SIAH1 modulates antiviral immune responses by targeting deubiquitinase USP19.

Tight control of the type I interferon (IFN) signaling pathway is critical for maintaining host innate immune responses, and the ubiquitination and deubiquitination of signaling molecules are essential for signal transduction. Deubiquitinase ubiquitin-specific protein 19 (USP19) is known to be involved in deubiquitinating Beclin1, TRAF3, and TRIF for downregulation of the type I IFN signaling. Here, we show that SIAH1, a cellular E3 ubiquitin ligase that is involved in multicellular pathway, is a potent positive regulator of virus-mediated type I IFN signaling that maintains homeostasis within the antiviral immune response by targeting USP19. In the early stages of virus infection, stabilized SIAH1 directly interacts with the USP19 and simultaneously mediates K27-linked ubiquitination of 489, 490, and 610 residues of USP19 for proteasomal degradation. Additionally, we found that USP19 specifically interacts with MAVS and deubiquitinates K63-linked ubiquitinated MAVS for negative regulation of type I IFN signaling. Ultimately, we identified that SIAH1-mediated degradation of USP19 reversed USP19-mediated deubiquitination of MAVS, Beclin1, TRAF3, and TRIF, resulting in the activation of antiviral immune responses. Taken together, these findings provide new insights into the molecular mechanism of USP19 and SIAH1, and suggest a critical role of SIAH1 in antiviral immune response and homeostasis.

A Detailed Protocol for Constructing a Human Single-Chain Variable Fragment (scFv) Library and Downstream Screening via Phage Display.

The development of monoclonal antibodies (mAbs) represents a significant milestone in both basic research and clinical applications due to their target specificity and versatility in therapeutic and diagnostic applications. The innovative strategy of mAb screening, utilizing phage display, facilitates the in vitro screening of antibodies with high affinity to target antigens. The single-chain variable fragment (scFv) is a subset of mAb derivatives, known for its high binding affinity and smaller size-just one-third of that of human IgG. This report outlines a detailed and comprehensive procedure for constructing a scFv phagemid library derived from human patients, followed by screening via phage display affinity selection. The protocol utilizes 348 primer combinations spanning the entire human antibody repertoire to minimize sequence bias and maintain library diversity during polymerase chain reaction (PCR) for scFv generation, resulting in a library size greater than 1 × 108. Furthermore, we describe a high-throughput phage display screening protocol using enzyme-linked immunosorbent assay (ELISA) to evaluate more than 1200 scFv candidates. The generation of a highly diverse scFv library, coupled with the implementation of a phage display screening methodology, is expected to provide a valuable resource for researchers in pursuit of scFvs with high affinity for target antigens, thus advancing both research and clinical endeavors.

TXNIP-mediated crosstalk between oxidative stress and glucose metabolism.

Thioredoxin-interacting protein (TXNIP) has emerged as a key player in cancer and diabetes since it targets thioredoxin (TRX)-mediated redox regulation and glucose transporter (GLUT)-mediated metabolism. TXNIP consists of two arrestin (ARR, N-ARR and C-ARR) domains at its amino-terminus and two PPxY (PY) motifs and a di-leucine (LL) motif for endocytosis at its carboxyl-terminus. Here, we report that TXNIP shuffles between TRX and GLUTs to regulate homeostasis of intracellular oxidative stress and glucose metabolism. While TXNIP functions as a gatekeeper of TRX by default, it robustly interacted with class I GLUTs through its C-ARR domain upon increase of intracellular reactive oxygen species. This interaction prompted the surface expression downregulation and lysosomal degradation of GLUTs by its carboxyl-terminal LL endocytic signaling motif to attenuate glucose uptake. Consequently, TXNIP expression significantly limited glucose uptake, leading to the suppression of glycolysis, hexosamine biosynthesis, and the pentose phosphate pathway. Our findings establish a fundamental link between ROS and glucose metabolism through TXNIP and provide a promising target for the drug development against GLUT-related metabolic disorders.

Current Progress of Severe Fever with Thrombocytopenia Syndrome Virus (SFTSV) Vaccine Development.

