoVCW3) does not require CD300lf expression on IECs for infection. However, deletion of CD300lf in myelomonocytic cells (LysM Cre+) partially reduces CW3 viral load in lymphoid and intestinal tissues. Disruption of CD300lf expression on B cells (CD19 Cre), neutrophils (Mrp8 Cre), and dendritic cells (CD11c Cre) did not affect MNoVCW3 viral RNA levels. Finally, we show that the transcription factor STAT1, which is critical for the innate immune response, partially restricts the cell tropism of MNoVCW3 to LysM+ cells. Taken together, these data demonstrate that CD300lf expression on tuft cells is essential for MNoVCR6, that myelomonocytic cells are a major, but not exclusive, target cell of MNoVCW3, and that STAT1 signaling restricts the cellular tropism of MNoVCW3 This provides the first genetic system to study the cell type-specific role of CD300lf in norovirus pathogenesis.IMPORTANCE Human noroviruses (HuNoVs) are a leading cause of gastroenteritis resulting in up to 200,000 deaths each year. The receptor and cell tropism of HuNoV in immunocompetent humans are unclear. We use murine norovirus (MNoV) as a model for HuNoV. We recently identified CD300lf as the sole physiologic receptor for MNoV. Here, we leverage this finding to generate a Cd300lf conditional knockout mouse to decipher the contributions of specific cell types to MNoV infection. We demonstrate that persistent MNoVCR6 requires CD300lf expression on tuft cells. In contrast, multiple CD300lf+ cell types, dominated by myelomonocytic cells, are sufficient for non-persistent MNoVCW3 infection. CD300lf expression on epithelial cells, B cells, neutrophils, and dendritic cells is not critical for MNoVCW3 infection. Mortality associated with MNoVCW3 strain in Stat1-/- mice does not require CD300lf expression on LysM+ cells, highlighting that both CD300lf receptor expression and innate immunity regulate MNoV cell tropism in vivo.

2.
J Virol ; 94(21)2020 Oct 14.
Artigo em Inglês | MEDLINE | ID: mdl-32847859

RESUMO

Interferon (IFN) family cytokines stimulate genes (interferon-stimulated genes [ISGs]) that are integral to antiviral host defense. Type I IFNs act systemically, whereas type III IFNs act preferentially at epithelial barriers. Among barrier cells, intestinal epithelial cells (IECs) are particularly dependent on type III IFN for the control and clearance of virus infection, but the physiological basis of this selective IFN response is not well understood. Here, we confirm that type III IFN treatment elicits robust and uniform ISG expression in neonatal mouse IECs and inhibits the replication of IEC-tropic rotavirus. In contrast, type I IFN elicits a marginal ISG response in neonatal mouse IECs and does not inhibit rotavirus replication. In vitro treatment of IEC organoids with type III IFN results in ISG expression that mirrors the in vivo type III IFN response. However, IEC organoids have increased expression of the type I IFN receptor relative to neonate IECs, and the response of IEC organoids to type I IFN is strikingly increased in magnitude and scope relative to type III IFN. The expanded type I IFN-specific response includes proapoptotic genes and potentiates toxicity triggered by tumor necrosis factor alpha (TNF-α). The ISGs stimulated in common by type I and III IFNs have strong interferon-stimulated response element (ISRE) promoter motifs, whereas the expanded set of type I IFN-specific ISGs, including proapoptotic genes, have weak ISRE motifs. Thus, the preferential responsiveness of IECs to type III IFN in vivo enables selective ISG expression during infection that confers antiviral protection but minimizes disruption of intestinal homeostasis.IMPORTANCE Enteric viral infections are a major cause of gastroenteritis worldwide and have the potential to trigger or exacerbate intestinal inflammatory diseases. Prior studies have identified specialized innate immune responses that are active in the intestinal epithelium following viral infection, but our understanding of the benefits of such an epithelium-specific response is incomplete. Here, we show that the intestinal epithelial antiviral response is programmed to enable protection while minimizing epithelial cytotoxicity that can often accompany an inflammatory response. Our findings offer new insight into the benefits of a tailored innate immune response at the intestinal barrier and suggest how dysregulation of this response could promote inflammatory disease.

