Inhibition of interferon signaling by dengue virus

Jorge Muñoz-Jordán

Gilma Sánchez-Burgos

Maudry Laurent-Rolle

Adolfo García-Sastre

Department of Microbiology, Mount Sinai School of Medicine, One Gustave Levy Place, New York, NY 10029

^{To whom correspondence should be addressed. E-mail:}ude.mssm@ertsas-aicrag.ofloda.

Edited by Peter Palese, Mount Sinai School of Medicine, New York, NY

Received 2003 Aug 12; Accepted 2003 Sep 25.

Abstract

Dengue virus is a worldwide-distributed mosquito-borne flavivirus with a positive strand RNA genome. Its transcribed polyprotein is cleaved by host- and virus-encoded peptidases into 10 proteins, some of which are of unknown function. Although dengue virus-infected cells seem to be resistant to the antiviral action of IFN, the viral products that mediate this resistance are unknown. Therefore, we have analyzed the ability of the 10 dengue virus-encoded proteins to antagonize the IFN response. We found that expression in human A549 cells of the dengue virus nonstructural proteins NS2A, NS4A, or NS4B enhances replication of an IFN-sensitive virus. Moreover, expression of NS4B and, to a lesser extent, of NS2A and NS4A proteins results in down-regulation of IFN-β-stimulated gene expression. Cells expressing NS4B or infected with dengue virus do not exhibit nuclear signal transducer and activator of transcription (STAT) 1 on treatment with IFN-β or IFN-γ, indicating that NS4B might be involved in blocking IFN signaling during dengue virus infections. This protein, encoded by a positive strand RNA virus, is implicated as an IFN-signaling inhibitor.

Abstract

Dengue virus (DEN) classifies in the family Flaviviridae (genus Flavivirus) of which >50 other members including West Nile (WN) and yellow fever (YF) viruses have been identified. Transmitted by the mosquito Aedes aegypti, DEN is the most prevalent arthropod-borne virus affecting humans, with >50 million new cases per year. The dengue fever is a relatively mild febrile illness with rash whereas the dengue hemorrhagic fever is a more severe, sometimes lethal disease.

The DEN virion contains a positive strand RNA molecule with an ≈10-kb-long ORF flanked by 5′ and 3′ nontranslated regions. After endocytosis and release of the viral nucleocapsid into the cytosol, a 3,391-aa-long polyprotein is translated from the viral RNA at the surface of the endoplasmic reticulum (ER). The combined activity of host and virus peptidases results in the cleavage of three structural (C, prM, and E) and seven nonstructural (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) proteins. During viral RNA replication, catalyzed by NS5/NS3 proteins, a negative strand RNA intermediate is generated that is found in association with the genomic positive strand RNA (1, 2).

The onset of the IFN-α/β response in virus-infected cells presumably occurs on viral entry and release/synthesis of viral components, including double-stranded RNA (dsRNA) intermediates. The transcription factors IFN regulatory factor (IRF)-3, IRF-7, NF-κB, and activating transcription factor 2 (ATF2)/c-Jun are activated by some of these viral components and trigger the expression of IFN-α/β (3-7). Secreted IFN-α/β binds to the IFN alpha receptor (IFNAR) on the surface of infected and neighboring cells, resulting in activation of the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathway and transcription of numerous genes from promoters containing IFN-stimulated regulatory elements (ISRE). About 100 genes are induced by IFN-α/β, resulting in the induction of an antiviral state (8). The pivotal role of IFN-α/β in protecting against viral infections has been demonstrated by the finding that STAT1^{mice are extremely sensitive to viral infections (}9-11).

The antiviral IFN-mediated response is often hindered by counteracting mechanisms developed by viruses. Experimentally, pretreatment with IFN-α/β or IFN-γ protects human cells against DEN infection (12). However, IFN treatment after DEN infection does not inhibit viral replication, indicating that DEN infection circumvents the action of IFN (12). In addition, the high pathogenicity of DEN in patients exhibiting high titers of IFN suggests that DEN is capable of antagonizing the IFN response (13). Interestingly, multiple anti-IFN mechanisms have been documented for the hepatitis C virus (HCV), a virus related to DEN. HCV NS5A inhibits the eukaryotic translation initiation factor 2α (eIF-2α) kinase PKR (protein kinase R), an IFN-induced protein with antiviral activity (14). The HCV envelope protein E2 also inhibits PKR by competing with eIF-2α as a PKR substrate (15). In addition, HCV NS3/4A protease inhibits activation of IRF-3 and IFN-α/β synthesis (16). Moreover, HCV seems to block the IFN-induced JAK/STAT pathway by mechanisms not yet elucidated (17). Pestiviruses, also related to DEN, express a protein, N^{, that inhibits IFN-α/β synthesis (}18). In contrast, the mechanism(s) used by arthropod-born human flaviviruses, including DEN virus, remain unknown. Although HCV and DEN have common structural features, they do not share enough sequence similarity to allow predictions about the possible function of many of their genes. In addition, the N^{gene of pestivirus is not present in DEN and other members of the genus}Flavivirus.

We have analyzed the ability of the 10 proteins encoded by DEN type 2 (DEN-2) to block the IFN system. Our results show that NS4B strongly blocks the IFN-induced signal-transduction cascade by interfering with STAT1 function. In addition, NS4A and, to a lesser extent, NS2A also block IFN signaling, and the cumulative effect of the three proteins inhibits IFN signaling completely. This inhibition is also observed in DEN-infected cells, suggesting a role for NS4B in DEN pathogenicity.

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Acknowledgments

We thank Georg Kochs and members of the Palese, García-Sastre, and Basler laboratories for valuable critiques, comments, and reagents. E. Nistal, R. Cadagan, and N. Morales contributed technical support. R. Kinney provided the DEN-2 infectious clone and valuable protocols. M. Wathelet provided the ISRE₄-9-27-CAT plasmid. A. Ting provided the pEAK-8-IFκBαDN. This work was supported by a National Institutes of Health grant fellowship (to J.L.M.-J.), a Fulbright fellowship (to G.G.S.-B.), and National Institutes of Health research grants (to A.G.-S).

Acknowledgments

Notes

This paper was submitted directly (Track II) to the PNAS office.

Abbreviations: DEN, dengue virus; ISRE, IFN-stimulated response element; FL, firefly luciferase; NDV, Newcastle disease virus; RL, Renilla luciferase; SeV, Sendai virus; STAT, signal transducer and activator of transcription; ER, endoplasmic reticulum; IRF, IFN regulatory factor; JAK, Janus kinase; HCV, hepatitis C virus; HA, hemagglutinin; CAT, chloramphenicol acetyl transferase; TNF, tumor necrosis factor; CEF, chicken embryo fibroblast.

Notes

This paper was submitted directly (Track II) to the PNAS office.

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