SFTSV is an emerging tick-borne virus causing hemorrhagic fever with a case fatality rate (CFR) that can reach up to 27%. With endemic infection in East Asia and the recent spread of the vector tick to more than 20 states in the United States, the SFTSV outbreak is a globally growing public health concern. However, there is currently no targeted antiviral therapy or licensed vaccine against SFTSV. Considering the age-dependent SFTS pathogenesis and disease outcome, a sophisticated vaccine development approach is required to safeguard the elderly population from lethal SFTSV infection. Given the recent emergence of SFTSV, the establishment of animal models to study immunogenicity and protection from SFTS symptoms has only occurred recently. The latest research efforts have applied diverse vaccine development approaches-including live-attenuated vaccine, DNA vaccine, whole inactivated virus vaccine, viral vector vaccine, protein subunit vaccine, and mRNA vaccine-in the quest to develop a safe and effective vaccine against SFTSV. This review aims to outline the current progress in SFTSV vaccine development and suggest future directions to enhance the safety and efficacy of these vaccines, ensuring their suitability for clinical application.

Respiratory distress in SARS-CoV-2 exposed uninfected neonates followed in the COVID Outcomes in Mother-Infant Pairs (COMP) Study.

Respiratory distress (RD) has been reported in SARS-CoV-2 exposed uninfected (SEU) term neonates. Prior studies suggest that prenatal exposure to Coronavirus Disease 19 (COVID-19) may activate an inflammatory cascade in the newborn airway. In this study, we examine the relationship between maternal COVID-19 vaccination and neonatal RD using a longitudinal cohort of mother-infant pairs in Los Angeles, CA. Two-hundred and twenty-one mothers with laboratory confirmed SARS-CoV-2 during pregnancy and 227 exposed fetuses are enrolled in our study. Maternal disease severity and neonatal RD variables were defined based on current accepted clinical criteria. To explore the multifactorial associations between maternal COVID-19 parameters and infant RD, we utilize a multivariable logistic regression model and a proteomic sub-analysis to propose a pathway for the development of RD following in utero exposure to SARS-CoV-2. Unusually high rates of RD are observed in SEU infants (17%). The odds ratio of RD is 3.06 (95% CI:1.08-10.21) in term neonates born to unvaccinated individuals versus those born to individuals vaccinated prior to maternal infection. Proteomic analysis reveals a robust inflammatory response associated with ciliary dysregulation and enhanced IgE production among SEU neonates with RD. Maternal vaccination against COVID-19 reduces the frequency of neonatal RD.

Virology-the path forward.

In the United States (US), biosafety and biosecurity oversight of research on viruses is being reappraised. Safety in virology research is paramount and oversight frameworks should be reviewed periodically. Changes should be made with care, however, to avoid impeding science that is essential for rapidly reducing and responding to pandemic threats as well as addressing more common challenges caused by infectious diseases. Decades of research uniquely positioned the US to be able to respond to the COVID-19 crisis with astounding speed, delivering life-saving vaccines within a year of identifying the virus. We should embolden and empower this strength, which is a vital part of protecting the health, economy, and security of US citizens. Herein, we offer our perspectives on priorities for revised rules governing virology research in the US.

Gadd45ß is critical for regulation of type I interferon signaling by facilitating G3BP-mediated stress granule formation.

Stress granules (SGs) constitute a signaling hub that plays a critical role in type I interferon responses. Here, we report that growth arrest and DNA damage-inducible beta (Gadd45ß) act as a positive regulator of SG-mediated interferon signaling by targeting G3BP upon RNA virus infection. Gadd45ß deficiency markedly impairs SG formation and SG-mediated activation of interferon signaling in vitro. Gadd45ß knockout mice are highly susceptible to RNA virus infection, and their ability to produce interferon and cytokines is severely impaired. Specifically, Gadd45ß interacts with the RNA-binding domain of G3BP, leading to conformational expansion of G3BP1 via dissolution of its autoinhibitory electrostatic intramolecular interaction. The acidic loop 1- and RNA-binding properties of Gadd45ß markedly increase the conformational expansion and RNA-binding affinity of the G3BP1-Gadd45ß complex, thereby promoting assembly of SGs. These findings suggest a role for Gadd45ß as a component and critical regulator of G3BP1-mediated SG formation, which facilitates RLR-mediated interferon signaling.

SFTSV Gn-Head mRNA vaccine confers efficient protection against lethal viral challenge.