3.
Cell ; 179(3): 644-658.e13, 2019 10 17.
Artigo em Inglês | MEDLINE | ID: mdl-31607511

RESUMO

Rotavirus (RV) encounters intestinal epithelial cells amidst diverse microbiota, opening possibilities of microbes influencing RV infection. Although RV clearance typically requires adaptive immunity, we unintentionally generated RV-resistant immunodeficient mice, which, we hypothesized, reflected select microbes protecting against RV. Accordingly, such RV resistance was transferred by co-housing and fecal transplant. RV-protecting microbiota were interrogated by heat, filtration, and antimicrobial agents, followed by limiting dilution transplant to germ-free mice and microbiome analysis. This approach revealed that segmented filamentous bacteria (SFB) were sufficient to protect mice against RV infection and associated diarrhea. Such protection was independent of previously defined RV-impeding factors, including interferon, IL-17, and IL-22. Colonization of the ileum by SFB induced changes in host gene expression and accelerated epithelial cell turnover. Incubation of RV with SFB-containing feces reduced infectivity in vitro, suggesting direct neutralization of RV. Thus, independent of immune cells, SFB confer protection against certain enteric viral infections and associated diarrheal disease.


Assuntos
Imunidade Adaptativa/genética , Diarreia/microbiologia , Mucosa Intestinal/microbiologia , Infecções por Rotavirus/microbiologia , Animais , Anti-Infecciosos/farmacologia , Bactérias/genética , Bactérias/metabolismo , Diarreia/prevenção & controle , Diarreia/virologia , Fezes/microbiologia , Regulação da Expressão Gênica/genética , Humanos , Íleo/microbiologia , Íleo/patologia , Íleo/virologia , Interferons/genética , Interleucina-17/genética , Interleucinas/genética , Mucosa Intestinal/patologia , Mucosa Intestinal/virologia , Camundongos , Microbiota/genética , Rotavirus/patogenicidade , Infecções por Rotavirus/prevenção & controle , Infecções por Rotavirus/virologia
4.
PLoS Pathog ; 15(7): e1007940, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31329638

RESUMO

Human norovirus (HNoV) is the leading cause of acute gastroenteritis and is spread by fecal shedding that can often persist for weeks to months after the resolution of symptoms. Elimination of persistent viral reservoirs has the potential to prevent outbreaks. Similar to HNoV, murine norovirus (MNV) is spread by persistent shedding in the feces and provides a tractable model to study molecular mechanisms of enteric persistence. Previous studies have identified non-structural protein 1 (NS1) from the persistent MNV strain CR6 as critical for persistent infection in intestinal epithelial cells (IECs), but its mechanism of action remains unclear. We now find that the function of CR6 NS1 is regulated by apoptotic caspase cleavage. Following induction of apoptosis in infected cells, caspases cleave the precursor NS1/2 protein, and this cleavage is prevented by mutation of caspase target motifs. These mutations profoundly compromise CR6 infection of IECs and persistence in the intestine. Conversely, NS1/2 cleavage is not strictly required for acute replication in extra-intestinal tissues or in cultured myeloid cells, suggesting an IEC-centric role. Intriguingly, we find that caspase cleavage of CR6 NS1/2 reciprocally promotes caspase activity, potentiates cell death, and amplifies spread among cultured IEC monolayers. Together, these data indicate that the function of CR6 NS1 is regulated by apoptotic caspases, and suggest that apoptotic cell death enables epithelial spread and persistent shedding.


Assuntos
Mucosa Intestinal/virologia , Norovirus/patogenicidade , Proteínas não Estruturais Virais/metabolismo , Animais , Apoptose , Infecções por Caliciviridae/etiologia , Infecções por Caliciviridae/patologia , Infecções por Caliciviridae/virologia , Caspases/metabolismo , Células Cultivadas , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Células Epiteliais/virologia , Feminino , Gastroenterite/etiologia , Gastroenterite/patologia , Gastroenterite/virologia , Interações entre Hospedeiro e Microrganismos , Humanos , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patologia , Masculino , Camundongos , Camundongos Knockout , Modelos Biológicos , Células Mieloides/metabolismo , Células Mieloides/patologia , Células Mieloides/virologia , Norovirus/genética , Norovirus/fisiologia , Proteínas não Estruturais Virais/genética , Replicação Viral , Eliminação de Partículas Virais
5.
Nat Microbiol ; 4(10): 1737-1749, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31182797

RESUMO

Products derived from bacterial members of the gut microbiota evoke immune signalling pathways of the host that promote immunity and barrier function in the intestine. How immune reactions to enteric viruses support intestinal homeostasis is unknown. We recently demonstrated that infection by murine norovirus (MNV) reverses intestinal abnormalities following depletion of bacteria, indicating that an intestinal animal virus can provide cues to the host that are typically attributed to the microbiota. Here, we elucidate mechanisms by which MNV evokes protective responses from the host. We identify an important role for the viral protein NS1/2 in establishing local replication and a type I interferon (IFN-I) response in the colon. We further show that IFN-I acts on intestinal epithelial cells to increase the proportion of CCR2-dependent macrophages and interleukin (IL)-22-producing innate lymphoid cells, which in turn promote pSTAT3 signalling in intestinal epithelial cells and protection from intestinal injury. In addition, we demonstrate that MNV provides a striking IL-22-dependent protection against early-life lethal infection by Citrobacter rodentium. These findings demonstrate novel ways in which a viral member of the microbiota fortifies the intestinal barrier during chemical injury and infectious challenges.


