Dengue Virus Targets Nrf2 for NS2B3-Mediated Degradation Leading to Enhanced Oxidative Stress and Viral Replication.

Dengue virus (DENV) is a mosquito-borne virus that infects upward of 300 million people annually and has the potential to cause fatal hemorrhagic fever and shock. While the parameters contributing to dengue immunopathogenesis remain unclear, the collapse of redox homeostasis and the damage induced by oxidative stress have been correlated with the development of inflammation and progression toward the more severe forms of disease. In the present study, we demonstrate that the accumulation of reactive oxygen species (ROS) late after DENV infection (>24 hpi) resulted from a disruption in the balance between oxidative stress and the nuclear factor erythroid 2-related factor 2 (Nrf2)-dependent antioxidant response. The DENV NS2B3 protease complex strategically targeted Nrf2 for degradation in a proteolysis-independent manner; NS2B3 licensed Nrf2 for lysosomal degradation. Impairment of the Nrf2 regulator by the NS2B3 complex inhibited the antioxidant gene network and contributed to the progressive increase in ROS levels, along with increased virus replication and inflammatory or apoptotic gene expression. By 24 hpi, when increased levels of ROS and antiviral proteins were observed, it appeared that the proviral effect of ROS overcame the antiviral effects of the interferon (IFN) response. Overall, these studies demonstrate that DENV infection disrupts the regulatory interplay between DENV-induced stress responses, Nrf2 antioxidant signaling, and the host antiviral immune response, thus exacerbating oxidative stress and inflammation in DENV infection.IMPORTANCE Dengue virus (DENV) is a mosquito-borne pathogen that threatens 2.5 billion people in more than 100 countries annually. Dengue infection induces a spectrum of clinical symptoms, ranging from classical dengue fever to severe dengue hemorrhagic fever or dengue shock syndrome; however, the complexities of DENV immunopathogenesis remain controversial. Previous studies have reported the importance of the transcription factor Nrf2 in the control of redox homeostasis and antiviral/inflammatory or death responses to DENV. Importantly, the production of reactive oxygen species and the subsequent stress response have been linked to the development of inflammation and progression toward the more severe forms of the disease. Here, we demonstrate that DENV uses the NS2B3 protease complex to strategically target Nrf2 for degradation, leading to a progressive increase in oxidative stress, inflammation, and cell death in infected cells. This study underlines the pivotal role of the Nrf2 regulatory network in the context of DENV infection.

SIRT1 Modulates the Sensitivity of Prostate Cancer Cells to Vesicular Stomatitis Virus Oncolysis.

Oncolytic virotherapy represents a promising experimental anticancer strategy, based on the use of genetically modified viruses to selectively infect and kill cancer cells. Vesicular stomatitis virus (VSV) is a prototypic oncolytic virus (OV) that induces cancer cell death through activation of the apoptotic pathway, although intrinsic resistance to oncolysis is found in some cell lines and many primary tumors, as a consequence of residual innate immunity to the virus. In the effort to improve OV therapeutic efficacy, we previously demonstrated that different agents, including histone deacetylase inhibitors (HDIs), functioned as reversible chemical switches to dampen the innate antiviral response and improve the susceptibility of resistant cancer cells to VSV infection. In the present study, we demonstrated that the NAD+-dependent histone deacetylase SIRT1 (silent mating type information regulation 2 homolog 1) plays a key role in the permissivity of prostate cancer PC-3 cells to VSVΔM51 replication and oncolysis. HDI-mediated enhancement of VSVΔM51 infection and cancer cell killing directly correlated with a decrease of SIRT1 expression. Furthermore, pharmacological inhibition as well as silencing of SIRT1 by small interfering RNA (siRNA) was sufficient to sensitize PC-3 cells to VSVΔM51 infection, resulting in augmentation of virus replication and spread. Mechanistically, HDIs such as suberoylanilide hydroxamic acid (SAHA; Vorinostat) and resminostat upregulated the microRNA miR-34a that regulated the level of SIRT1. Taken together, our findings identify SIRT1 as a viral restriction factor that limits VSVΔM51 infection and oncolysis in prostate cancer cells.IMPORTANCE The use of nonpathogenic viruses to target and kill cancer cells is a promising strategy in cancer therapy. However, many types of human cancer are resistant to the oncolytic (cancer-killing) effects of virotherapy. In this study, we identify a host cellular protein, SIRT1, that contributes to the sensitivity of prostate cancer cells to infection by a prototypical oncolytic virus. Knockout of SIRT1 activity increases the sensitivity of prostate cancer cells to virus-mediated killing. At the molecular level, SIRT1 is controlled by a small microRNA termed miR-34a. Altogether, SIRT1 and/or miR-34a levels may serve as predictors of response to oncolytic-virus therapy.

