PKR and RNase L Contribute to Protection against Lethal West Nile Virus Infection by Controlling Early Viral Spread in the Periphery and Replication in Neurons

ABSTRACT West Nile virus (WNV) is a neurotropic, mosquito-borne flavivirus that can cause lethal meningoencephalitis. Type I interferon (IFN) plays a critical role in controlling WNV replication, spread, and tropism. In this study, we begin to examine the effector mechanisms by which type I IFN inhibits WNV infection. Mice lacking both the interferon-induced, double-stranded-RNA-activated protein kinase (PKR) and the endoribonuclease of the 2′,5′-oligoadenylate synthetase-RNase L system (PKR−/− × RL−/−) were highly susceptible to subcutaneous WNV infection, with a 90% mortality rate compared to the 30% mortality rate observed in congenic wild-type mice. PKR−/− × RL−/− mice had increased viral loads in their draining lymph nodes, sera, and spleens, which led to early viral entry into the central nervous system (CNS) and higher viral burden in neuronal tissues. Although mice lacking RNase L showed a higher CNS viral burden and an increased mortality, they were less susceptible than the PKR−/− × RL−/− mice; thus, we also infer an antiviral role for PKR in the control of WNV infection. Notably, a deficiency in both PKR and RNase L resulted in a decreased ability of type I IFN to inhibit WNV in primary macrophages and cortical neurons. In contrast, the peripheral neurons of the superior cervical ganglia of PKR−/− × RL−/− mice showed no deficiency in the IFN-mediated inhibition of WNV. Our data suggest that PKR and RNase L contribute to IFN-mediated protection in a cell-restricted manner and control WNV infection in peripheral tissues and some neuronal subtypes.

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Interferon Regulatory Factor IRF-7 Induces the Antiviral Alpha Interferon Response and Protects against Lethal West Nile Virus Infection

Journal of Virology ◽

10.1128/jvi.00918-08 ◽

2008 ◽

Vol 82 (17) ◽

pp. 8465-8475 ◽

Cited By ~ 114

Author(s):

Stephane Daffis ◽

Melanie A. Samuel ◽

Mehul S. Suthar ◽

Brian C. Keller ◽

Michael Gale ◽

...

Keyword(s):

West Nile Virus ◽

Cortical Neurons ◽

Interferon Regulatory Factor ◽

Interferon Response ◽

Type I ◽

Type I Ifn ◽

Regulatory Factor ◽

West Nile

ABSTRACT Type I interferon (IFN-α/β) comprises a family of immunomodulatory cytokines that are critical for controlling viral infections. In cell culture, many RNA viruses trigger IFN responses through the binding of RNA recognition molecules (RIG-I, MDA5, and TLR-3) and induction of interferon regulatory factor IRF-3-dependent gene transcription. Recent studies with West Nile virus (WNV) have shown that type I IFN is essential for restricting infection and that a deficiency of IRF-3 results in enhanced lethality. However, IRF-3 was not required for optimal systemic IFN production in vivo or in vitro in macrophages. To begin to define the transcriptional factors that regulate type I IFN after WNV infection, we evaluated IFN induction and virus control in IRF-7−/− mice. Compared to congenic wild-type mice, IRF-7−/− mice showed increased lethality after WNV infection and developed early and elevated WNV burdens in both peripheral and central nervous system tissues. As a correlate, a deficiency of IRF-7 blunted the systemic type I IFN response in mice. Consistent with this, IFN-α gene expression and protein production were reduced and viral titers were increased in IRF-7−/− primary macrophages, fibroblasts, dendritic cells, and cortical neurons. In contrast, in these cells the IFN-β response remained largely intact. Our data suggest that the early protective IFN-α response against WNV occurs through an IRF-7-dependent transcriptional signal.

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Measure and Countermeasure: Type I IFN (IFN-α/β) Antiviral Response against West Nile Virus

Journal of Innate Immunity ◽

10.1159/000226248 ◽

2009 ◽

Vol 1 (5) ◽

pp. 435-445 ◽

Cited By ~ 21

Author(s):

Stephane Daffis ◽

Mehul S. Suthar ◽

Michael Gale, Jr. ◽

Michael S. Diamond

Keyword(s):

West Nile Virus ◽

Antiviral Response ◽

Type I ◽

Type I Ifn ◽

West Nile ◽

Ifn Alpha ◽

Alpha Beta

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Single Nucleotide Polymorphisms in Genes for 2′‐5′‐Oligoadenylate Synthetase and RNase L in Patients Hospitalized with West Nile Virus Infection

The Journal of Infectious Diseases ◽

10.1086/497340 ◽

2005 ◽

Vol 192 (10) ◽

pp. 1741-1748 ◽

Cited By ~ 93

Author(s):

Imtiaz Yakub ◽

Kristy M. Lillibridge ◽

Ana Moran ◽

Omar Y. Gonzalez ◽

John Belmont ◽

...

