Lipid nanoparticle composition for adjuvant formulation modulates disease after influenza virus infection in quadrivalent influenza vaccine vaccinated mice.

There are considerable avenues through which currently licensed influenza vaccines could be optimized. We tested influenza vaccination in a mouse model with two adjuvants: Sendai virus-derived defective interfering (SDI) RNA, a RIG-I agonist; and an amphiphilic imidazoquinoline (IMDQ-PEG-Chol), a TLR7/8 agonist. The negatively charged SDI RNA was formulated into lipid nanoparticles (LNPs) facilitating direct delivery of SDI RNA to the cytosol, where RIG-I sensing induces inflammatory and type I interferon responses. We previously tested SDI RNA and IMDQ-PEG-Chol as standalone and combination adjuvants for influenza and SARS-CoV-2 vaccines. Here, we tested two different ionizable lipids, K-Ac7-Dsa and S-Ac7-Dog, for LNP formulations. The LNPs were incorporated with SDI RNA to determine its potential as a combination adjuvant with IMDQ-PEG-Chol by evaluating the host immune response to vaccination and infection in immunized BALB/c mice. Adjuvanticity of IMDQ-PEG-Chol with and without empty or SDI-loaded LNPs was validated with quadrivalent inactivated influenza vaccine (QIV), showing robust induction of antibody titers and T-cell responses. Depending on the adjuvant combination and LNP formulation, humoral and cellular vaccine responses could be tailored towards type 1 or type 2 host responses with specific cytokine profiles that correlated with the protective responses to viral infection. The extent of protection conferred by different vaccine/LNP/adjuvant combinations was tested by challenging mice with a vaccine-matched strain of influenza A virus A/Singapore/gp1908/2015 IVR-180 (H1N1). Groups that received either LNP formulated with SDI or IMDQ-PEG-Chol, or both, showed very low levels of viral replication in their lungs at 5 days post-infection (DPI). These studies provide evidence that the combination of vaccines with LNPs and/or adjuvants promote antigen-specific cellular responses that can contribute to protection upon infection. Interestingly, we observed differences in humoral and cellular responses to vaccination between different groups receiving K-Ac7-Dsa or S-Ac7-Dog lipids in LNP formulations. The differences were also reflected in inflammatory responses in lungs of vaccinated animals to infection, depending on LNP formulations. Therefore, this study suggests that the composition of the LNPs, particularly the ionizable lipid, plays an important role in inducing inflammatory responses in vivo, which is important for vaccine safety and to prevent adverse effects upon viral exposure.

ZNF205 positively regulates RLR antiviral signaling by targeting RIG-I.

Retinoic acid-inducible gene I (RIG-I) is a cytosolic viral RNA receptor. Upon viral infection, the protein recognizes and then recruits adapter protein mitochondrial antiviral signaling (MAVS) protein, initiating the production of interferons and proinflammatory cytokines to establish an antiviral state. In the present study, we identify zinc finger protein 205 (ZNF205) which associates with RIG-I and promotes the Sendai virus (SeV)-induced antiviral innate immune response. Overexpression of ZNF205 facilitates interferon-beta (IFN-ß) introduction, whereas ZNF205 deficiency restricts its introduction. Mechanistically, the C-terminal zinc finger domain of ZNF205 interacts with the N-terminal tandem caspase recruitment domains (CARDs) of RIG-I; this interaction markedly promotes K63 ubiquitin-linked polyubiquitination of RIG-I, which is crucial for RIG-I activation. Thus, our results demonstrate that ZNF205 is a positive regulator of the RIG-I-mediated innate antiviral immune signaling pathway.

SARS-CoV-2 NSP5 and N protein counteract the RIG-I signaling pathway by suppressing the formation of stress granules.