Activation of Latent HIV-1 T Cell Reservoirs with a Combination of Innate Immune and Epigenetic Regulators.

The presence of T cell reservoirs in which human immunodeficiency virus (HIV) establishes latency by integrating into the host genome represents a major obstacle to an HIV cure and has prompted the development of strategies aimed at the eradication of HIV from latently infected cells. The "shock-and-kill" strategy is one of the most pursued approaches to the elimination of viral reservoirs. Although several latency-reversing agents (LRAs) have shown promising reactivation activity, they have failed to eliminate the cellular reservoir. In this study, we evaluated a novel immune system-mediated approach to clearing the HIV reservoir, based on a combination of innate immune stimulation and epigenetic reprogramming. The combination of the STING agonist cGAMP (cyclic GMP-AMP) and the FDA-approved histone deacetylase inhibitor resminostat resulted in a significant increase in HIV proviral reactivation and specific apoptosis in HIV-infected cells in vitro Reductions in the proportion of HIV-harboring cells and the total amount of HIV DNA were also observed in CD4+ central memory T (TCM) cells, a primary cell model of latency, where resminostat alone or together with cGAMP induced high levels of selective cell death. Finally, high levels of cell-associated HIV RNA were detected ex vivo in peripheral blood mononuclear cells (PBMCs) and CD4+ T cells from individuals on suppressive antiretroviral therapy (ART). Although synergism was not detected in PBMCs with the combination, viral RNA expression was significantly increased in CD4+ T cells. Collectively, these results represent a promising step toward HIV eradication by demonstrating the potential of innate immune activation and epigenetic modulation for reducing the viral reservoir and inducing specific death of HIV-infected cells.IMPORTANCE One of the challenges associated with HIV-1 infection is that despite antiretroviral therapies that reduce HIV-1 loads to undetectable levels, proviral DNA remains dormant in a subpopulation of T lymphocytes. Numerous strategies to clear residual virus by reactivating latent virus and eliminating the reservoir of HIV-1 (so-called "shock-and-kill" strategies) have been proposed. In the present study, we use a combination of small molecules that activate the cGAS-STING antiviral innate immune response (the di-cyclic nucleotide cGAMP) and epigenetic modulators (histone deacetylase inhibitors) that induce reactivation and HIV-infected T cell killing in cell lines, primary T lymphocytes, and patient samples. These studies represent a novel strategy for HIV eradication by reducing the viral reservoir and inducing specific death of HIV-infected cells.

Alternate NF-κB-Independent Signaling Reactivation of Latent HIV-1 Provirus.

Current combination antiretroviral therapies (cART) are unable to eradicate HIV-1 from infected individuals because of the establishment of proviral latency in long-lived cellular reservoirs. The shock-and-kill approach aims to reactivate viral replication from the latent state (shock) using latency-reversing agents (LRAs), followed by the elimination of reactivated virus-producing cells (kill) by specific therapeutics. The NF-κB RelA/p50 heterodimer has been characterized as an essential component of reactivation of the latent HIV-1 long terminal repeat (LTR). Nevertheless, prolonged NF-κB activation contributes to the development of various autoimmune, inflammatory, and malignant disorders. In the present study, we established a cellular model of HIV-1 latency in J-Lat CD4+ T cells that stably expressed the NF-κB superrepressor IκB-α 2NΔ4 and demonstrate that conventional treatments with bryostatin-1 and hexamethylenebisacetamide (HMBA) or ionomycin synergistically reactivated HIV-1 from latency, even under conditions where NF-κB activation was repressed. Using specific calcineurin phosphatase, p38, and MEK1/MEK2 kinase inhibitors or specific short hairpin RNAs, c-Jun was identified to be an essential factor binding to the LTR enhancer κB sites and mediating the combined synergistic reactivation effect. Furthermore, acetylsalicylic acid (ASA), a potent inhibitor of the NF-κB activator kinase IκB kinase ß (IKK-ß), did not significantly diminish reactivation in a primary CD4+ T central memory (TCM) cell latency model. The present work demonstrates that the shock phase of the shock-and-kill approach to reverse HIV-1 latency may be achieved in the absence of NF-κB, with the potential to avoid unwanted autoimmune- and or inflammation-related side effects associated with latency-reversing strategies.IMPORTANCE The shock-and-kill approach consists of the reactivation of HIV-1 replication from latency using latency-reversing agents (LRAs), followed by the elimination of reactivated virus-producing cells. The cellular transcription factor NF-κB is considered a master mediator of HIV-1 escape from latency induced by LRAs. Nevertheless, a systemic activation of NF-κB in HIV-1-infected patients resulting from the combined administration of different LRAs could represent a potential risk, especially in the case of a prolonged treatment. We demonstrate here that conventional treatments with bryostatin-1 and hexamethylenebisacetamide (HMBA) or ionomycin synergistically reactivate HIV-1 from latency, even under conditions where NF-κB activation is repressed. Our study provides a molecular proof of concept for the use of anti-inflammatory drugs, like aspirin, capable of inhibiting NF-κB in patients under combination antiretroviral therapy during the shock-and-kill approach, to avoid potential autoimmune and inflammatory disorders that can be elicited by combinations of LRAs.

