Identification of Anti-Influenza A Compounds Inhibiting the Viral Non-Structural Protein 1 (NS1) Using a Type I Interferon-Driven Screening Strategy.

There is an urgent need to identify efficient antiviral compounds to combat existing and emerging RNA virus infections, particularly those related to seasonal and pandemic influenza outbreaks. While inhibitors of the influenza viral integral membrane proton channel protein (M2), neuraminidase (NA), and cap-dependent endonuclease are available, circulating influenza viruses acquire resistance over time. Thus, the need for the development of additional anti-influenza drugs with novel mechanisms of action exists. In the present study, a cell-based screening assay and a small molecule library were used to screen for activities that antagonized influenza A non-structural protein 1 (NS1), a highly conserved, multifunctional accessory protein that inhibits the type I interferon response against influenza. Two potential anti-influenza agents, compounds 157 and 164, were identified with anti-NS1 activity, resulting in the reduction of A/PR/8/34(H1N1) influenza A virus replication and the restoration of IFN-ß expression in human lung epithelial A549 cells. A 3D pharmacophore modeling study of the active compounds provided a glimpse of the structural motifs that may contribute to anti-influenza virus activity. This screening approach is amenable to a broader analysis of small molecule compounds to inhibit other viral targets.

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.

Co-Infections and Superinfections between HIV-1 and Other Human Viruses at the Cellular Level.

Co-infection or superinfection of the host by two or more virus species is a common event, potentially leading to viral interference, viral synergy, or neutral interaction. The simultaneous presence of two or more viruses, even distantly related, within the same cell depends upon viral tropism, i.e., the entry of viruses via receptors present on the same cell type. Subsequently, productive infection depends on the ability of these viruses to replicate efficiently in the same cellular environment. HIV-1 initially targets CCR5-expressing tissue memory CD4+ T cells, and in the absence of early cART initiation, a co-receptor switch may occur, leading to the infection of naïve and memory CXCR4-expressing CD4+ T cells. HIV-1 infection of macrophages at the G1 stage of their cell cycle also occurs in vivo, broadening the possible occurrence of co-infections between HIV-1 and other viruses at the cellular level. Moreover, HIV-1-infected DCs can transfer the virus to CD4+ T cells via trans-infection. This review focuses on the description of reported co-infections within the same cell between HIV-1 and other human pathogenic, non-pathogenic, or low-pathogenic viruses, including HIV-2, HTLV, HSV, HHV-6/-7, GBV-C, Dengue, and Ebola viruses, also discussing the possible reciprocal interactions in terms of virus replication and virus pseudotyping.

A cellular screening platform, stably expressing DENV2 NS5, defines a novel anti-DENV mechanism of action of Apigenin based on STAT2 activation.

Type I interferon (IFN-I) evasion by Dengue virus (DENV) is key in DENV pathogenesis. The non-structural protein 5 (NS5) antagonizes IFN-I response through the degradation of the signal transducer and activator of transcription 2 (STAT2). We developed a K562 cell-based platform, for high throughput screening of compounds potentially counteracting the NS5-mediated antagonism of IFN-I signaling. Upon a screening with a library of 1220 approved drugs, 3 compounds previously linked to DENV inhibition (Apigenin, Chrysin, and Luteolin) were identified. Luteolin and Apigenin determined a significant inhibition of DENV2 replication in Huh7 cells and the restoration of STAT2 phosphorylation in both cell systems. Apigenin and Luteolin were able to stimulate STAT2 even in the absence of infection. Despite the "promiscuous" and "pan-assay-interfering" nature of Luteolin, Apigenin promotes STAT2 Tyr 689 phosphorylation and activation, highlighting the importance of screening for compounds able to interact with host factors, to counteract viral proteins capable of dampening innate immune responses.

New Monoclonal Antibodies Specific for Different Epitopes of the Spike Protein of SARS-CoV-2 and Its Major Variants: Additional Tools for a More Specific COVID-19 Diagnosis.

The emergence of the new pathogen SARS-CoV-2 determined a rapid need for monoclonal antibodies (mAbs) to detect the virus in biological fluids as a rapid tool to identify infected individuals to be treated or quarantined. The majority of commercially available antigenic tests for SARS-CoV-2 rely on the detection of N antigen in biologic fluid using anti-N antibodies, and their capacity to specifically identify subjects infected by SARS-CoV-2 is questionable due to several structural analogies among the N proteins of different coronaviruses. In order to produce new specific antibodies, BALB/c mice were immunized three times at 20-day intervals with a recombinant spike (S) protein. The procedure used was highly efficient, and 40 different specific mAbs were isolated, purified and characterized, with 13 ultimately being selected for their specificity and lack of cross reactivity with other human coronaviruses. The specific epitopes recognized by the selected mAbs were identified through a peptide library and/or by recombinant fragments of the S protein. In particular, the selected mAbs recognized different linear epitopes along the S1, excluding the receptor binding domain, and along the S2 subunits of the S protein of SARS-CoV-2 and its major variants of concern. We identified combinations of anti-S mAbs suitable for use in ELISA or rapid diagnostic tests, with the highest sensitivity and specificity coming from proof-of-concept tests using recombinant antigens, SARS-CoV-2 or biological fluids from infected individuals, that represent important additional tools for the diagnosis of COVID-19.

