Flaviviruses continue to emerge as global health threats. There are currently no Food and Drug Administration (FDA) approved antiviral treatments for flaviviral infections. Therefore, there is a ...pressing need to identify host and viral factors that can be targeted for effective therapeutic intervention. Type I interferon (IFN-I) production in response to microbial products is one of the host's first line of defense against invading pathogens. Cytidine/uridine monophosphate kinase 2 (CMPK2) is a type I interferon-stimulated gene (ISG) that exerts antiviral effects. However, the molecular mechanism by which CMPK2 inhibits viral replication is unclear. Here, we report that CMPK2 expression restricts Zika virus (ZIKV) replication by specifically inhibiting viral translation and that IFN-I- induced CMPK2 contributes significantly to the overall antiviral response against ZIKV. We demonstrate that expression of CMPK2 results in a significant decrease in the replication of other pathogenic flaviviruses including dengue virus (DENV-2), Kunjin virus (KUNV) and yellow fever virus (YFV). Importantly, we determine that the N-terminal domain (NTD) of CMPK2, which lacks kinase activity, is sufficient to restrict viral translation. Thus, its kinase function is not required for CMPK2's antiviral activity. Furthermore, we identify seven conserved cysteine residues within the NTD as critical for CMPK2 antiviral activity. Thus, these residues may form an unknown functional site in the NTD of CMPK2 contributing to its antiviral function. Finally, we show that mitochondrial localization of CMPK2 is required for its antiviral effects. Given its broad antiviral activity against flaviviruses, CMPK2 is a promising potential pan-flavivirus inhibitor.
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.
To successfully establish infection, flaviviruses have to overcome the antiviral state induced by type I interferon (IFN-I). The nonstructural NS5 proteins of several flaviviruses antagonize IFN-I ...signaling. Here we show that yellow fever virus (YFV) inhibits IFN-I signaling through a unique mechanism that involves binding of YFV NS5 to the IFN-activated transcription factor STAT2 only in cells that have been stimulated with IFN-I. This NS5-STAT2 interaction requires IFN-I-induced tyrosine phosphorylation of STAT1 and the K63-linked polyubiquitination at a lysine in the N-terminal region of YFV NS5. We identified TRIM23 as the E3 ligase that interacts with and polyubiquitinates YFV NS5 to promote its binding to STAT2 and trigger IFN-I signaling inhibition. Our results demonstrate the importance of YFV NS5 in overcoming the antiviral action of IFN-I and offer a unique example of a viral protein that is activated by the same host pathway that it inhibits.
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•Yellow fever virus (YFV) NS5 antagonizes type I interferon (IFN-I) signaling•NS5 binds STAT2 only in IFN-I-stimulated cells•NS5-STAT2 interaction requires NS5 lysine 6 ubiquitination and STAT1 phosphorylation•The E3 ligase TRIM23 interacts with and ubiquitinates YFV NS5 lysine 6
To establish infection, yellow fever virus (YFV) has to antagonize IFN-I’s antiviral effects. Laurent-Rolle et al. find that the YFV NS5 protein binds the IFN-activated transcription factor STAT2, but only in IFN-I-stimulated cells. IFN-I-induced STAT1 phosphorylation and NS5 polyubiquitination were both required for STAT2 binding and IFN antagonism by NS5.
The Eastern Equine Encephalitis Virus (EEEV) is an emerging public health threat, with the number of reported cases in the US increasing in recent years. EEEV is a BSL3 pathogen, and the North ...American strain is a US Federal Select Agent (SA). These restrictions make experiments with EEEV difficult to perform, as high-tech equipment is often unavailable in BSL3 spaces and due to concerns about generating aerosols during manipulations. Therefore, a range of inactivation methods suitable for different downstream analysis methods are essential for advancing research on EEEV. We used heat, chemical, and ultraviolet (UV)-based methods for the inactivation of infected cells and supernatants infected with the non-select agent Madariaga virus (MADV). Although the MADV and EEEV strains are genetically distinct, differing by 8-11% at the amino acid level, they are expected to be similarly susceptible to various inactivation methods. We determined the following to be effective methods of inactivation: heat, TRIzol LS, 4% PFA, 10% formalin, and UV radiation for infected supernatants; TRIzol, 2.5% SDS with BME, 0.2% NP40, 4% PFA, and 10% formalin for infected cells. Our results have the potential to expand the types and complexity of experiments and analyses performed by EEEV researchers.