An optimized retinoic acid-inducible gene I agonist M8 induces immunogenic cell death markers in human cancer cells and dendritic cell activation.

RIG-I is a cytosolic RNA sensor that recognizes short 5' triphosphate RNA, commonly generated during virus infection. Upon activation, RIG-I initiates antiviral immunity, and in some circumstances, induces cell death. Because of this dual capacity, RIG-I has emerged as a promising target for cancer immunotherapy. Previously, a sequence-optimized RIG-I agonist (termed M8) was generated and shown to stimulate a robust immune response capable of blocking viral infection and to function as an adjuvant in vaccination strategies. Here, we investigated the potential of M8 as an anti-cancer agent by analyzing its ability to induce cell death and activate the immune response. In multiple cancer cell lines, M8 treatment strongly activated caspase 3-dependent apoptosis, that relied on an intrinsic NOXA and PUMA-driven pathway that was dependent on IFN-I signaling. Additionally, cell death induced by M8 was characterized by the expression of markers of immunogenic cell death-related damage-associated molecular patterns (ICD-DAMP)-calreticulin, HMGB1 and ATP-and high levels of ICD-related cytokines CXCL10, IFNß, CCL2 and CXCL1. Moreover, M8 increased the levels of HLA-ABC expression on the tumor cell surface, as well as up-regulation of genes involved in antigen processing and presentation. M8 induction of the RIG-I pathway in cancer cells favored dendritic cell phagocytosis and induction of co-stimulatory molecules CD80 and CD86, together with increased expression of IL12 and CXCL10. Altogether, these results highlight the potential of M8 in cancer immunotherapy, with the capacity to induce ICD-DAMP on tumor cells and activate immunostimulatory signals that synergize with current therapies.

The Coronavirus pandemic - 2022: Viruses, variants & vaccines.

Since the beginning of the COVID-19 pandemic in 2019-2020, Cytokine & Growth Factor Reviews has published several Special Issues focused on the biology, pathogenesis and therapeutic options in the treatment of COVID-19 infection, including articles on the involvement of the chemokine system in the cytokine storm in COVID-19, intervention in the early stages of COVID-19 pneumonia, the therapeutic value of corticosteroid treatment, early clinical intervention with type 1 interferons, progress in vaccine development, and organ specific complications of COVID-19. By 2022, multiple highly efficacious vaccines are available and are being administered in countries around the world, therapeutic options have been clinically evaluated and approved, and SARS-CoV-2 has arguably become the most thoroughly studied virus in history. But, with progress has also come unanticipated problems - misinformation, anti-vaxxers, opposition to protective masks, and politically motivated interference disguised as knowledge. With this issue of CGFR, we continue to document the global coronavirus pandemic and provide an update on the emergence of viral variants, the global effort to administer vaccines and the impediments to progress posed by misinformation and anti-vaccine sentiment.

Virus-like particle - mediated delivery of the RIG-I agonist M8 induces a type I interferon response and protects cells against viral infection.

Virus-Like Particles (VLPs) are nanostructures that share conformation and self-assembly properties with viruses, but lack a viral genome and therefore the infectious capacity. In this study, we produced VLPs by co-expression of VSV glycoprotein (VSV-G) and HIV structural proteins (Gag, Pol) that incorporated a strong sequence-optimized 5'ppp-RNA RIG-I agonist, termed M8. Treatment of target cells with VLPs-M8 generated an antiviral state that conferred resistance against multiple viruses. Interestingly, treatment with VLPs-M8 also elicited a therapeutic effect by inhibiting ongoing viral replication in previously infected cells. Finally, the expression of SARS-CoV-2 Spike glycoprotein on the VLP surface retargeted VLPs to ACE2 expressing cells, thus selectively blocking viral infection in permissive cells. These results highlight the potential of VLPs-M8 as a therapeutic and prophylactic vaccine platform. Overall, these observations indicate that the modification of VLP surface glycoproteins and the incorporation of nucleic acids or therapeutic drugs, will permit modulation of particle tropism, direct specific innate and adaptive immune responses in target tissues, and boost immunogenicity while minimizing off-target effects.