Assuntos
Microbioma Gastrointestinal/imunologia , Interferon Tipo I/metabolismo , Interleucinas/metabolismo , Intestinos/imunologia , Intestinos/virologia , Animais , Antibacterianos/toxicidade , Proliferação de Células , Citrobacter rodentium/fisiologia , Colo/citologia , Colo/imunologia , Colo/metabolismo , Colo/virologia , Sulfato de Dextrana/toxicidade , Infecções por Enterobacteriaceae/prevenção & controle , Interleucinas/genética , Mucosa Intestinal/citologia , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/imunologia , Mucosa Intestinal/metabolismo , Intestinos/citologia , Intestinos/efeitos dos fármacos , Linfócitos/citologia , Linfócitos/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Mutação , Norovirus/imunologia , Norovirus/fisiologia , Transdução de Sinais/genética , Organismos Livres de Patógenos Específicos , Proteínas não Estruturais Virais/genética , Replicação Viral
6.
Cell Host Microbe ; 25(6): 845-857.e5, 2019 06 12.
Artigo em Inglês | MEDLINE | ID: mdl-31130511

RESUMO

Murine norovirus (MNoV) infects a low percentage of enteric tuft cells and can persist in these cells for months following acute infection. Both tuft-cell tropism and resistance to interferon-λ (IFN-λ)-mediated clearance during persistent infection requires the viral nonstructural protein 1/2 (NS1/2). We show that processing of NS1/2 yields NS1, an unconventionally secreted viral protein that is central for IFN-λ resistance. MNoV infection globally suppresses intestinal IFN-λ responses, which is attributable to secreted NS1. MNoV NS1 secretion is triggered by caspase-3 cleavage of NS1/2, and a secreted form of human NoV NS1 is also observed. NS1 secretion is essential for intestinal infection and resistance to IFN-λ in vivo. NS1 vaccination alone protects against MNoV challenge, despite the lack of induction of neutralizing anti-capsid antibodies previously shown to confer protection. Thus, despite infecting a low number of tuft cells, NS1 secretion allows MNoV to globally suppress IFN responses and promote persistence.


Assuntos
Infecções por Caliciviridae/patologia , Infecções por Caliciviridae/virologia , Citocinas/antagonistas & inibidores , Evasão da Resposta Imune , Norovirus/crescimento & desenvolvimento , Norovirus/patogenicidade , Proteínas não Estruturais Virais/metabolismo , Animais , Modelos Animais de Doenças , Gastroenterite/patologia , Gastroenterite/virologia , Humanos , Camundongos , Fatores de Virulência/metabolismo
7.
Cell Host Microbe ; 24(5): 665-676.e4, 2018 11 14.
Artigo em Inglês | MEDLINE | ID: mdl-30392829

RESUMO

Viral persistence can contribute to chronic disease and promote virus dissemination. Prior work demonstrated that timely clearance of systemic murine norovirus (MNV) infection depends on cell-intrinsic type I interferon responses and adaptive immunity. We now find that the capsid of the systemically replicating MNV strain CW3 promotes lytic cell death, release of interleukin-1α, and increased inflammatory cytokine release. Correspondingly, inflammatory monocytes and neutrophils are recruited to sites of infection in a CW3-capsid-dependent manner. Recruited monocytes and neutrophils are subsequently infected, representing a majority of infected cells in vivo. Systemic depletion of inflammatory monocytes or neutrophils from persistently infected Rag1-/- mice reduces viral titers in a tissue-specific manner. These data indicate that the CW3 capsid facilitates lytic cell death, inflammation, and recruitment of susceptible cells to promote persistence. Infection of continuously recruited inflammatory cells may be a mechanism of persistence broadly utilized by lytic viruses incapable of establishing latency.


Assuntos
Infecções por Caliciviridae/imunologia , Gastroenterite/imunologia , Células Mieloides/imunologia , Células Mieloides/virologia , Norovirus/imunologia , Norovirus/patogenicidade , Imunidade Adaptativa , Animais , Infecções por Caliciviridae/virologia , Capsídeo/imunologia , Morte Celular , Citocinas/metabolismo , Modelos Animais de Doenças , Feminino , Gastroenterite/virologia , Genes Virais/genética , Células HEK293 , Proteínas de Homeodomínio/genética , Interações Hospedeiro-Patógeno , Humanos , Inflamação/imunologia , Interferon Tipo I/imunologia , Interleucina-1alfa/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Monócitos/imunologia , Monócitos/virologia , Neutrófilos/imunologia , Neutrófilos/virologia , Norovirus/genética , Carga Viral
8.
J Virol ; 92(23)2018 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-30209176

