A single amino acid substitution in the novel H7N9 influenza A virus NS1 protein increases CPSF30 binding and virulence

Although an effective interferon antagonist in human and avian cells, the novel H7N9 influenza virus NS1 protein is defective at inhibiting CPSF30. An I106M substitution in H7N9 NS1 can restore CPSF30 binding together with the ability to block host gene expression. Furthermore, a recombinant virus expressing H7N9 NS1-I106M replicates to higher titers in vivo, and is subtly more virulent, than parental. Natural polymorphisms in H7N9 NS1 that enhance CPSF30 binding may be cause for concern

Positive regulatory role of the e3 ubiquitin ligase TRIM65 downstream the IFNβ induction pathway.

Introduction: Viral infection triggers a fast and effective cellular response mediated primarily by the production of interferon β (IFNβ) that induces an anti-viral state through complex signal cascades. Therefore, the regulation of its induction and subsequent IFNβ signaling needs to be tightly controlled. There is growing evidence implicating the members of Tripartite-motif (TRIM) protein family of E3 ligases as critical players in this regulation. However, the exact role, mechanism of action, and the physiological relevance of their activity in vivo still remain poorly investigated. Previous work in our lab revealed that an unprecedented large number of TRIMs play critical roles as enhancers in the regulation of innate immune signaling pathways. Methods: To study the role of TRIM65 in innate immune signaling we have used luciferase assays, overexpression in A549 and 293T cells, transient knock down using siRNAs in 293T cells, TRIM65 CRISPR knock out cell lines, Western blots, RT-qPCR, PR8-GLuc antiviral assays and immunofluorescence. Results: Our recent studies focused on TRIM65 showed that this protein possesses antiviral activity comparable to TRIM25 in a PR8-GLuc antiviral assay. We have also demonstrated that its overexpression strongly increased not only the 2CARD-RIG-I- but also the IRF3-dependent activation of the INFβ and ISRE reporters. Consequently, IFNβ, ISG54 as well as other cytokines and ISGs mRNA levels are decreased in TRIM65 knock down and knock out (KO) cells upon infection compared to infected/treated control cells. Re-constitution assays on TRIM65 KO cells reverted the inhibition of IFNβ and ISG54 confirming the phenotype. Altogether, these data indicates a stimulatory role for TRIM65 downstream the interferon induction pathway. Since the E3 ubiquitin ligase activity of many TRIMs has been linked to their antiviral functions, we have identified IRF3 and IRF7 as TRIM65 interacting factors and putative substrates. Immunofluorescence experiments suggest that IRF3 binds TRIM65 in the nucleus and in vitro ubiquitination assays indicates that TRIM65 is able to ubiquitinate IRF3 in a RING-independent manner. Our current studies are focused on completely delineate the molecular mechanism by which TRIM65-mediated ubiquitination or TRIM65 E3 Ub ligase-independent function could regulate the response to viral infection. Conclusion: A better understanding of positive regulatory networks of the IFN response will provide new knowledge that will help to design more effective therapeutics

Host-Specific NS5 Ubiquitination Determines Yellow Fever Virus Tropism

The recent yellow fever virus (YFV) epidemic in Brazil in 2017 and Zika virus (ZIKV) epidemic in 2015 serve to remind us of the importance of flaviviruses as emerging human pathogens. With the current global flavivirus threat, there is an urgent need for antivirals and vaccines to curb the spread of these viruses. However, the lack of suitable animal models limits the research questions that can be answered. A common trait of all flaviviruses studied thus far is their ability to antagonize interferon (IFN) signaling so as to enhance viral replication and dissemination. Previously, we reported that YFV NS5 requires the presence of type I IFN (IFN-α/β) for its engagement with human signal transducer and activator of transcription 2 (hSTAT2). In this manuscript, we report that like the NS5 proteins of ZIKV and dengue virus (DENV), YFV NS5 protein is able to bind hSTAT2 but not murine STAT2 (mSTAT2). Contrary to what has been demonstrated with ZIKV NS5 and DENV NS5, replacing mSTAT2 with hSTAT2 cannot rescue the YFV NS5-STAT2 interaction, as YFV NS5 is also unable to interact with hSTAT2 in murine cells. We show that the IFN-α/β-dependent ubiquitination of YFV NS5 that is required for STAT2 binding in human cells is absent in murine cells. In addition, we demonstrate that mSTAT2 restricts YFV replication in vivo These data serve as further impetus for the development of an immunocompetent mouse model that can serve as a disease model for multiple flaviviruses.IMPORTANCE Flaviviruses such as yellow fever virus (YFV), Zika virus (ZIKV), and dengue virus (DENV) are important human pathogens. A common flavivirus trait is the antagonism of interferon (IFN) signaling to enhance viral replication and spread. We report that like ZIKV NS5 and DENV NS5, YFV NS5 binds human STAT2 (hSTAT2) but not mouse STAT2 (mSTAT2), a type I IFN (IFN-α/β) pathway component. Additionally, we show that contrary to what has been demonstrated with ZIKV NS5 and DENV NS5, YFV NS5 is unable to interact with hSTAT2 in murine cells. We demonstrate that mSTAT2 restricts YFV replication in mice and that this correlates with a lack of IFN-α/β-induced YFV NS5 ubiquitination in murine cells. The lack of suitable animal models limits flavivirus pathogenesis, vaccine, and drug research. These data serve as further impetus for the development of an immunocompetent mouse model that can serve as a disease model for multiple flaviviruses

