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 virusinfected 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 The DEN virion contains a positive strand RNA molecule with an Ϸ10-kb-long ORF flanked by 5Ј and 3Ј 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 NS5͞NS3 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-␣͞␤ response in virus-infected cells presumably occurs on viral entry and release͞synthesis of viral components, including double-stranded RNA (dsRNA) intermediates. The transcription factors IFN regulatory factor (IRF)-3, IRF-7, NF-B, and activating transcription factor 2 (ATF2)͞c-Jun are activated by some of these viral components and trigger the expression of IFN-␣͞␤ (3-7). Secreted IFN-␣͞␤ 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-␣͞␤, resulting in the induction of an antiviral state (8). The pivotal role of IFN-␣͞␤ in protecting against viral infections has been demonstrated by the finding that STAT1 Ϫ/Ϫ 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-␣͞␤ or IFN-␥ 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 2␣ (eIF-2␣) kinase PKR (protein kinase R), an IFN-induced protein with antiviral activity (14). The H...

The mammalian interferon (IFN) signaling pathway is a primary component of the innate antiviral response. As such, viral pathogens have devised multiple mechanisms to antagonize this pathway and thus facilitate infection. Dengue virus (DENV) encodes several proteins (NS2a, NS4a, and NS4b) that have been shown individually to inhibit the IFN response. In addition, DENV infection results in reduced levels of expression of STAT2, which is required for IFN signaling (M. Jones, A. Davidson, L. Hibbert, P. Gruenwald, J. Schlaak, S. Ball, G. R. Foster, and M. Jacobs, J. Virol. 79:5414-5420, 2005). Translation of the DENV genome results in a single polypeptide, which is processed by viral and host proteases into at least 10 separate proteins. To date, no single DENV protein has been implicated in the targeting of STAT2 for decreased levels of expression. We demonstrate here that the polymerase of the virus, NS5, binds to STAT2 and is necessary and sufficient for its reduced level of expression. The decrease in protein level observed requires ubiquitination and proteasome activity, strongly suggesting an active degradation process. Furthermore, we show that the degradation of but not binding to STAT2 is dependent on the expression of the polymerase in the context of a polyprotein that undergoes proteolytic processing for NS5 maturation. Thus, the mature form of NS5, when not expressed as a precursor, was able to bind to STAT2 but was unable to target it for degradation, establishing a unique role for viral polyprotein processing in providing an additional function to a viral polypeptide. Therefore, we have identified both a novel mechanism by which DENV evades the innate immune response and a potential target for antiviral therapeutics.Dengue virus (DENV) is the causative agent of dengue fever, dengue hemorrhagic fever, and dengue shock syndrome (2). The virus and its arthropod vector, Aedes aegypti (21), are endemic to over 100 countries around the world including the United States. It is responsible for an estimated 50 million to 100 million infections annually, with over 24,000 deaths resulting, predominantly in children under 14 years of age (25). The virus exists in four serotypes (DEN1 to DEN4) and is grouped into the flavivirus genus along with a number of additional human pathogens including West Nile virus (WNV), Japanese encephalitis virus (JEV), and tick-borne encephalitis virus (TBEV). These viruses have a positive-strand, nonsegmented genome of ϳ11 kb (5, 55), the organization of which is highly conserved, encoding, in order, three structural proteins (C, M, and E), followed by seven nonstructural proteins (NS1, NS2a, NS2b, NS3, NS4a, NS4b, and NS5). The genome is translated as a single endoplasmic reticulum (ER)-bound polyprotein, which is co-and posttranslationally processed by both viral (NS2b and NS3) and cellular proteases (8,44).A critical component of the human antiviral response is the type 1 interferon (IFN) pathway, which acts to delay virus replication and to stimulate the activation of antiviral ...

Flaviviruses are insect-borne, positive-strand RNA viruses that have been disseminated worldwide. Their genome is translated into a polyprotein, which is subsequently cleaved by a combination of viral and host proteases to produce three structural proteins and seven nonstructural proteins. The nonstructural protein NS4B of dengue 2 virus partially blocks activation of STAT1 and interferon-stimulated response element (ISRE) promoters in cells stimulated with interferon (IFN). We have found that this function of NS4B is conserved in West Nile and yellow fever viruses. Deletion analysis shows that that the first 125 amino acids of dengue virus NS4B are sufficient for inhibition of alpha/beta IFN (IFN-␣/␤) signaling. The cleavable signal peptide at the N terminus of NS4B, a peptide with a molecular weight of 2,000, is required for IFN antagonism but can be replaced by an unrelated signal peptide. Coexpression of dengue virus NS4A and NS4B together results in enhanced inhibition of ISRE promoter activation in response to IFN-␣/␤. In contrast, expression of the precursor NS4A/B fusion protein does not cause an inhibition of IFN signaling unless this product is cleaved by the viral peptidase NS2B/NS3, indicating that proper viral polyprotein processing is required for anti-interferon function.The arthropod-borne flaviviruses are important human pathogens. Dengue viruses (DEN) are the causative agents of the most prevalent insect-borne viral illness, dengue fever, characterized by high fever, chills, body aches, and skin rash. More than 50 to 100 million cases of dengue fever are reported yearly in over 80 countries where the mosquito vector Aedes aegypti is endemic, and approximately 500,000 patients suffer from the more debilitating and often lethal illnesses known as dengue hemorrhagic fever and dengue shock syndrome. Japanese encephalitis virus is the leading cause of arboviral encephalitis in Asia, accounting for 30,000 to 50,000 cases annually. St. Louis encephalitis virus causes sporadic epidemic encephalitis in the Americas. West Nile virus (WNV), previously unknown in the Western Hemisphere, has caused more than 9,000 cases in North America since 1999. Many infections are asymptomatic, and a small proportion of infected people develop a mild febrile syndrome; but 1% of cases are at a high risk of developing potentially fatal encephalitis. There is currently no specific treatment for dengue-and West Nile-related diseases or available vaccines to prevent human infection. The yellow fever virus (YFV) is largely under control due to the effectiveness of the yellow fever vaccine; nonetheless, the disease continues to occur intermittently in tropical South America and sub-Saharan Africa with a high fatality rate in infants.Flaviviruses contain a positive-strand RNA molecule with a Ϸ10.7-kb-long open reading frame. After receptor-mediated endocytosis and release of the nucleocapsid in the cytoplasm (3,18,19), an interaction of ribosomes with the nucleocapsid brings the viral RNA to the cytoplasmic side of the rough en...