As a highly pathogenic human coronavirus, SARS-CoV-2 has to counteract an intricate network of antiviral host responses to establish infection and spread. The nucleic acid-induced stress response is an essential component of antiviral defense and is closely related to antiviral innate immunity. However, whether SARS-CoV-2 regulates the stress response pathway to achieve immune evasion remains elusive. In this study, SARS-CoV-2 NSP5 and N protein were found to attenuate antiviral stress granule (avSG) formation. Moreover, NSP5 and N suppressed IFN expression induced by infection of Sendai virus or transfection of a synthetic mimic of dsRNA, poly (I:C), inhibiting TBK1 and IRF3 phosphorylation, and restraining the nuclear translocalization of IRF3. Furthermore, HEK293T cells with ectopic expression of NSP5 or N protein were less resistant to vesicular stomatitis virus infection. Mechanistically, NSP5 suppressed avSG formation and disrupted RIG-I-MAVS complex to attenuate the RIG-I-mediated antiviral immunity. In contrast to the multiple targets of NSP5, the N protein specifically targeted cofactors upstream of RIG-I. The N protein interacted with G3BP1 to prevent avSG formation and to keep the cofactors G3BP1 and PACT from activating RIG-I. Additionally, the N protein also affected the recognition of dsRNA by RIG-I. This study revealed the intimate correlation between SARS-CoV-2, the stress response, and innate antiviral immunity, shedding light on the pathogenic mechanism of COVID-19.

Atopic Neutrophils Prevent Postviral Airway Disease.

Respiratory syncytial virus (RSV) infection in infancy is associated with increased risk of asthma, except in those with allergic disease at the time of infection. Using house dust mite allergen, we examined the effect of pre-existing atopy on postviral airway disease using Sendai virus in mice, which models RSV infection in humans. Sendai virus drives postviral airway disease in nonatopic mice; however, pre-existing atopy protected against the development of airway disease. This protection depended upon neutrophils, as depletion of neutrophils at the time of infection restored the susceptibility of atopic mice to postviral airway disease. Associated with development of atopy was an increase in polymorphonuclear neutrophil-dendritic cell hybrid cells that develop in Th2 conditions and demonstrated increased viral uptake. Systemic inhibition of IL-4 reversed atopic protection against postviral airway disease, suggesting that increased virus uptake by neutrophils was IL-4 dependent. Finally, human neutrophils from atopic donors were able to reduce RSV infection of human airway epithelial cells in vitro, suggesting these findings could apply to the human. Collectively our data support the idea that pre-existing atopy derives a protective neutrophil response via potential interaction with IL-4, preventing development of postviral airway disease.

Type II alveolar cell MHCII improves respiratory viral disease outcomes while exhibiting limited antigen presentation.

Type II alveolar cells (AT2s) are critical for basic respiratory homeostasis and tissue repair after lung injury. Prior studies indicate that AT2s also express major histocompatibility complex class II (MHCII) molecules, but how MHCII expression by AT2s is regulated and how it contributes to host defense remain unclear. Here we show that AT2s express high levels of MHCII independent of conventional inflammatory stimuli, and that selective loss of MHCII from AT2s in mice results in modest worsening of respiratory virus disease following influenza and Sendai virus infections. We also find that AT2s exhibit MHCII presentation capacity that is substantially limited compared to professional antigen presenting cells. The combination of constitutive MHCII expression and restrained antigen presentation may position AT2s to contribute to lung adaptive immune responses in a measured fashion, without over-amplifying damaging inflammation.

USP18 positively regulates innate antiviral immunity by promoting K63-linked polyubiquitination of MAVS.

Activation of MAVS, an adaptor molecule in Rig-I-like receptor (RLR) signaling, is indispensable for antiviral immunity, yet the molecular mechanisms modulating MAVS activation are not completely understood. Ubiquitination has a central function in regulating the activity of MAVS. Here, we demonstrate that a mitochondria-localized deubiquitinase USP18 specifically interacts with MAVS, promotes K63-linked polyubiquitination and subsequent aggregation of MAVS. USP18 upregulates the expression and production of type I interferon following infection with Sendai virus (SeV) or Encephalomyocarditis virus (EMCV). Mice with a deficiency of USP18 are more susceptible to RNA virus infection. USP18 functions as a scaffold protein to facilitate the re-localization of TRIM31 and enhances the interaction between TRIM31 and MAVS in mitochondria. Our results indicate that USP18 functions as a post-translational modulator of MAVS-mediated antiviral signaling.