Keyword(s):

West Nile Virus ◽

Virus Infection ◽

Single Nucleotide Polymorphisms ◽

West Nile Virus Infection ◽

Rnase L ◽

Nucleotide Polymorphisms ◽

Oligoadenylate Synthetase ◽

West Nile ◽

Single Nucleotide

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RNase L Plays a Role in the Antiviral Response to West Nile Virus

Journal of Virology ◽

10.1128/jvi.80.6.2987-2999.2006 ◽

2006 ◽

Vol 80 (6) ◽

pp. 2987-2999 ◽

Cited By ~ 101

Author(s):

Svetlana V. Scherbik ◽

Jayashree M. Paranjape ◽

Bronislava M. Stockman ◽

Robert H. Silverman ◽

Margo A. Brinton

Keyword(s):

West Nile Virus ◽

Antiviral Response ◽

Dominant Negative ◽

Activity Levels ◽

Rnase L ◽

Genomic Rna ◽

Oligoadenylate Synthetase ◽

Susceptible Mouse ◽

West Nile ◽

Cellular Antiviral Response

ABSTRACT Alleles at the Flv locus determine disease outcome after a flavivirus infection in mice. Although comparable numbers of congenic resistant and susceptible mouse embryo fibroblasts (MEFs) are infected by the flavivirus West Nile virus (WNV), resistant MEFs produce ∼100- to 150-fold lower titers than susceptible ones and flavivirus titers in the brains of resistant and susceptible animals can differ by >10,000-fold. The Flv locus was previously identified as the 2′-5′ oligoadenylate synthetase 1b (Oas1b) gene. Oas gene expression is up-regulated by interferon (IFN), and after activation by double-stranded RNA, some mouse synthetases produce 2-5A, which activates latent RNase L to degrade viral and cellular RNAs. To determine whether the lower levels of intracellular flavivirus genomic RNA from resistant mice detected in cells at all times after infection were mediated by RNase L, RNase L activity levels in congenic resistant and susceptible cells were compared. Similar moderate levels of RNase L activation by transfected 2-5A were observed in both types of uninfected cells. After WNV infection, the mRNAs of IFN-β and three Oas genes were up-regulated to similar levels in both types of cells. However, significant levels of RNase L activity were not detected until 72 h after WNV infection and the patterns of viral RNA cleavage products generated were similar in both types of cells. When RNase L activity was down-regulated in resistant cells via stable expression of a dominant negative RNase L mutant, ∼5- to 10-times-higher yields of WNV were produced. Similarly, about ∼5- to 10-times-higher virus yields were produced by susceptible C57BL/6 RNase L−/− cells compared to RNase L+/+ cells that were either left untreated or pretreated with IFN and/or poly(I) · poly(C). The data indicate that WNV genomic RNA is susceptible to RNase L cleavage and that RNase L plays a role in the cellular antiviral response to flaviviruses. The results suggest that RNase L activation is not a major component of the Oas1b-mediated flavivirus resistance phenotype.

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Activation of 2′ 5′-Oligoadenylate Synthetase by Stem Loops at the 5′-End of the West Nile Virus Genome

PLoS ONE ◽

10.1371/journal.pone.0092545 ◽

2014 ◽

Vol 9 (3) ◽

pp. e92545 ◽

Cited By ~ 30

Author(s):

Soumya Deo ◽

Trushar R. Patel ◽

Edis Dzananovic ◽

Evan P. Booy ◽

Khalid Zeid ◽

...

Keyword(s):

West Nile Virus ◽

Virus Genome ◽

Oligoadenylate Synthetase ◽

The West ◽

West Nile

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Heterozygous OAS1 gain-of-function variants cause an autoinflammatory immunodeficiency

Science Immunology ◽

10.1126/sciimmunol.abf9564 ◽

2021 ◽

Vol 6 (60) ◽

pp. eabf9564

Author(s):

Thomas Magg ◽

Tsubasa Okano ◽

Lars M. Koenig ◽

Daniel F.R. Boehmer ◽

Samantha L. Schwartz ◽

...