RESUMO

The linear ubiquitin chain assembly complex (LUBAC), composed of heme-oxidized IRP2 ubiquitin ligase 1 (HOIL1), HOIL1-interacting protein (HOIP), and SHANK-associated RH domain-interacting protein (SHARPIN), is a crucial regulator of multiple immune signaling pathways. In humans, HOIL1 or HOIP deficiency is associated with an immune disorder involving autoinflammation, immunodeficiency, and inflammatory bowel disease (IBD)-like symptoms. During viral infection, LUBAC is reported to inhibit the induction of interferon (IFN) by the cytosolic RNA sensor retinoic acid-inducible gene I (RIG-I). Surprisingly, we found that HOIL1 is essential for the induction of both type I and type III IFNs, as well as the phosphorylation of IFN regulatory factor 3 (IRF3), during murine norovirus (MNoV) infection in cultured dendritic cells. The RIG-I-like receptor, melanoma differentiation-associated protein 5 (MDA5), is also required for IFN induction and IRF3 phosphorylation during MNoV infection. Furthermore, HOIL1 and MDA5 were required for IFN induction after Theiler's murine encephalomyelitis virus infection and poly(I·C) transfection, but not Sendai virus or vesicular stomatitis virus infection, indicating that HOIL1 and LUBAC are required selectively for MDA5 signaling. Moreover, Hoil1 - / - mice exhibited defective control of acute and persistent murine norovirus infection and defective regulation of MNoV persistence by the microbiome as also observed previously for mice deficient in interferon lambda (IFN-λ) receptor, signal transducer and activator of transcription factor 1 (STAT1), and IRF3. These data indicate that LUBAC plays a critical role in IFN induction to control RNA viruses sensed by MDA5.IMPORTANCE Human noroviruses are a leading cause of gastroenteritis throughout the world but are challenging to study in vivo and in vitro Murine norovirus (MNoV) provides a tractable genetic and small-animal model to study norovirus biology and immune responses. Interferons are critical mediators of antiviral immunity, but excessive expression can dysregulate the immune system. IFN-λ plays an important role at mucosal surfaces, including the gastrointestinal tract, and both IFN-λ and commensal enteric bacteria are important modulators of persistent MNoV infection. LUBAC, of which HOIL1 is a component, is reported to inhibit type I IFN induction after RIG-I stimulation. We show, in contrast, that HOIL1 is critical for type I and III IFN induction during infection with MNoV, a virus that preferentially activates MDA5. Moreover, HOIL1 regulates MNoV infection in vivo These data reveal distinct functions for LUBAC in these closely related signaling pathways and in modulation of IFN expression.


Assuntos
Infecções por Caliciviridae/virologia , Interferon Tipo I/metabolismo , Helicase IFIH1 Induzida por Interferon/metabolismo , Interferons/metabolismo , Norovirus/patogenicidade , Ubiquitina-Proteína Ligases/fisiologia , Animais , Infecções por Caliciviridae/genética , Infecções por Caliciviridae/metabolismo , Infecções por Caliciviridae/microbiologia , Células Cultivadas , Células Dendríticas/metabolismo , Células Dendríticas/microbiologia , Células Dendríticas/virologia , Fibroblastos/metabolismo , Fibroblastos/microbiologia , Fibroblastos/virologia , Genoma Viral , Fator Regulador 3 de Interferon/genética , Fator Regulador 3 de Interferon/metabolismo , Interferon Tipo I/genética , Helicase IFIH1 Induzida por Interferon/genética , Interferons/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microbiota , Norovirus/genética , Fosforilação
9.
Trends Microbiol ; 26(6): 510-524, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29157967

RESUMO

Persistent viral infections result from evasion or avoidance of sterilizing immunity, extend the timeframe of virus transmission, and can trigger disease. Prior studies in mouse models of persistent infection have suggested that ineffective adaptive immune responses are necessary for persistent viral infection. However, recent work in the murine norovirus (MNV) model of persistent infection demonstrates that innate immunity can control both early and persistent viral replication independently of adaptive immune effector functions. Interferons (IFNs) are central to the innate control of persistent MNV, apart from a role in modulating adaptive immunity. Furthermore, subtypes of IFN play distinct tissue-specific roles in innate control of persistent MNV infection. Type I IFN (IFN-α/ß) controls systemic replication, and type III IFN (IFN-λ) controls MNV persistence in the intestinal epithelium. In this article, we review recent findings in the MNV model, highlighting the role of IFNs and innate immunity in clearing persistent viral infection, and discussing the broader implications of these findings for control of persistent human infections.