HSPBP1 facilitates cellular RLR-mediated antiviral response by inhibiting the K48-linked ubiquitination of RIG-I.

Retinoic acid-inducible gene I (RIG-I) plays a critical role in the recognition of intracytoplasmic viral RNA. Upon binding to the RNA of invading viruses, the activated RIG-I translocates to mitochondria, where it recruits adapter protein MAVS, causing a series of signaling cascades. In this study, we demonstrated that Hsp70 binding protein 1 (HSPBP1) promotes RIG-I-mediated signal transduction. The overexpression of HSPBP1 can increase the stability of RIG-I protein by inhibiting its K48-linked ubiquitination, and promote the activation of IRF3 and the production of IFN-ß induced by Sendai virus. Knockdown and knockout of HSPBP1 leads to down-regulation of virus-induced RIG-I expression, inhibits IRF3 activation, and reduces the production of IFNB1. These results indicate that HSPBP1 positively regulates the antiviral signal pathway induced by inhibiting the K48-linked ubiquitination of RIG-I.

The BRCA1 Pseudogene Negatively Regulates Antitumor Responses through Inhibition of Innate Immune Defense Mechanisms.

Innate immune defense mechanisms play a pivotal role in antitumor responses. Recent evidence suggests that antiviral innate immunity is regulated not only by exogenous non-self-RNA but also by host-derived pseudogene RNAs. A growing body of evidence also indicates a biological role for pseudogenes as gene expression regulators or immune modulators. Here, we report an important role for BRCA1P1, the pseudogene of the BRCA1 tumor-suppressor gene, in regulating innate immune defense mechanisms in breast cancer cells. BRCA1P1 expresses a long-noncoding RNA (lncRNA) in breast cancer cells through divergent transcription. Expression of lncRNA-BRCA1P1 is increased in breast tumors compared with normal breast tissues. Depletion of BRCA1P1 induces an antiviral defense-like program, including the expression of antiviral genes in breast cancer cells. Furthermore, BRCA1P1-deficient cancer cells mimic virus-infected cells by stimulating cytokines and inducing cell apoptosis. Accordingly, depletion of BRCA1P1 increases host innate immune responses and restricts virus replication. In converse, overexpression of BRCA1P1 reduces cytokine expression in breast cancer cells. Mechanistically, lncRNA-BRCA1P1 is localized in the nucleus, binds to the NF-κB subunit RelA, and negatively regulates antiviral gene expression. Finally, in a xenograft mouse model of breast cancer, depletion of BRCA1P1 stimulates cytokine expression and local immunity, and suppresses tumor growth. Our results suggest an important role for BRCA1P1 in innate immune defense mechanisms and antitumor responses. This mechanism of antiviral immunity regulated by a host-derived pseudogene RNA may guide the development of novel therapies targeting immune responses in breast cancer. SIGNIFICANCE: This study identifies a novel mechanism of innate immunity driven by a host pseudogene RNA that inhibits innate immune defense mechanisms and antitumor responses through regulation of antiviral gene expression.

CD8 T Cells Show Protection against Highly Pathogenic Simian Immunodeficiency Virus (SIV) after Vaccination with SIV Gene-Expressing BCG Prime and Vaccinia Virus/Sendai Virus Vector Boosts.