A serological assay to detect SARS-CoV-2 seroconversion in humans

Development of an enzyme-linked immunosorbent assay to detect antibodies to the SARS-CoV-2 spike protein in human sera and plasma. Here, we describe a serological enzyme-linked immunosorbent assay for the screening and identification of human SARS-CoV-2 seroconverters. This assay does not require the handling of infectious virus, can be adjusted to detect different antibody types in serum and plasma and is amenable to scaling. Serological assays are of critical importance to help define previous exposure to SARS-CoV-2 in populations, identify highly reactive human donors for convalescent plasma therapy and investigate correlates of protection.Peer reviewe

Unanchored K48-Linked Polyubiquitin Synthesized by the E3-Ubiquitin Ligase TRIM6 Stimulates the Interferon-IKKε Kinase-Mediated Antiviral Response.

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, interacted with IKKε and promoted induction of IKKε-dependent ISGs. TRIM6 and the E2-ubiquitin conjugase UbE2K cooperated in the synthesis of unanchored K48-linked polyubiquitin chains, which activated IKKε for subsequent STAT1 phosphorylation. Our work attributes a previously unrecognized activating role of K48-linked unanchored polyubiquitin chains in kinase activation and identifies the UbE2K-TRIM6-ubiquitin axis as critical for IFN signaling and antiviral response

Impact of SARS-CoV-2 ORF6 and its variant polymorphisms on host responses and viral pathogenesis

: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) encodes several proteins that inhibit host interferon responses. Among these, ORF6 antagonizes interferon signaling by disrupting nucleocytoplasmic trafficking through interactions with the nuclear pore complex components Nup98-Rae1. However, the roles and contributions of ORF6 during physiological infection remain unexplored. We assessed the role of ORF6 during infection using recombinant viruses carrying a deletion or loss-of-function (LoF) mutation in ORF6. ORF6 plays key roles in interferon antagonism and viral pathogenesis by interfering with nuclear import and specifically the translocation of IRF and STAT transcription factors. Additionally, ORF6 inhibits cellular mRNA export, resulting in the remodeling of the host cell proteome, and regulates viral protein expression. Interestingly, the ORF6:D61L mutation that emerged in the Omicron BA.2 and BA.4 variants exhibits reduced interactions with Nup98-Rae1 and consequently impairs immune evasion. Our findings highlight the role of ORF6 in antagonizing innate immunity and emphasize the importance of studying the immune evasion strategies of SARS-CoV-2

Autoantibodies against type I IFNs in patients with critical influenza pneumonia

In an international cohort of 279 patients with hypoxemic influenza pneumonia, we identified 13 patients (4.6%) with autoantibodies neutralizing IFN-alpha and/or -omega, which were previously reported to underlie 15% cases of life-threatening COVID-19 pneumonia and one third of severe adverse reactions to live-attenuated yellow fever vaccine. Autoantibodies neutralizing type I interferons (IFNs) can underlie critical COVID-19 pneumonia and yellow fever vaccine disease. We report here on 13 patients harboring autoantibodies neutralizing IFN-alpha 2 alone (five patients) or with IFN-omega (eight patients) from a cohort of 279 patients (4.7%) aged 6-73 yr with critical influenza pneumonia. Nine and four patients had antibodies neutralizing high and low concentrations, respectively, of IFN-alpha 2, and six and two patients had antibodies neutralizing high and low concentrations, respectively, of IFN-omega. The patients' autoantibodies increased influenza A virus replication in both A549 cells and reconstituted human airway epithelia. The prevalence of these antibodies was significantly higher than that in the general population for patients 70 yr of age (3.1 vs. 4.4%, P = 0.68). The risk of critical influenza was highest in patients with antibodies neutralizing high concentrations of both IFN-alpha 2 and IFN-omega (OR = 11.7, P = 1.3 x 10(-5)), especially those <70 yr old (OR = 139.9, P = 3.1 x 10(-10)). We also identified 10 patients in additional influenza patient cohorts. Autoantibodies neutralizing type I IFNs account for similar to 5% of cases of life-threatening influenza pneumonia in patients <70 yr old

A SARS-CoV-2 protein interaction map reveals targets for drug repurposing

The novel coronavirus SARS-CoV-2, the causative agent of COVID-19 respiratory disease, has infected over 2.3 million people, killed over 160,000, and caused worldwide social and economic disruption1,2. There are currently no antiviral drugs with proven clinical efficacy, nor are there vaccines for its prevention, and these efforts are hampered by limited knowledge of the molecular details of SARS-CoV-2 infection. To address this, we cloned, tagged and expressed 26 of the 29 SARS-CoV-2 proteins in human cells and identified the human proteins physically associated with each using affinity-purification mass spectrometry (AP-MS), identifying 332 high-confidence SARS-CoV-2-human protein-protein interactions (PPIs). Among these, we identify 66 druggable human proteins or host factors targeted by 69 compounds (29 FDA-approved drugs, 12 drugs in clinical trials, and 28 preclinical compounds). Screening a subset of these in multiple viral assays identified two sets of pharmacological agents that displayed antiviral activity: inhibitors of mRNA translation and predicted regulators of the Sigma1 and Sigma2 receptors. Further studies of these host factor targeting agents, including their combination with drugs that directly target viral enzymes, could lead to a therapeutic regimen to treat COVID-19