Assuntos
Imunidade Adaptativa , Infecções por Caliciviridae/imunologia , Imunidade Inata , Interferons/imunologia , Animais , Modelos Animais de Doenças , Interações entre Hospedeiro e Microrganismos/imunologia , Humanos , Interferon Tipo I/imunologia , Camundongos , Norovirus/imunologia , Replicação Viral
10.
Immunity ; 47(4): 723-738.e5, 2017 10 17.
Artigo em Inglês | MEDLINE | ID: mdl-29031786

RESUMO

Noroviruses can establish chronic infections with active viral shedding in healthy humans but whether persistence is associated with adaptive immune dysfunction is unknown. We used genetically engineered strains of mouse norovirus (MNV) to investigate CD8+ T cell differentiation during chronic infection. We found that chronic infection drove MNV-specific tissue-resident memory (Trm) CD8+ T cells to a differentiation state resembling inflationary effector responses against latent cytomegalovirus with only limited evidence of exhaustion. These MNV-specific Trm cells remained highly functional yet appeared ignorant of ongoing viral replication. Pre-existing MNV-specific Trm cells provided partial protection against chronic infection but largely ceased to detect virus within 72 hours of challenge, demonstrating rapid sequestration of viral replication away from T cells. Our studies revealed a strategy of immune evasion by MNV via the induction of a CD8+ T cell program normally reserved for latent pathogens and persistence in an immune-privileged enteric niche.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Infecções por Caliciviridae/imunologia , Diferenciação Celular/imunologia , Gastroenterite/imunologia , Norovirus/imunologia , Animais , Linfócitos T CD8-Positivos/metabolismo , Linfócitos T CD8-Positivos/virologia , Infecções por Caliciviridae/genética , Infecções por Caliciviridae/virologia , Diferenciação Celular/genética , Linhagem Celular , Microambiente Celular/genética , Microambiente Celular/imunologia , Gastroenterite/genética , Gastroenterite/virologia , Perfilação da Expressão Gênica/métodos , Ontologia Genética , Células HEK293 , Interações Hospedeiro-Patógeno/imunologia , Humanos , Memória Imunológica/genética , Memória Imunológica/imunologia , Camundongos Endogâmicos C57BL , Norovirus/fisiologia , Análise de Sequência com Séries de Oligonucleotídeos/métodos
11.
Cell Host Microbe ; 22(4): 449-459.e4, 2017 Oct 11.
Artigo em Inglês | MEDLINE | ID: mdl-28966054

RESUMO

Cellular tropism during persistent viral infection is commonly conferred by the interaction of a viral surface protein with a host receptor complex. Norovirus, the leading global cause of gastroenteritis, can be persistently shed during infection, but its in vivo cellular tropism and tropism determinants remain unidentified. Using murine norovirus (MNoV), we determine that a small number of intestinal epithelial cells (IECs) serve as the reservoir for fecal shedding and persistence. The viral non-structural protein NS1, rather than a viral surface protein, determines IEC tropism. Expression of NS1 from a persistent MNoV strain is sufficient for an acute MNoV strain to target IECs and persist. In addition, interferon-lambda (IFN-λ) is a key host determinant blocking MNoV infection in IECs. The inability of acute MNoV to shed and persist is rescued in Ifnlr1-/- mice, suggesting that NS1 evades IFN-λ-mediated antiviral immunity. Thus, NS1 and IFN-λ interactions govern IEC tropism and persistence of MNoV.


Assuntos
Infecções por Caliciviridae/virologia , Citocinas/metabolismo , Norovirus/fisiologia , Proteínas não Estruturais Virais/metabolismo , Tropismo Viral , Animais , Infecções por Caliciviridae/imunologia , Linhagem Celular , Interações Hospedeiro-Patógeno , Humanos , Imunidade Inata/imunologia , Intestinos/citologia , Intestinos/imunologia , Intestinos/virologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Norovirus/genética , Proteínas não Estruturais Virais/genética , Eliminação de Partículas Virais
12.
mBio ; 8(4)2017 07 11.
Artigo em Inglês | MEDLINE | ID: mdl-28698274