Toward development of a dual vaccine for human immunodeficiency virus type 1 (HIV-1) and tuberculosis infections, we developed a urease-deficient bacillus Calmette-Guérin (BCG) strain Tokyo172 (BCGΔurease) to enhance its immunogenicity. BCGΔurease expressing a simian immunodeficiency virus (SIV) Gag induced BCG antigen-specific CD4+ and CD8+ T cells more efficiently and more Gag-specific CD8+ T cells. We evaluated its protective efficacy against SIV infection in cynomolgus monkeys of Asian origin, shown to be as susceptible to infection with SIVmac251 as Indian rhesus macaques. Priming with recombinant BCG (rBCG) expressing SIV genes was followed by a boost with SIV gene-expressing LC16m8Δ vaccinia virus and a second boost with SIV Env-expressing Sendai virus. Eight weeks after the second boost, monkeys were repeatedly challenged with a low dose of SIVmac251 intrarectally. Two animals out of 6 vaccinees were protected, whereas all 7 control animals were infected without any early viral controls. In one vaccinated animal, which had the most potent CD8+ T cells in an in vitro suppression activity (ISA) assay of SIVmac239 replication, plasma viremia was undetectable throughout the follow-up period. Protection was confirmed by the lack of anamnestic antibody responses and detectable cell-associated provirus in various organs. Another monkey with a high ISA acquired a small amount of SIV, but it later became suppressed below the detection limit. Moreover, the ISA score correlated with SIV acquisition. On the other hand, any parameter relating anti-Env antibody was not correlated with the protection.IMPORTANCE Because both AIDS and tuberculosis are serious health threats in middle/low-income countries, development of a dual vaccine against them would be highly beneficial. To approach the goal, here we first assessed a urease-deficient bacillus Calmette-Guérin (BCG) for improvement of immunogenicity against both Mycobacterium tuberculosis and SIV. Second, we demonstrated the usefulness of Asian-origin cynomolgus monkeys for development of a preclinical AIDS vaccine by direct comparison with Indian rhesus macaques as the only validated hosts that identically mirror the outcomes of clinical trials, since the availability of Indian rhesus macaques is limited in countries other than the United States. Finally, we report the protective effect of a vaccination regimen comprising BCG, the highly attenuated vaccinia virus LC16m8Δ strain, and nontransmissible Sendai virus as safe vectors expressing SIV genes using repeated mucosal challenge with highly pathogenic SIVmac251. Identification of CD8+ T cells as a protective immunity suggests a future direction of AIDS vaccine development.

Hemagglutinating virus of Japan-envelope containing programmed cell death-ligand 1 siRNA inhibits immunosuppressive activities and elicits antitumor immune responses in glioma.

The programmed cell death-1/programmed cell death-ligand 1 (PD-1/PD-L1) pathway is involved in preventing immune system-mediated destruction of malignant tumors including glioblastoma. However, the therapeutic influence of PD-1/PD-L1 inhibition alone in glioblastoma is limited. To develop effective combination therapy involving PD-1/PD-L1 inhibition, we used a non-replicating virus-derived vector, hemagglutinating virus of Japan-envelope (HVJ-E), to inhibit tumor cell PD-L1 expression by delivering siRNA targeting PD-L1. HVJ-E is a promising vector for efficient delivery of enclosed substances to the target cells. Moreover, HVJ-E provokes robust antitumoral immunity by activating natural killer (NK) cells and cytotoxic T lymphocytes (CTLs), and by suppressing regulatory T lymphocytes (Treg). We hypothesized that we could efficiently deliver PD-L1-inhibiting siRNAs to tumor cells using HVJ-E, and that synergistic activation of antitumoral immunity would occur due to the immunostimulating effects of HVJ-E and PD-1/PD-L1 inhibition. We used artificially induced murine glioma stem-like cells, TS, to create mouse (C57BL/6N) glioblastoma models. Intratumoral injection of HVJ-E containing siRNA targeting PD-L1 (siPDL1/HVJ-E) suppressed the expression of tumor cell PD-L1 and significantly suppressed tumor growth in subcutaneous models and prolonged overall survival in brain tumor models. Flow cytometric analyses of brain tumor models showed that the proportions of brain-infiltrating CTL and NK cells were significantly increased after giving siPDL1/HVJ-E; in contrast, the rate of Treg/CD4+ cells was significantly decreased in HVJ-E-treated tumors. CD8 depletion abrogated the therapeutic effect of siPDL1/HVJ-E, indicating that CD8+ T lymphocytes mainly mediated this therapeutic effect. We believe that this non-replicating immunovirotherapy may be a novel therapeutic alternative to treat patients with glioblastoma.