Severe fever with thrombocytopenia syndrome virus (SFTSV) is an emerging tick-borne virus, causing thrombocytopenia and hemorrhagic fever, with a fatality rate ranging from 12% to 30%. SFTSV possesses Gn and Gc glycoproteins, which are responsible for host cell receptor attachment and membrane fusion, respectively, to infect host cells. We have previously reported a protein subunit vaccine candidate (sGn-H-FT) of the SFTSV soluble Gn head region (sGn-H) fused with self-assembling ferritin (FT) nanoparticles, displaying strong protective immunogenicity. In this study, we present messenger RNA (mRNA) vaccine candidates encoding sGn-H or sGn-H-FT, both of which exhibit potent in vivo immunogenicity and protection capacity. Mice immunized with either sGn-H or sGn-H-FT mRNA lipid nanoparticle (LNP) vaccine produced strong total antibodies and neutralizing antibodies (NAbs) against sGn-H. Importantly, NAb titers remained high for an extended period. Finally, mice immunized with sGn-H or sGn-H-FT mRNA LNP vaccine were fully protected from a lethal dose of SFTSV challenge, showing no fatality. These findings underscore the promise of sGn-H and sGn-H-FT as vaccine antigen candidates capable of providing protective immunity against SFTSV infection.

Viral codon optimization on SARS-CoV-2 Spike boosts immunity in the development of COVID-19 mRNA vaccines.

Life-long persistent herpesviruses carry "trans-inducers" to overcome the unusual codon usage of their glycoproteins for efficient expression. Strikingly, this "trans-inducibility" can be achieved by simply changing the codon-usage of acute virus glycoproteins to that of persistent herpesvirus glycoproteins with herpesviral trans-inducer. Here, we apply the "persistent viral codon-usage-trans-inducer" principle to SARS-CoV-2 Spike mRNA vaccine platform, in which the codon-usage of Spike is changed to that of Herpes Simplex Virus-1 (HSV-1) glycoprotein B (gB) with its "trans-inducer" ICP27. The HSVgB-ICP27-codon-optimized Spike mRNA vaccine induced markedly high antigen expression and stability, total IgG, neutralizing antibody, and T cell response, ultimately enhancing protection against lethal SARS-CoV-2 challenge. Moreover, the HSVgB- codon-optimized Delta (B.1.617.2) strain Spike mRNA vaccine provided significant enhancements in antigen expression and long-term protection against SARS-CoV-2 challenge. Thus, we report a novel persistent viral codon-usage-trans-inducer mRNA vaccine platform for enhanced antigen expression and long-term protection against lethal viral infection.

Self-assembling Gn head ferritin nanoparticle vaccine provides full protection from lethal challenge of Dabie bandavirus in aged ferrets.

IMPORTANCE: Dabie bandavirus (DBV) is an emerging tick-borne virus that causes severe fever with thrombocytopenia syndrome (SFTS) in infected patients. Human SFTS symptoms progress from fever, fatigue, and muscle pain to the depletion of white blood cells and platelets with fatality rates up to 30%. The recent spread of its vector tick to over 20 states in the United States increases the potential for outbreaks of the SFTS beyond the East Asia. Thus, the development of vaccine to control this rapidly emerging virus is a high priority. In this study, we applied self-assembling ferritin (FT) nanoparticle to enhance the immunogenicity of DBV Gn head domain (GnH) as a vaccine target. Mice immunized with the GnH-FT nanoparticle vaccine induced potent antibody responses and cellular immunity. Immunized aged ferrets were fully protected from the lethal challenge of DBV. Our study describes the GnH-FT nanoparticle vaccine candidate that provides protective immunity against the emerging DBV infection.

SARS-CoV-2 variants with NSP12 P323L/G671S mutations display enhanced virus replication in ferret upper airways and higher transmissibility.

With the emergence of multiple predominant SARS-CoV-2 variants, it becomes important to have a comprehensive assessment of their viral fitness and transmissibility. Here, we demonstrate that natural temperature differences between the upper (33°C) and lower (37°C) respiratory tract have profound effects on SARS-CoV-2 replication and transmissibility. Specifically, SARS-CoV-2 variants containing the NSP12 mutations P323L or P323L/G671S exhibit enhanced RNA-dependent RNA polymerase (RdRp) activity at 33°C compared with 37°C and high transmissibility. Molecular dynamics simulations and microscale thermophoresis demonstrate that the NSP12 P323L and P323L/G671S mutations stabilize the NSP12-NSP7-NSP8 complex through hydrophobic effects, leading to increased viral RdRp activity. Furthermore, competitive transmissibility assay reveals that reverse genetic (RG)-P323L or RG-P323L/G671S NSP12 outcompetes RG-WT (wild-type) NSP12 for replication in the upper respiratory tract, allowing markedly rapid transmissibility. This suggests that NSP12 P323L or P323L/G671S mutation of SARS-CoV-2 is associated with increased RdRp complex stability and enzymatic activity, promoting efficient transmissibility.