An estimated 50 million dengue virus (DENV) infections occur annually and more than forty percent of the human population is currently at risk of developing dengue fever (DF) or dengue hemorrhagic fever (DHF). Despite the prevalence and potential severity of DF and DHF, there are no approved vaccines or antiviral therapeutics available. An improved understanding of DENV immune evasion is pivotal for the rational development of anti-DENV therapeutics. Antagonism of type I interferon (IFN-I) signaling is a crucial mechanism of DENV immune evasion. DENV NS5 protein inhibits IFN-I signaling by mediating proteasome-dependent STAT2 degradation. Only proteolytically-processed NS5 can efficiently mediate STAT2 degradation, though both unprocessed and processed NS5 bind STAT2. Here we identify UBR4, a 600-kDa member of the N-recognin family, as an interacting partner of DENV NS5 that preferentially binds to processed NS5. Our results also demonstrate that DENV NS5 bridges STAT2 and UBR4. Furthermore, we show that UBR4 promotes DENV-mediated STAT2 degradation, and most importantly, that UBR4 is necessary for efficient viral replication in IFN-I competent cells. Our data underscore the importance of NS5-mediated STAT2 degradation in DENV replication and identify UBR4 as a host protein that is specifically exploited by DENV to inhibit IFN-I signaling via STAT2 degradation.

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Summary Dengue virus encodes several interferon antagonists. Among these the NS5 protein binds STAT2, a necessary component of the type-I interferon signaling pathway, and targets it for degradation. We now demonstrate that the ability of dengue NS5 to associate with and degrade STAT2 is species specific. Thus, NS5 is able to bind and degrade human STAT2 but not mouse STAT2. This difference was exploited to demonstrate, absent manipulation of the viral genome, that NS5 mediated IFN antagonism is essential for efficient virus replication. Moreover, we demonstrate that differences in NS5 mediated binding and degradation between human and mouse STAT2 maps to a region within the STAT2 coiled-coil domain. By using STAT2−/− mice, we also demonstrate that mouse STAT2 restricts early dengue virus replication in vivo. These results suggest that overcoming this restriction through transgenic mouse technology may help in the development of a long-sought immune-competent mouse model of dengue virus infection.

Summary Type-I interferons (IFN-I) are essential antiviral cytokines produced upon microbial infection. IFN-I elicits this activity through the upregulation of hundreds of IFN-I stimulated genes (ISGs). The full breadth of ISG induction demands activation of a number of cellular factors including the IκB kinase epsilon (IKKε). However, the mechanism of IKKε activation upon IFN receptor signaling has remained elusive. Here we show that TRIM6, a member of the E3-ubiquitin ligase tripartite motif (TRIM) family of proteins, interacts with IKKε and promotes induction of IKKε-dependent ISGs. TRIM6 and the E2-ubiquitin conjugase UbE2K cooperate in the synthesis of unanchored K48-linked poly-ubiquitin chains, which activate IKKε for subsequent STAT1 phosphorylation. Our work attributes a previously unrecognized activating role of K48-linked unanchored poly-ubiquitin chains in kinase activation and identifies the UbE2K-TRIM6-ubiquitin axis as critical for IFN signaling and antiviral response.

The ongoing COVID-19 pandemic has caused an unprecedented global health crisis. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of COVID-19. Subversion of host protein synthesis is a common strategy that pathogenic viruses use to replicate and propagate in their host. In this study, we show that SARS-CoV-2 is able to shut down host protein synthesis and that SARS-CoV-2 nonstructural protein NSP14 exerts this activity. We show that the translation inhibition activity of NSP14 is conserved in human coronaviruses. NSP14 is required for virus replication through contribution of its exoribonuclease (ExoN) and N7-methyltransferase (N7-MTase) activities. Mutations in the ExoN or N7-MTase active sites of SARS-CoV-2 NSP14 abolish its translation inhibition activity. In addition, we show that the formation of NSP14−NSP10 complex enhances translation inhibition executed by NSP14. Consequently, the translational shutdown by NSP14 abolishes the type I interferon (IFN-I)-dependent induction of interferon-stimulated genes (ISGs). Together, we find that SARS-CoV-2 shuts down host innate immune responses via a translation inhibitor, providing insights into the pathogenesis of SARS-CoV-2.