ß-arrestin 2 as an activator of cGAS-STING signaling and target of viral immune evasion.

Virus infection may induce excessive interferon (IFN) responses that can lead to host tissue injury or even death. ß-arrestin 2 regulates multiple cellular events through the G protein-coupled receptor (GPCR) signaling pathways. Here we demonstrate that ß-arrestin 2 also promotes virus-induced production of IFN-ß and clearance of viruses in macrophages. ß-arrestin 2 interacts with cyclic GMP-AMP synthase (cGAS) and increases the binding of dsDNA to cGAS to enhance cyclic GMP-AMP (cGAMP) production and the downstream stimulator of interferon genes (STING) and innate immune responses. Mechanistically, deacetylation of ß-arrestin 2 at Lys171 facilitates the activation of the cGAS-STING signaling and the production of IFN-ß. In vitro, viral infection induces the degradation of ß-arrestin 2 to facilitate immune evasion, while a ß-blocker, carvedilol, rescues ß-arrestin 2 expression to maintain the antiviral immune response. Our results thus identify a viral immune-evasion pathway via the degradation of ß-arrestin 2, and also hint that carvedilol, approved for treating heart failure, can potentially be repurposed as an antiviral drug candidate.

The Ubiquitin E3 Ligase Parkin Inhibits Innate Antiviral Immunity Through K48-Linked Polyubiquitination of RIG-I and MDA5.

Innate immunity is the first-line defense against antiviral or antimicrobial infection. RIG-I and MDA5, which mediate the recognition of pathogen-derived nucleic acids, are essential for production of type I interferons (IFN). Here, we identified mitochondrion depolarization inducer carbonyl cyanide 3-chlorophenylhydrazone (CCCP) inhibited the response and antiviral activity of type I IFN during viral infection. Furthermore, we found that the PTEN-induced putative kinase 1 (PINK1) and the E3 ubiquitin-protein ligase Parkin mediated mitophagy, thus negatively regulating the activation of RIG-I and MDA5. Parkin directly interacted with and catalyzed the K48-linked polyubiquitination and subsequent degradation of RIG-I and MDA5. Thus, we demonstrate that Parkin limits RLR-triggered innate immunity activation, suggesting Parkin as a potential therapeutic target for the control of viral infection.

Immune Modulation to Improve Survival of Viral Pneumonia in Mice.

Viral pneumonias remain global health threats, as exemplified in the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic, requiring novel treatment strategies both early and late in the disease process. We have reported that mice treated before or soon after infection with a combination of inhaled Toll-like receptor (TLR) 2/6 and 9 agonists (Pam2-ODN) are broadly protected against microbial pathogens including respiratory viruses, but the mechanisms remain incompletely understood. The objective of this study was to validate strategies for immune modulation in a preclinical model of viral pneumonia and determine their mechanisms. Mice were challenged with the Sendai paramyxovirus in the presence or absence of Pam2-ODN treatment. Virus burden and host immune responses were assessed to elucidate Pam2-ODN mechanisms of action and to identify additional opportunities for therapeutic intervention. Enhanced survival of Sendai virus pneumonia with Pam2-ODN treatment was associated with reductions in lung virus burden and with virus inactivation before internalization. We noted that mortality in sham-treated mice corresponded with CD8+ T-cell lung inflammation on days 11-12 after virus challenge, after the viral burden had declined. Pam2-ODN blocked this injurious inflammation by minimizing virus burden. As an alternative intervention, depleting CD8+ T cells 8 days after viral challenge also decreased mortality. Stimulation of local innate immunity within the lungs by TLR agonists early in disease or suppression of adaptive immunity by systemic CD8+ T-cell depletion late in disease improves outcomes of viral pneumonia in mice. These data reveal opportunities for targeted immunomodulation to protect susceptible human subjects.

Porcine deltacoronavirus (PDCoV) infection antagonizes interferon-λ1 production.