Analogs of the Catechol Derivative Dynasore Inhibit HIV-1 Ribonuclease H, SARS-CoV-2 nsp14 Exoribonuclease, and Virus Replication.

Viral replication often depends on RNA maturation and degradation processes catalyzed by viral ribonucleases, which are therefore candidate targets for antiviral drugs. Here, we synthesized and studied the antiviral properties of a novel nitrocatechol compound (1c) and other analogs that are structurally related to the catechol derivative dynasore. Interestingly, compound 1c strongly inhibited two DEDD box viral ribonucleases, HIV-1 RNase H and SARS-CoV-2 nsp14 3'-to-5' exoribonuclease (ExoN). While 1c inhibited SARS-CoV-2 ExoN activity, it did not interfere with the mRNA methyltransferase activity of nsp14. In silico molecular docking placed compound 1c in the catalytic pocket of the ExoN domain of nsp14. Finally, 1c inhibited SARS-CoV-2 replication but had no toxicity to human lung adenocarcinoma cells. Given its simple chemical synthesis from easily available starting materials, these results suggest that 1c might be a lead compound for the design of new antiviral compounds that target coronavirus nsp14 ExoN and other viral ribonucleases.

Self-assembling Gn head ferritin nanoparticle vaccine provides full protection from lethal challenge of Dabie Bandavirus in aged ferrets.

Dabie Bandavirus (DBV), previously known as Severe Fever with Thrombocytopenia Syndrome (SFTS) Virus, induces a characteristic thrombocytopenia with a mortality rate ranging from 12% to as high as 30%. The sero-prevalence of DBV in healthy people is not significantly different among age groups, but clinically diagnosed SFTS patients are older than ~50 years, suggesting that age is the critical risk factor for SFTS morbidity and mortality. Accordingly, our immune-competent ferret model demonstrates an age (>4 years old)-dependent DBV infection and pathogenesis that fully recapitulates human clinical manifestation. To protect the aged population from DBV-induced SFTS, vaccine should carry robust immunogenicity with high safety profile. Previous studies have shown that glycoproteins Gn/Gc are the most effective antigens for inducing both neutralizing antibody (NAb)- and T cell-mediated immunity and, thereby, protection. Here, we report the development of a protein subunit vaccine with 24-mer self-assembling ferritin (FT) nanoparticle to present DBV Gn head region (GnH) for enhanced immunogenicity. Anion exchange chromatography and size exclusion chromatography readily purified the GnH-FT nanoparticles to homogeneity with structural integrity. Mice immunized with GnH-FT nanoparticles induced robust NAb response and T-cell immunity against DBV Gn. Furthermore, aged ferrets immunized with GnH-FT nanoparticles were fully protected from DBV challenge without SFTS symptoms such as body weight loss, thrombocytopenia, leukopenia, and fatality. This study demonstrates that DBV GnH-FT nanoparticles provide an efficient vaccine efficacy in mouse and aged ferret models and should be an outstanding vaccine candidate targeted for the aged population against fatal DBV infection.

Noncovalent antibody catenation on a target surface greatly increases the antigen-binding avidity.

Immunoglobulin G (IgG) antibodies are widely used for diagnosis and therapy. Given the unique dimeric structure of IgG, we hypothesized that, by genetically fusing a homodimeric protein (catenator) to the C-terminus of IgG, reversible catenation of antibody molecules could be induced on a surface where target antigen molecules are abundant, and that it could be an effective way to greatly enhance the antigen-binding avidity. A thermodynamic simulation showed that quite low homodimerization affinity of a catenator, e.g. dissociation constant of 100 µM, can enhance nanomolar antigen-binding avidity to a picomolar level, and that the fold enhancement sharply depends on the density of the antigen. In a proof-of-concept experiment where antigen molecules are immobilized on a biosensor tip, the C-terminal fusion of a pair of weakly homodimerizing proteins to three different antibodies enhanced the antigen-binding avidity by at least 110 or 304 folds from the intrinsic binding avidity. Compared with the mother antibody, Obinutuzumab(Y101L) which targets CD20, the same antibody with fused catenators exhibited significantly enhanced binding to SU-DHL5 cells. Together, the homodimerization-induced antibody catenation would be a new powerful approach to improve antibody applications, including the detection of scarce biomarkers and targeted anticancer therapies.