RESUMO

The Norovirus genus contains important human pathogens, but the role of host pathways in norovirus replication is largely unknown. Murine noroviruses provide the opportunity to study norovirus replication in cell culture and in small animals. The human norovirus nonstructural protein NS1/2 interacts with the host protein VAMP-associated protein A (VAPA), but the significance of the NS1/2-VAPA interaction is unexplored. Here we report decreased murine norovirus replication in VAPA- and VAPB-deficient cells. We characterized the role of VAPA in detail. VAPA was required for the efficiency of a step(s) in the viral replication cycle after entry of viral RNA into the cytoplasm but before the synthesis of viral minus-sense RNA. The interaction of VAPA with viral NS1/2 proteins is conserved between murine and human noroviruses. Murine norovirus NS1/2 directly bound the major sperm protein (MSP) domain of VAPA through its NS1 domain. Mutations within NS1 that disrupted interaction with VAPA inhibited viral replication. Structural analysis revealed that the viral NS1 domain contains a mimic of the phenylalanine-phenylalanine-acidic-tract (FFAT) motif that enables host proteins to bind to the VAPA MSP domain. The NS1/2-FFAT mimic region interacted with the VAPA-MSP domain in a manner similar to that seen with bona fide host FFAT motifs. Amino acids in the FFAT mimic region of the NS1 domain that are important for viral replication are highly conserved across murine norovirus strains. Thus, VAPA interaction with a norovirus protein that functionally mimics host FFAT motifs is important for murine norovirus replication.IMPORTANCE Human noroviruses are a leading cause of gastroenteritis worldwide, but host factors involved in norovirus replication are incompletely understood. Murine noroviruses have been studied to define mechanisms of norovirus replication. Here we defined the importance of the interaction between the hitherto poorly studied NS1/2 norovirus protein and the VAPA host protein. The NS1/2-VAPA interaction is conserved between murine and human noroviruses and was important for early steps in murine norovirus replication. Using structure-function analysis, we found that NS1/2 contains a short sequence that molecularly mimics the FFAT motif that is found in multiple host proteins that bind VAPA. This represents to our knowledge the first example of functionally important mimicry of a host FFAT motif by a microbial protein.


Assuntos
Interações Hospedeiro-Patógeno , Norovirus/fisiologia , Fenilalanina/química , Proteínas de Transporte Vesicular/metabolismo , Proteínas não Estruturais Virais/química , Replicação Viral , Motivos de Aminoácidos , Animais , Linhagem Celular , Células HEK293 , Humanos , Camundongos , Norovirus/genética , Células RAW 264.7 , RNA Viral/genética , Genética Reversa , Proteínas de Transporte Vesicular/genética , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/metabolismo
13.
Immunity ; 46(5): 768-770, 2017 05 16.
Artigo em Inglês | MEDLINE | ID: mdl-28514682

RESUMO

Type III interferon (IFNλ) and type I IFN (IFNα/ß) have overlapping antiviral activities in the lung. In this issue of Immunity, Galani et al. (2017) identify a critical early role for IFNλ, not shared by IFNα/ß, in protection of the lung following influenza virus infection.


Assuntos
Antivirais , Interferon-alfa , Humanos , Orthomyxoviridae , Infecções por Orthomyxoviridae/imunologia
15.
J Virol ; 91(7)2017 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-28077655

RESUMO

Lambda interferon (IFN-λ) has potent antiviral effects against multiple enteric viral pathogens, including norovirus and rotavirus, in both preventing and curing infection. Because the intestine includes a diverse array of cell types, however, the cell(s) upon which IFN-λ acts to exert its antiviral effects is unclear. Here, we sought to identify IFN-λ-responsive cells by generation of mice with lineage-specific deletion of the receptor for IFN-λ, Ifnlr1 We found that expression of IFNLR1 on intestinal epithelial cells (IECs) in the small intestine and colon is required for enteric IFN-λ antiviral activity. IEC Ifnlr1 expression also determines the efficacy of IFN-λ in resolving persistent murine norovirus (MNoV) infection and regulates fecal shedding and viral titers in tissue. Thus, the expression of Ifnlr1 by IECs is necessary for the response to both endogenous and exogenous IFN-λ. We further demonstrate that IEC Ifnlr1 expression is required for the sterilizing innate immune effects of IFN-λ by extending these findings in Rag1-deficient mice. Finally, we assessed whether our findings pertained to multiple viral pathogens by infecting mice specifically lacking IEC Ifnlr1 expression with reovirus. These mice phenocopied Ifnlr1-null animals, exhibiting increased intestinal tissue titers and enhanced reovirus fecal shedding. Thus, IECs are the critical cell type responding to IFN-λ to control multiple enteric viruses. This is the first genetic evidence that supports an essential role for IECs in IFN-λ-mediated control of enteric viral infection, and these findings provide insight into the mechanism of IFN-λ-mediated antiviral activity.IMPORTANCE Human noroviruses (HNoVs) are the leading cause of epidemic gastroenteritis worldwide. Type III interferons (IFN-λ) control enteric viral infections in the gut and have been shown to cure mouse norovirus, a small-animal model for HNoVs. Using a genetic approach with conditional knockout mice, we identified IECs as the dominant IFN-λ-responsive cells in control of enteric virus infection in vivo Upon murine norovirus or reovirus infection, Ifnlr1 depletion in IECs largely recapitulated the phenotype seen in Ifnlr1-/- mice of higher intestinal tissue viral titers and increased viral shedding in the stool. Moreover, IFN-λ-mediated sterilizing immunity against murine norovirus requires the capacity of IECs to respond to IFN-λ. These findings clarify the mechanism of action of this cytokine and emphasize the therapeutic potential of IFN-λ for treating mucosal viral infections.