Porcine deltacoronavirus (PDCoV) is a novel swine enteropathogenic coronavirus that causes watery diarrhea, vomiting and mortality in nursing piglets. Type III interferons (IFN-λs) are the major antiviral cytokines in intestinal epithelial cells, the target cells in vivo for PDCoV. In this study, we found that PDCoV infection remarkably inhibited Sendai virus-induced IFN-λ1 production by suppressing transcription factors IRF and NF-κB in IPI-2I cells, a line of porcine intestinal mucosal epithelial cells. We also confirmed that PDCoV infection impeded the activation of IFN-λ1 promoter stimulated by RIG-I, MDA5 and MAVS, but not by TBK1 and IRF1. Although the expression levels of IRF1 and MAVS were not changed, PDCoV infection resulted in reduction of the number of peroxisomes, the platform for MAVS to activate IRF1, and subsequent type III IFN production. Taken together, our study demonstrates that PDCoV suppresses type III IFN responses to circumvent the host's antiviral immunity.

Impaired HVJ-stimulated Interferon producing capacity in MPO-ANCA-associated vasculitis with rapidly progressive glomerulonephritis lead to susceptibility to infection.

ANCA-associated RPGN leads to renal failure through systemic vasculitis and diffuse crescentic glomerulonephritis. MPO-ANCA-RPGN patients are highly susceptible to infections. Our aim in this study was to uncover reasons why these patients were susceptible to infections. We analyzed various aspects of type I interferon system including HVJ-stimulated IFN-α producing capacity and plasmacytoid dendritic cell (pDC) number in whole blood in MPO-ANCA-RPGN patients. Compared with healthy subjects, MPO-ANCA-RPGN patients showed impaired HVJ-stimulated IFN-α producing capacity and lower pDC number with or without glucocorticoid treatment. Immuno-histological staining of MPO-ANCA-RPGN kidney samples revealed a few but apparent pDC in T cell infiltrating regions even in patients with low pDC number in their peripheral blood. Patients' low HVJ-stimulated IFN-α producing capacity and pDC numbers persisted even after patients underwent several years of treatment. Former infection was determined using patients' serum BPI, Lamp-2 and Calprotectin, since they are reflective of a history of infection. These markers were higher in MPO-ANCA-RPGN patients than in healthy subjects. These results indicate that impaired HVJ-stimulated IFN-α production as well as dysfunction of the IFN system might have resulted from a previous bout of infection and can be partially implicated in patients' long-term susceptibility and vulnerability to infection.

Porcine Deltacoronavirus Accessory Protein NS7a Antagonizes IFN-ß Production by Competing With TRAF3 and IRF3 for Binding to IKKε.

As an emerging swine enteropathogenic coronavirus, porcine deltacoronavirus (PDCoV) not only causes serious diarrhea in suckling piglets but also possesses the potential for cross-species transmission, which has sparked growing interest when studying this emerging virus. We previously identified a novel accessory protein NS7a encoded by PDCoV; however, the function of NS7a was not resolved. In this study, we demonstrated that PDCoV NS7a is an interferon antagonist. Overexpression of NS7a notably inhibited Sendai virus (SeV)-induced interferon-ß (IFN-ß) production and the activation of IRF3 rather than NF-κB. NS7a also inhibited IFN-ß promoter activity induced by RIG-I, MDA5, MAVS, TBK1, and IKKε, which are key components of the RIG-I-like receptor (RLR) signaling pathway but not IRF3, the transcription factor downstream of TBK1/IKKε. Surprisingly, NS7a specifically interacts with IKKε but not with the closely related TBK1. Furthermore, NS7a interacts simultaneously with the kinase domain (KD) and the scaffold dimerization domain (SDD) of IKKε, competing with TRAF3, and IRF3 for binding to IKKε, leading to the reduction of RLR-mediated IFN-ß production. The interactions of TRAF3-IKKε and IKKε-IRF3 are also attenuated in PDCoV-infected cells. Taken together, our results demonstrate that PDCoV NS7a inhibits IFN-ß production by disrupting the association of IKKε with both TRAF3 and IRF3, revealing a new mechanism utilized by a PDCoV accessory protein to evade the host antiviral innate immune response.