Lung-specific MCEMP1 functions as an adaptor for KIT to promote SCF-mediated mast cell proliferation.

Lung mast cells are important in host defense, and excessive proliferation or activation of these cells can cause chronic inflammatory disorders like asthma. Two parallel pathways induced by KIT-stem cell factor (SCF) and FcεRI-immunoglobulin E interactions are critical for the proliferation and activation of mast cells, respectively. Here, we report that mast cell-expressed membrane protein1 (MCEMP1), a lung-specific surface protein, functions as an adaptor for KIT, which promotes SCF-mediated mast cell proliferation. MCEMP1 elicits intracellular signaling through its cytoplasmic immunoreceptor tyrosine-based activation motif and forms a complex with KIT to enhance its autophosphorylation and activation. Consequently, MCEMP1 deficiency impairs SCF-induced peritoneal mast cell proliferation in vitro and lung mast cell expansion in vivo. Mcemp1-deficient mice exhibit reduced airway inflammation and lung impairment in chronic asthma mouse models. This study shows lung-specific MCEMP1 as an adaptor for KIT to facilitate SCF-mediated mast cell proliferation.

Glucosylceramide is essential for Heartland and Dabie bandavirus glycoprotein-induced membrane fusion.

Due to climate changes, there has been a large expansion of emerging tick-borne zoonotic viruses, including Heartland bandavirus (HRTV) and Dabie bandavirus (DBV). As etiologic agents of hemorrhagic fever with high fatality, HRTV and DBV have been recognized as dangerous viral pathogens that likely cause future wide epidemics. Despite serious health concerns, the mechanisms underlying viral infection are largely unknown. HRTV and DBV Gn and Gc are viral surface glycoproteins required for early entry events during infection. Glycosphingolipids, including galactosylceramide (GalCer), glucosylceramide (GlcCer) and lactosylceramide (LacCer), are a class of membrane lipids that play essential roles in membrane structure and viral lifecycle. Here, our genome-wide CRISPR/Cas9 knockout screen identifies that glycosphingolipid biosynthesis pathway is essential for HRTV and DBV infection. The deficiency of UDP-glucose ceramide glucosyltransferase (UGCG) that produces GlcCer resulted in the loss of infectivity of recombinant viruses pseudotyped with HRTV or DBV Gn/Gc glycoproteins. Conversely, exogenous supplement of GlcCer, but not GalCer or LacCer, recovered viral entry of UGCG-deficient cells in a dose-dependent manner. Biophysical analyses showed that GlcCer targeted the lipid-head-group binding pocket of Gc to form a stable protein-lipid complex, which allowed the insertion of Gc protein into host lysosomal membrane lipid bilayers for viral fusion. Mutagenesis showed that D841 residue at the Gc lipid binding pocket was critical for GlcCer interaction and thereby, viral entry. These findings reveal detailed mechanism of GlcCer glycosphingolipid in HRTV and DBV Gc-mediated membrane fusion and provide a potential therapeutic target for tickborne virus infection.

LIN28 and histone H3K4 methylase induce TLR4 to generate tumor-initiating stem-like cells.

Chemoresistance and plasticity of tumor-initiating stem-like cells (TICs) promote tumor recurrence and metastasis. The gut-originating endotoxin-TLR4-NANOG oncogenic axis is responsible for the genesis of TICs. This study investigated mechanisms as to how TICs arise through transcriptional, epigenetic, and post-transcriptional activation of oncogenic TLR4 pathways. Here, we expressed constitutively active TLR4 (caTLR4) in mice carrying pLAP-tTA or pAlb-tTA, under a tetracycline withdrawal-inducible system. Liver progenitor cell induction accelerated liver tumor development in caTLR4-expressing mice. Lentiviral shRNA library screening identified histone H3K4 methylase SETD7 as central to activation of TLR4. SETD7 combined with hypoxia induced TLR4 through HIF2 and NOTCH. LIN28 post-transcriptionally stabilized TLR4 mRNA via de-repression of let-7 microRNA. These results supported a LIN28-TLR4 pathway for the development of HCCs in a hypoxic microenvironment. These findings not only advance our understanding of molecular mechanisms responsible for TIC generation in HCC, but also represent new therapeutic targets for the treatment of HCC.