Inhibition of Glycolysis Impairs Retinoic Acid-Inducible Gene I-Mediated Antiviral Responses in Primary Human Dendritic Cells.

Dendritic cells (DCs) are important mediators of the induction and regulation of adaptive immune responses following microbial infection and inflammation. Sensing environmental danger signals including viruses, microbial products, or inflammatory stimuli by DCs leads to the rapid transition from a resting state to an activated mature state. DC maturation involves enhanced capturing and processing of antigens for presentation by major histocompatibility complex (MHC) class I and class II, upregulation of chemokines and their receptors, cytokines and costimulatory molecules, and migration to lymphoid tissues where they prime naive T cells. Orchestrating a cellular response to environmental threats requires a high bioenergetic cost that accompanies the metabolic reprogramming of DCs during activation. We previously demonstrated that DCs undergo a striking functional transition after stimulation of the retinoic acid-inducible gene I (RIG-I) pathway with a synthetic 5' triphosphate containing RNA (termed M8), consisting of the upregulation of interferon (IFN)-stimulated antiviral genes, increased DC phagocytosis, activation of a proinflammatory phenotype, and induction of markers associated with immunogenic cell death. In the present study, we set out to determine the metabolic changes associated with RIG-I stimulation by M8. The rate of glycolysis in primary human DCs was increased in response to RIG-I activation, and glycolytic reprogramming was an essential requirement for DC activation. Pharmacological inhibition of glycolysis in monocyte-derived dendritic cells (MoDCs) impaired type I IFN induction and signaling by disrupting the TBK1-IRF3-STAT1 axis, thereby countering the antiviral activity induced by M8. Functionally, the impaired IFN response resulted in enhanced viral replication of dengue, coronavirus 229E, and Coxsackie B5.

Type I Interferons in COVID-19 Pathogenesis.

Among the many activities attributed to the type I interferon (IFN) multigene family, their roles as mediators of the antiviral immune response have emerged as important components of the host response to Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection. Viruses likewise have evolved multiple immune evasion strategies to circumvent the host immune response and promote virus propagation and dissemination. Therefore, a thorough characterization of host-virus interactions is essential to understand SARS-CoV-2 pathogenesis. Here, we summarize the virus-mediated evasion of the IFN responses and the viral functions involved, the genetic basis of IFN production in SARS-CoV-2 infection and the progress of clinical trials designed to utilize type I IFN as a potential therapeutic tool.

Fighting HIV-1 Persistence: At the Crossroads of "Shoc-K and B-Lock".

Despite the success of highly active antiretroviral therapy (HAART), integrated HIV-1 proviral DNA cannot be eradicated from an infected individual. HAART is not able to eliminate latently infected cells that remain invisible to the immune system. Viral sanctuaries in specific tissues and immune-privileged sites may cause residual viral replication that contributes to HIV-1 persistence. The "Shock or Kick, and Kill" approach uses latency reversing agents (LRAs) in the presence of HAART, followed by cell-killing due to viral cytopathic effects and immune-mediated clearance. Different LRAs may be required for the in vivo reactivation of HIV-1 in different CD4+ T cell reservoirs, leading to the activation of cellular transcription factors acting on the integrated proviral HIV-1 LTR. An important requirement for LRA drugs is the reactivation of viral transcription and replication without causing a generalized immune activation. Toll-like receptors, RIG-I like receptors, and STING agonists have emerged recently as a new class of LRAs that augment selective apoptosis in reactivated T lymphocytes. The challenge is to extend in vitro observations to HIV-1 positive patients. Further studies are also needed to overcome the mechanisms that protect latently infected cells from reactivation and/or elimination by the immune system. The Block and Lock alternative strategy aims at using latency promoting/inducing agents (LPAs/LIAs) to block the ability of latent proviruses to reactivate transcription in order to achieve a long term lock down of potential residual virus replication. The Shock and Kill and the Block and Lock approaches may not be only alternative to each other, but, if combined together (one after the other), or given all at once [namely "Shoc-K(kill) and B(block)-Lock"], they may represent a better approach to a functional cure.

Dolutegravir-Based Regimen for Maintenance of Viral Suppression in People Living with HIV: 48-Week Results in Real-Life Setting.