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.

Isolation and Characterization of Mouse Monoclonal Antibodies That Neutralize SARS-CoV-2 and Its Variants of Concern Alpha, Beta, Gamma and Delta by Binding Conformational Epitopes of Glycosylated RBD With High Potency.

Antibodies targeting Receptor Binding Domain (RBD) of SARS-CoV-2 have been suggested to account for the majority of neutralizing activity in COVID-19 convalescent sera and several neutralizing antibodies (nAbs) have been isolated, characterized and proposed as emergency therapeutics in the form of monoclonal antibodies (mAbs). However, SARS-CoV-2 variants are rapidly spreading worldwide from the sites of initial identification. The variants of concern (VOC) B.1.1.7 (Alpha), B.1.351 (Beta), P.1 (Gamma) and B.1.167.2 (Delta) showed mutations in the SARS-CoV-2 spike protein potentially able to cause escape from nAb responses with a consequent reduction of efficacy of vaccines and mAbs-based therapy. We produced the recombinant RBD (rRBD) of SARS-CoV-2 spike glycoprotein from the Wuhan-Hu 1 reference sequence in a mammalian system, for mice immunization to isolate new mAbs with neutralizing activity. Here we describe four mAbs that were able to bind the rRBD in Enzyme-Linked Immunosorbent Assay and the transmembrane full-length spike protein expressed in HEK293T cells by flow cytometry assay. Moreover, the mAbs recognized the RBD in supernatants of SARS-CoV-2 infected VERO E6 cells by Western Blot under non-reducing condition or in supernatants of cells infected with lentivirus pseudotyped for spike protein, by immunoprecipitation assay. Three out of four mAbs lost their binding efficiency to completely N-deglycosylated rRBD and none was able to bind the same recombinant protein expressed in Escherichia coli, suggesting that the epitopes recognized by three mAbs are generated by the conformational structure of the glycosylated native protein. Of particular relevance, three mAbs were able to inhibit Wuhan SARS-CoV-2 infection of VERO E6 cells in a plaque-reduction neutralization test and the Wuhan SARS-CoV-2 as well as the Alpha, Beta, Gamma and Delta VOC in a pseudoviruses-based neutralization test. These mAbs represent important additional tools for diagnosis and therapy of COVID-19 and may contribute to the understanding of the functional structure of SARS-CoV-2 RBD.

IκB kinase-ε-mediated phosphorylation triggers IRF-1 degradation in breast cancer cells.

Interferon Regulatory Factors (IRFs) are key regulators of immunity, cell survival and apoptosis. IRF transcriptional activity and subcellular localization are tightly regulated by posttranscriptional modifications including phosphorylation. The IκB kinase family member IKK-ε is essential in regulating antiviral innate immunity mediated by IRFs but is now also recognized as an oncoprotein amplified and overexpressed in breast cancer cell lines and patient-derived tumors. In the present study, we report that the tumor suppressor IRF-1 is a specific target of IKK-ε in breast cancer cells. IKK-ε-mediated phosphorylation of IRF-1 dramatically decreases IRF-1 protein stability, accelerating IRF-1 degradation and quenching IRF-1 transcriptional activity. Chemical inhibition of IKK-ε activity, fully restores IRF-1 levels and function and positively correlates with inhibition of cell growth and proliferation of breast cancer cells. By using a breast cancer cell line stably expressing a dominant negative version of IRF-1 we were able to demonstrate that IKK-ε preferentially exerts its oncogenic potential in breast cancer through the regulation of IRF-1 and point to the IKK-ε-mediated phosphorylation of IRF-1 as a therapeutic target to overcome IKK-ε-mediated tumorigenesis.

A model of the three-dimensional structure of human interferon responsive factor 1 and its modifications upon phosphorylation or phosphorylation-mimicking mutations.

Interferon responsive factor 1 (IRF-1) is a pleiotropic transcription factor, possessing non-redundant biological activities that depend on its interaction with different protein partners and multiple post-translational modifications including phosphorylation. In particular, a 5'-SXXXSXS-3' motif of the protein represents the target of the IκB-related kinases, TANK-binding kinase (TBK)-1 and inhibitor of nuclear factor kappa-B kinase (IKK)-ε. Here, a 3D model of human IRF-1 was determined by using multi-template comparative modeling and molecular dynamics approaches. Models obtained through either phosphorylation or aspartate mutation of residues 215, 219 and 221 were also calculated and compared to the wild type. Calculations indicated that each of these modifications mainly induces a rigidification of the protein structure and only slightly changes in electrostatics and hydrophobicity of IRF-1 surface, resulting in the impairment of the capacity of IRF-1 containing as partate mutations (S221D and S215D/S219D/S221D) to synergize with tumour necrosis factor (TNF)-α stimulation in inducing interferon (IFN) promoter-mediated reporter gene activation. Therefore, these changes are qualitatively correlated to the amount of negative charge located on the 215-221 segments of IRF-1 by phosphorylation or aspartate mutation. Hypotheses on the structural mechanism that governs the phosphorylation-related damping of IRF-1 activity were also drawn. Communicated by Ramaswamy H. Sarma.