Assuntos
Células Epiteliais/metabolismo , Norovirus/fisiologia , Orthoreovirus de Mamíferos/fisiologia , Receptores de Interferon/metabolismo , Animais , Infecções por Caliciviridae/imunologia , Infecções por Caliciviridae/metabolismo , Infecções por Caliciviridae/virologia , Linhagem Celular , Células Epiteliais/imunologia , Células Epiteliais/virologia , Imunidade Inata , Mucosa Intestinal/imunologia , Mucosa Intestinal/metabolismo , Intestino Grosso/imunologia , Intestino Grosso/metabolismo , Intestino Grosso/virologia , Intestino Delgado/imunologia , Intestino Delgado/metabolismo , Intestino Delgado/virologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Especificidade de Órgãos , Infecções por Reoviridae/imunologia , Infecções por Reoviridae/metabolismo , Infecções por Reoviridae/virologia , Eliminação de Partículas Virais
16.
PLoS Pathog ; 12(6): e1005684, 2016 06.
Artigo em Inglês | MEDLINE | ID: mdl-27327515

RESUMO

In order for a virus to persist, there must be a balance between viral replication and immune clearance. It is commonly believed that adaptive immunity drives clearance of viral infections and, thus, dysfunction or viral evasion of adaptive immunity is required for a virus to persist. Type I interferons (IFNs) play pleiotropic roles in the antiviral response, including through innate control of viral replication. Murine norovirus (MNoV) replicates in dendritic cells (DCs) and type I IFN signaling in DCs is important for early control of MNoV replication. We show here that the non-persistent MNoV strain CW3 persists systemically when CD11c positive DCs are unable to respond to type I IFN. Persistence in this setting is associated with increased early viral titers, maintenance of DC numbers, increased expression of DC activation markers and an increase in CD8 T cell and antibody responses. Furthermore, CD8 T cell function is maintained during the persistent phase of infection and adaptive immune cells from persistently infected mice are functional when transferred to Rag1-/- recipients. Finally, increased early replication and persistence are also observed in mixed bone marrow chimeras where only half of the CD11c positive DCs are unable to respond to type I IFN. These findings demonstrate that increased early viral replication due to a cell-intrinsic innate immune deficiency is sufficient for persistence and a functional adaptive immune response is not sufficient for viral clearance.


Assuntos
Infecções por Caliciviridae/imunologia , Células Dendríticas/imunologia , Células Dendríticas/virologia , Interferon Tipo I/imunologia , Receptor de Interferon alfa e beta/imunologia , Imunidade Adaptativa/imunologia , Animais , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Imunidade Inata/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Norovirus , Reação em Cadeia da Polimerase em Tempo Real , Receptor de Interferon alfa e beta/deficiência , Replicação Viral/fisiologia
17.
Immunity ; 43(1): 15-28, 2015 Jul 21.
Artigo em Inglês | MEDLINE | ID: mdl-26200010

RESUMO

When type III interferon (IFN-λ; also known as interleukin-28 [IL-28] and IL-29) was discovered in 2003, its antiviral function was expected to be analogous to that of type I IFNs (IFN-α and IFN-ß) via the induction of IFN-stimulated genes (ISGs). Although IFN-λ stimulates expression of antiviral ISGs preferentially in cells of epithelial origin, recent studies have defined additional antiviral mechanisms in other cell types and tissues. Viral infection models using mice lacking IFN-λ signaling and SNP associations with human disease have expanded our understanding of the contribution of IFN-λ to the antiviral response at anatomic barriers and the immune response beyond these barriers. In this review, we highlight recent insights into IFN-λ functions, including its ability to restrict virus spread into the brain and to clear chronic viral infections in the gastrointestinal tract. We also discuss how IFN-λ modulates innate and adaptive immunity, autoimmunity, and tumor progression and its possible therapeutic applications in human disease.