The ORF6, ORF8 and nucleocapsid proteins of SARS-CoV-2 inhibit type I interferon signaling pathway.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a novel human coronavirus causing the pandemic of severe pneumonia (Coronavirus Disease 2019, COVID-19). SARS-CoV-2 is highly pathogenic in human, having posed immeasurable public health challenges to the world. Innate immune response is critical for the host defense against viral infection and the dysregulation of the host innate immune responses probably aggravates SARS-CoV-2 infection, contributing to the high morbidity and lethality of COVID-19. It has been reported that some coronavirus proteins play an important role in modulating innate immunity of the host, but few studies have been conducted on SARS-CoV-2. In this study, we screened the viral proteins of SARS-CoV-2 and found that the viral ORF6, ORF8 and nucleocapsid proteins were potential inhibitors of type I interferon signaling pathway, a key component for antiviral response of host innate immune. All the three proteins showed strong inhibition on type I interferon (IFN-ß) and NF-κB-responsive promoter, further examination revealed that these proteins were able to inhibit the interferon-stimulated response element (ISRE) after infection with Sendai virus, while only ORF6 and ORF8 proteins were able to inhibit the ISRE after treatment with interferon beta. These findings would be helpful for the further study of the detailed signaling pathway and unveil the key molecular player that may be targeted.

SUMOylation of DDX39A Alters Binding and Export of Antiviral Transcripts to Control Innate Immunity.

The RNA helicase DDX39A plays an important role in the RNA splicing/export process. In our study, human DDX39A facilitated RNA virus escape from innate immunity to promote virus proliferation by trapping TRAF3, TRAF6, and MAVS mRNAs in the HEK293T cell nucleus. DDX39A was a target for SUMOylation. SUMO1, 2, and 3 modifications were found on immunoprecipitated DDX39A. However, only the SUMO1 modification decreased in vesicular stomatitis virus-infected HEK293T cells. Further studies have found that viral infection reduced SUMO1 modification of DDX39A and enhanced its ability to bind innate immunity-associated mRNAs by regulating the abundance of RanBP2 with SUMO1 E3 ligase activity. RanBP2 acted as an E3 SUMO ligase of DDX39A, which enhanced SUMO1 modification of DDX39A and attenuated its ability to bind RNA. This work described that specific mRNAs encoding antiviral signaling components were bound and sequestered in the nucleus by DDX39A to limit their expression, which proposed a new protein SUMOylation model to regulate innate immunity in viral infection.

Intratumoral and s.c. injection of inactivated hemagglutinating virus of Japan envelope (GEN0101) in metastatic castration-resistant prostate cancer.

Inactivated hemagglutinating virus of Japan envelope (HVJ-E) has an antitumor effect and tumor immunity. We undertook an open-label, phase I, dose-escalation study in patients with castration-resistant prostate cancer (CRPC) to determine the safety and efficacy of intratumoral and s.c. injection of HVJ-E (GEN0101). Patients with CRPC, who were resistant to or unable to receive standard of care, were included. GEN0101 was injected directly into the prostate and s.c. in two 28-day treatment cycles. The primary end-points were to evaluate the safety and tolerability of GEN0101 and determine its recommended dose. The secondary end-points were to analyze the antitumor effect and tumor immunity. Three patients received 30 000 mNAU GEN0101 and 6 received 60 000 mNAU. There was no dose-limiting toxicity, and the recommended dose of GEN0101 was defined as 60 000 mNAU. Radiographically, 1 patient had stable disease and 2 had progressive disease in the low-dose group, whereas 5 patients had stable disease and 1 had progressive disease in the high-dose group. Three patients in the high-dose group showed reduction in lymph node metastasis. Prostate-specific antigen increase rates in the high-dose group were suppressed more than those in the low-dose group. Natural killer cell activity was enhanced in 2 patients of the low-dose group and in 5 patients in the high-dose group. In conclusion, intratumoral and s.c. injections of GEN0101 were well-tolerated and feasible to use. The study is registered with the UMIN Clinical Trials Registry (no. UMIN000017092).