Keyword(s):

B Cells ◽

Hematopoietic Cell Transplantation ◽

De Novo ◽

Dynamics Simulation ◽

Recurrent Fever ◽

Type I ◽

Rnase L ◽

Oligoadenylate Synthetase ◽

Gain Of Function ◽

Translational Arrest

Analysis of autoinflammatory and immunodeficiency disorders elucidates human immunity and fosters the development of targeted therapies. Oligoadenylate synthetase 1 is a type I interferon–induced, intracellular double-stranded RNA (dsRNA) sensor that generates 2′-5′-oligoadenylate to activate ribonuclease L (RNase L) as a means of antiviral defense. We identified four de novo heterozygous OAS1 gain-of-function variants in six patients with a polymorphic autoinflammatory immunodeficiency characterized by recurrent fever, dermatitis, inflammatory bowel disease, pulmonary alveolar proteinosis, and hypogammaglobulinemia. To establish causality, we applied genetic, molecular dynamics simulation, biochemical, and cellular functional analyses in heterologous, autologous, and inducible pluripotent stem cell–derived macrophages and/or monocytes and B cells. We found that upon interferon-induced expression, OAS1 variant proteins displayed dsRNA-independent activity, which resulted in RNase L–mediated RNA cleavage, transcriptomic alteration, translational arrest, and dysfunction and apoptosis of monocytes, macrophages, and B cells. RNase L inhibition with curcumin modulated and allogeneic hematopoietic cell transplantation cured the disorder. Together, these data suggest that human OAS1 is a regulator of interferon-induced hyperinflammatory monocyte, macrophage, and B cell pathophysiology.

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Viral determinants in the NS3 helicase and 2K peptide that promote West Nile virus resistance to antiviral action of 2′,5′-oligoadenylate synthetase 1b

Virology ◽

10.1016/j.virol.2009.12.036 ◽

2010 ◽

Vol 399 (1) ◽

pp. 176-185 ◽

Cited By ~ 21

Author(s):

Eva Mertens ◽

Anna Kajaste-Rudnitski ◽

Shessy Torres ◽

Anneke Funk ◽

Marie-Pascale Frenkiel ◽

...

Keyword(s):

West Nile Virus ◽

Virus Resistance ◽

Antiviral Action ◽

Oligoadenylate Synthetase ◽

West Nile ◽

Viral Determinants

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The Host Response to West Nile Virus Infection Limits Viral Spread through the Activation of the Interferon Regulatory Factor 3 Pathway

Journal of Virology ◽

10.1128/jvi.78.14.7737-7747.2004 ◽

2004 ◽

Vol 78 (14) ◽

pp. 7737-7747 ◽

Cited By ~ 111

Author(s):

Brenda L. Fredericksen ◽

Maria Smith ◽

Michael G. Katze ◽

Pei-Yong Shi ◽

Michael Gale

Keyword(s):

West Nile Virus ◽

Virus Replication ◽

Host Response ◽

Molecular Mechanisms ◽

Target Genes ◽

West Nile Virus Infection ◽

Null Mouse ◽

Regulatory Factor ◽

West Nile ◽

Viral Spread

ABSTRACT Recent outbreaks of West Nile Virus (WNV) have been associated with an increase in morbidity and mortality in humans, birds, and many other species. We have initiated studies to define the molecular mechanisms by which a recent pathogenic isolate of WNV evades the host cell innate antiviral response. Biochemical and microarray analyses demonstrated that WNV induced the expression of beta interferon (IFN-β) and several IFN-stimulated genes late in infection of cultured human cells. The late expression of these antiviral genes was due to the delayed activation of the transcription factor IFN regulatory factor 3 (IRF-3). Despite this host response, WNV was still able to replicate efficiently. The effect of the IRF-3 pathway on WNV replication was assessed by examining virus replication and spread in cultures of wild-type or IRF-3-null mouse embryo fibroblasts. The absence of IRF-3 was marked by a significant increase in plaque size and a sustained production of infectious particles. Although the activation of the IRF-3 pathway was not sufficient to block virus replication, our results suggest that IRF-3 target genes function to constrain WNV infection and limit cell-to-cell virus spread.

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