IFN Regulatory Factors and Antiviral Innate Immunity: How Viruses Can Get Better.

The interferon regulatory factor (IRF) family consists of transcriptional regulators that exert multifaceted and versatile functions in multiple biological processes. Their crucial role as central mediators in the establishment and execution of host immunity in response to pathogen-derived signals downstream pattern recognition receptors (PRRs) makes IRFs a hallmark of the host antiviral response. They function as hub molecules at the crossroad of different signaling pathways for the induction of interferon (IFN) and inflammatory cytokines, as well as of antiviral and immunomodulatory genes even in an IFN-independent manner. By regulating the development and activity of immune cells, IRFs also function as a bridge between innate and adaptive responses. As such, IRFs represent attractive and compulsive targets in viral strategies to subvert antiviral signaling. In this study, we discuss current knowledge on the wide array of strategies put in place by pathogenic viruses to evade, subvert, and/or hijack these essential components of host antiviral immunity.

HIV-1 Tat Recruits HDM2 E3 Ligase To Target IRF-1 for Ubiquitination and Proteasomal Degradation.

In addition to its ability to regulate HIV-1 promoter activation, the viral transactivator Tat also functions as a determinant of pathogenesis and disease progression by directly and indirectly modulating the host anti-HIV response, largely through the capacity of Tat to interact with and modulate the activities of multiple host proteins. We previously demonstrated that Tat modulated both viral and host transcriptional machinery by interacting with the cellular transcription factor interferon regulatory factor 1 (IRF-1). In the present study, we investigated the mechanistic basis and functional significance of Tat-IRF-1 interaction and demonstrate that Tat dramatically decreased IRF-1 protein stability. To accomplish this, Tat exploited the cellular HDM2 (human double minute 2 protein) ubiquitin ligase to accelerate IRF-1 proteasome-mediated degradation, resulting in a quenching of IRF-1 transcriptional activity during HIV-1 infection. These data identify IRF-1 as a new target of Tat-induced modulation of the cellular protein machinery and reveal a new strategy developed by HIV-1 to evade host immune responses. IMPORTANCE: Current therapies have dramatically reduced morbidity and mortality associated with HIV infection and have converted infection from a fatal pathology to a chronic disease that is manageable via antiretroviral therapy. Nevertheless, HIV-1 infection remains a challenge, and the identification of useful cellular targets for therapeutic intervention remains a major goal. The cellular transcription factor IRF-1 impacts various physiological functions, including the immune response to viral infection. In this study, we have identified a unique mechanism by which HIV-1 evades IRF-1-mediated host immune responses and show that the viral protein Tat accelerates IRF-1 proteasome-mediated degradation and inactivates IRF-1 function. Restoration of IRF-1 functionality may thus be regarded as a potential strategy to reinstate both a direct antiviral response and a more broadly acting immune regulatory circuit.

Type I IFN--a blunt spear in fighting HIV-1 infection.

For more than 50 years, Type I Interferon (IFN) has been recognized as critical in controlling viral infections. IFN is produced downstream germ-line encoded pattern recognition receptors (PRRs) upon engagement by pathogen-associated molecular patterns (PAMPs). As a result, hundreds of different interferon-stimulated genes (ISGs) are rapidly induced, acting in both autocrine and paracrine manner to build a barrier against viral replication and spread. ISGs encode proteins with direct antiviral and immunomodulatory activities affecting both innate and adaptive immune responses. During infection with viruses, as HIV-1, that can establish a persistent infection, IFN although produced, is not able to block the initial infection and a chronic IFN-mediated immune activation/inflammation becomes a pathogenic mechanism of disease progression. This review will briefly summarize when and how IFN is produced during HIV-1 infection and the way this innate immune response is manipulated by the virus to its own advantage to drive chronic immune activation and progression to AIDS.

IκB kinase ε targets interferon regulatory factor 1 in activated T lymphocytes.

IκB kinase ε (IKK-ε) has an essential role as a regulator of innate immunity, functioning downstream of pattern recognition receptors to modulate NF-κB and interferon (IFN) signaling. In the present study, we investigated IKK-ε activation following T cell receptor (TCR)/CD28 stimulation of primary CD4(+) T cells and its role in the stimulation of a type I IFN response. IKK-ε was activated following TCR/CD28 stimulation of primary CD4(+) T cells; however, in T cells treated with poly(I·C), TCR/CD28 costimulation blocked induction of IFN-ß transcription. We demonstrated that IKK-ε phosphorylated the transcription factor IFN regulatory factor 1 (IRF-1) at amino acid (aa) 215/219/221 in primary CD4(+) T cells and blocked its transcriptional activity. At the mechanistic level, IRF-1 phosphorylation impaired the physical interaction between IRF-1 and the NF-κB RelA subunit and interfered with PCAF-mediated acetylation of NF-κB RelA. These results demonstrate that TCR/CD28 stimulation of primary T cells stimulates IKK-ε activation, which in turn contributes to suppression of IFN-ß production.