Assuntos
Encéfalo/imunologia , Trato Gastrointestinal/imunologia , Interferon gama/imunologia , Neoplasias/imunologia , Viroses/imunologia , Imunidade Adaptativa/imunologia , Animais , Antivirais/imunologia , Autoimunidade/imunologia , Encéfalo/virologia , Trato Gastrointestinal/virologia , Humanos , Imunidade Inata/imunologia , Camundongos , Transdução de Sinais/imunologia
18.
Science ; 347(6219): 269-73, 2015 Jan 16.
Artigo em Inglês | MEDLINE | ID: mdl-25431489

RESUMO

Norovirus gastroenteritis is a major public health burden worldwide. Although fecal shedding is important for transmission of enteric viruses, little is known about the immune factors that restrict persistent enteric infection. We report here that although the cytokines interferon-α (IFN-α) and IFN-ß prevented the systemic spread of murine norovirus (MNoV), only IFN-λ controlled persistent enteric infection. Infection-dependent induction of IFN-λ was governed by the MNoV capsid protein and correlated with diminished enteric persistence. Treatment of established infection with IFN-λ cured mice in a manner requiring nonhematopoietic cell expression of the IFN-λ receptor, Ifnlr1, and independent of adaptive immunity. These results suggest the therapeutic potential of IFN-λ for curing virus infections in the gastrointestinal tract.


Assuntos
Infecções por Caliciviridae/tratamento farmacológico , Infecções por Caliciviridae/imunologia , Citocinas/imunologia , Citocinas/uso terapêutico , Gastroenterite/imunologia , Norovirus/imunologia , Norovirus/fisiologia , Imunidade Adaptativa , Animais , Infecções por Caliciviridae/virologia , Proteínas do Capsídeo/imunologia , Proteínas do Capsídeo/metabolismo , Células Cultivadas , Citocinas/biossíntese , Fezes/virologia , Gastroenterite/tratamento farmacológico , Gastroenterite/virologia , Imunidade Inata , Interferon-alfa/biossíntese , Interferon-alfa/imunologia , Interferon beta/biossíntese , Interferon beta/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Replicação Viral , Eliminação de Partículas Virais
19.
Science ; 347(6219): 266-9, 2015 Jan 16.
Artigo em Inglês | MEDLINE | ID: mdl-25431490

RESUMO

The capacity of human norovirus (NoV), which causes >90% of global epidemic nonbacterial gastroenteritis, to infect a subset of people persistently may contribute to its spread. How such enteric viruses establish persistent infections is not well understood. We found that antibiotics prevented persistent murine norovirus (MNoV) infection, an effect that was reversed by replenishment of the bacterial microbiota. Antibiotics did not prevent tissue infection or affect systemic viral replication but acted specifically in the intestine. The receptor for the antiviral cytokine interferon-λ, Ifnlr1, as well as the transcription factors Stat1 and Irf3, were required for antibiotics to prevent viral persistence. Thus, the bacterial microbiome fosters enteric viral persistence in a manner counteracted by specific components of the innate immune system.


Assuntos
Infecções por Caliciviridae/virologia , Citocinas/fisiologia , Gastroenterite/virologia , Intestinos/microbiologia , Microbiota , Norovirus/fisiologia , Simbiose , Animais , Antibacterianos/farmacologia , Infecções por Caliciviridae/tratamento farmacológico , Infecções por Caliciviridae/imunologia , Infecções por Caliciviridae/microbiologia , Feminino , Gastroenterite/tratamento farmacológico , Gastroenterite/imunologia , Gastroenterite/microbiologia , Intestinos/virologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microbiota/efeitos dos fármacos , Norovirus/imunologia , Receptores de Citocinas/genética , Receptores de Citocinas/metabolismo , Transdução de Sinais , Carga Viral , Replicação Viral , Eliminação de Partículas Virais
20.
Cell Host Microbe ; 17(1): 85-97, 2015 Jan 14.
Artigo em Inglês | MEDLINE | ID: mdl-25482432

RESUMO

The host immune system functions constantly to maintain chronic commensal and pathogenic organisms in check. The consequences of these immune responses on host physiology are as yet unexplored, and may have long-term implications in health and disease. We show that chronic viral infection increases epithelial turnover in multiple tissues, and the antiviral cytokines type I interferons (IFNs) mediate this response. Using a murine model with persistently elevated type I IFNs in the absence of exogenous viral infection, the Irgm1(-/-) mouse, we demonstrate that type I IFNs act through nonepithelial cells, including macrophages, to promote increased epithelial turnover and wound repair. Downstream of type I IFN signaling, the highly related IFN-stimulated genes Apolipoprotein L9a and b activate epithelial proliferation through ERK activation. Our findings demonstrate that the host immune response to chronic viral infection has systemic effects on epithelial turnover through a myeloid-epithelial circuit.


Assuntos
Células Epiteliais/fisiologia , Epitélio/imunologia , Interferon Tipo I/metabolismo , Viroses/imunologia , Animais , Células Epiteliais/efeitos dos fármacos , Epitélio/fisiologia , Feminino , Proteínas de Ligação ao GTP/deficiência , Perfilação da Expressão Gênica , Masculino , Camundongos Knockout , Dados de Sequência Molecular , Análise de Sequência de DNA , Transdução de Sinais
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