To evaluate the efficacy, safety, and tolerability of switching to a dolutegravir (DTG)-based regimen in a cohort of virological suppressed HIV-infected patients who have previously been treated with different antiretroviral combination. The dynamics of total HIV-DNA and levels of high-sensitivity c-reactive protein, interleukin-6, soluble-CD14, and D-Dimer were also analyzed. Ninety-six individuals who switched to a DTG-containing regimen were followed up for 48 weeks. HIV RNA, CD4+ T cell count, weight, and levels of laboratory parameters were recorded at baseline, after 24 and 48 weeks of treatment for all study participants. In a subgroup of patients, HIV DNA and inflammation/coagulation marker levels were also analyzed until week 24. Ninety-three out of 96 patients maintained virological suppression, including patients who switched to dual-therapy from triple-drug combination. Eighteen out of 96 patients had residual viremia at baseline, of which 13 reached the maximal viral suppression at W48. Serum creatinine levels showed a significant increase at weeks 24 and 48. A progressive reduction of total cholesterol was observed from week 24 and up to week 48. No variation in body mass index was detected. HIV DNA, inflammation, and coagulation marker levels did not significantly change during follow-up. Switching to a DTG-based regimen may be a key option for achieving and maintaining maximal virological suppression, even in patients showing residual viremia at baseline. Furthermore, the improvement in blood lipid profile and the overall tolerability observed in this study strongly support the use of these regimens in the aging HIV population.

Antiviral Potential of the Antimicrobial Drug Atovaquone against SARS-CoV-2 and Emerging Variants of Concern.

The antimicrobial medication malarone (atovaquone/proguanil) is used as a fixed-dose combination for treating children and adults with uncomplicated malaria or as chemoprophylaxis for preventing malaria in travelers. It is an inexpensive, efficacious, and safe drug frequently prescribed around the world. Following anecdotal evidence from 17 patients in the provinces of Quebec and Ontario, Canada, suggesting that malarone/atovaquone may present some benefits in protecting against COVID-19, we sought to examine its antiviral potential in limiting the replication of SARS-CoV-2 in cellular models of infection. In VeroE6 expressing human TMPRSS2 and human lung Calu-3 epithelial cells, we show that the active compound atovaquone at micromolar concentrations potently inhibits the replication of SARS-CoV-2 and other variants of concern including the alpha, beta, and delta variants. Importantly, atovaquone retained its full antiviral activity in a primary human airway epithelium cell culture model. Mechanistically, we demonstrate that the atovaquone antiviral activity against SARS-CoV-2 is partially dependent on the expression of TMPRSS2 and that the drug can disrupt the interaction of the spike protein with the viral receptor, ACE2. Additionally, spike-mediated membrane fusion was also reduced in the presence of atovaquone. In the United States, two clinical trials of atovaquone administered alone or in combination with azithromycin were initiated in 2020. While we await the results of these trials, our findings in cellular infection models demonstrate that atovaquone is a potent antiviral FDA-approved drug against SARS-CoV-2 and other variants of concern in vitro.

The global impact of the coronavirus pandemic.

The coronavirus pandemic has engulfed the nations of the world for the first five months of 2020 and altered the pace, fabric and nature of our lives. In this overview accompanying the Special Issue of Cytokine & Growth Factor Reviews, we examine some of the many social and scientific issues impacted by SARS-CoV2 - personal lives, economy, scientific communication, the environment. International members of Istituto Pasteur in Rome and INITIATE, the Marie Curie Training Network reflect on the lasting global impact of the coronavirus pandemic.

Dengue Virus Immunopathogenesis: Lessons Applicable to the Emergence of Zika Virus.

Dengue is the leading mosquito-transmitted viral infection in the world. There are more than 390 million new infections annually; while the majority of infected individuals are asymptomatic or develop a self-limited dengue fever, up to 1 million clinical cases develop severe manifestations, including dengue hemorrhagic fever and shock syndrome, resulting in ~25,000 deaths annually, mainly in children. Gaps in our understanding of the mechanisms that contribute to dengue infection and immunopathogenesis have hampered the development of vaccines and antiviral agents. Some of these limitations are highlighted by the explosive re-emergence of another arthropod-borne flavivirus-Zika virus-spread by the same vector, the Aedes aegypti mosquito, that also carries dengue, yellow fever and chikungunya viruses. This review will discuss the early virus-host interactions in dengue infection, with emphasis on the interrelationship between oxidative stress and innate immune pathways, and will provide insight as to how lessons learned from dengue research may expedite therapeutic strategies for Zika virus.