Innate immune responses induce hundreds of interferon-stimulated genes (ISGs). Viperin, a member of the radical S-adenosyl methionine (SAM) superfamily of enzymes, is the product of one such ISG that ...restricts the replication of a broad spectrum of viruses. Here, we report a previously unknown antiviral mechanism in which viperin activates a ribosome collision-dependent pathway that inhibits both cellular and viral RNA translation. We found that the radical SAM activity of viperin is required for translation inhibition and that this is mediated by viperin’s enzymatic product, 3′-deoxy-3′,4′-didehydro-CTP (ddhCTP). Viperin triggers ribosome collisions and activates the MAPKKK ZAK pathway that in turn activates the GCN2 arm of the integrated stress response pathway to inhibit translation. The study illustrates the importance of translational repression in the antiviral response and identifies viperin as a translation regulator in innate immunity.
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•Viperin regulates cellular translation to restrict viral translation and replication•Viperin activates the GCN2-arm of the ISR•Viperin induces ribosome collision-mediated translation inhibition•Viperin’s product ddhCTP induces ribosome collision
Hsu et al. show that viperin, a broad-spectrum antiviral protein, inhibits the viral replication of two medically relevant flaviviruses by limiting viral protein production. Viperin synthesizes a nucleoside derivative, ddhCTP, which induces ribosome collisions, leading to the activation of the integrated stress response and consequently translation inhibition.
Limited therapeutic options exist for coronavirus disease 2019 (COVID-19). COVID-19 convalescent plasma (CCP) is a potential therapeutic, but there is limited data for patients with ...moderate-to-severe disease. What are outcomes associated with administration of CCP in patients with moderate-to-severe COVID-19 infection? We conducted a propensity score-matched analysis of patients with moderate-to-severe COVID-19. The primary endpoints were in-hospital mortality. Secondary endpoints were number of days alive and ventilator-free at 30 days; length of hospital stay; and change in WHO scores from CCP administration (or index date) to discharge. Of 151 patients who received CCP, 132 had complete follow-up data. Patients were transfused after a median of 6 hospital days; thus, we investigated the effect of convalescent plasma before and after this timepoint with 77 early (within 6 days) and 55 late (after 6 days) recipients. Among 3,217 inpatients who did not receive CCP, 2,551 were available for matching. Early CCP recipients, of whom 31 (40%) were on mechanical ventilation, had lower 14-day (15% vs 23%) and 30-day (38% vs 49%) mortality compared to a matched unexposed cohort, with nearly 50% lower likelihood of in-hospital mortality (HR 0.52, 95% CI 0.28-0.96; P = 0.036). Early plasma recipients had more days alive and ventilator-free at 30 days (+3.3 days, 95% CI 0.2 to 6.3 days; P = 0.04) and improved WHO scores at 7 days (-0.8, 95% CI: -1.2 to -0.4; P = 0.0003) and hospital discharge (-0.9, 95% CI: -1.5 to -0.3; P = 0.004) compared to the matched unexposed cohort. No clinical differences were observed in late plasma recipients.
Inhibition of Interferon Signaling by Dengue Virus Muñoz-Jordán, Jorge L.; Sánchez-Burgos, Gilma G.; Laurent-Rolle, Maudry ...
Proceedings of the National Academy of Sciences - PNAS,
11/2003, Letnik:
100, Številka:
24
Journal Article
Recenzirano
Odprti dostop
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.
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