Inhibition of dengue virus replication by novel inhibitors of RNA-dependent RNA polymerase and protease activities

Dengue virus (DENV) is the leading mosquito-transmitted viral infection in the world. With more than 390 million new infections annually, and up to 1 million clinical cases with severe disease manifestations, there continues to be a need to develop new antiviral agents against dengue infection. In addition, there is no approved anti-DENV agents for treating DENV-infected patients. In the present study, we identified new compounds with anti-DENV replication activity by targeting viral replication enzymes – NS5, RNA-dependent RNA polymerase (RdRp) and NS3 protease, using cell-based reporter assay. Subsequently, we performed an enzyme-based assay to clarify the action of these compounds against DENV RdRp or NS3 protease activity. Moreover, these compounds exhibited anti-DENV activity in vivo in the ICR-suckling DENV-infected mouse model. Combination drug treatment exhibited a synergistic inhibition of DENV replication. These results describe novel prototypical small anti-DENV molecules for further development through compound modification and provide potential antivirals for treating DENV infection and DENV-related diseases

Microarray comparative genomic hybridisation analysis of intraocular uveal melanomas identifies distinctive imbalances associated with loss of chromosome 3

Defining regions of genomic imbalance can identify genes involved in tumour development. Conventional cytogenetics has identified several nonrandom copy number alterations (CNA) in uveal melanomas (UVM), which include monosomy 3, chromosome 6 abnormalities and gain of 8q. To gain further insight into the CNAs and define the regions involved more precisely we analysed 18 primary UVMs using 1 Mb BAC microarray comparative genomic hybridisation (CGH). Our analysis showed that the most common genomic imbalances were 8q gain (78%), 6p gain (67%) and monosomy 3 (56%). Two distinct CGH profiles could be delineated on the basis of the chromosome 3 status. The most common genetic changes in monosomy 3 tumours, in our study, were gain of 8q11.21–q24.3, 6p25.1–p21.2, 21q21.2–q21.3 and 21q22.13–q22.3 and loss of 1p36.33–p34.3, 1p31.1–p21.2, 6q16.2–q25.3 and 8p23.3–p11.23. In contrast, disomy 3 tumours showed recurrent gains of only 6p25.3–p22.3 and 8q23.2–q24.3. Our approach allowed definition of the smallest overlapping regions of imbalance, which may be important in the development of UVM

Human Bocavirus NS1 and NS1-70 Proteins Inhibit TNF-α-Mediated Activation of NF-κB by targeting p65.

Human bocavirus (HBoV), a parvovirus, is a single-stranded DNA etiologic agent causing lower respiratory tract infections in young children worldwide. Nuclear factor kappa B (NF-κB) transcription factors play crucial roles in clearance of invading viruses through activation of many physiological processes. Previous investigation showed that HBoV infection could significantly upregulate the level of TNF-α which is a strong NF-κB stimulator. Here we investigated whether HBoV proteins modulate TNF-α-mediated activation of the NF-κB signaling pathway. We showed that HBoV NS1 and NS1-70 proteins blocked NF-κB activation in response to TNF-α. Overexpression of TNF receptor-associated factor 2 (TRAF2)-, IκB kinase alpha (IKKα)-, IκB kinase beta (IKKβ)-, constitutively active mutant of IKKβ (IKKβ SS/EE)-, or p65-induced NF-κB activation was inhibited by NS1 and NS1-70. Furthermore, NS1 and NS1-70 didn't interfere with TNF-α-mediated IκBα phosphorylation and degradation, nor p65 nuclear translocation. Coimmunoprecipitation assays confirmed the interaction of both NS1 and NS1-70 with p65. Of note, NS1 but not NS1-70 inhibited TNF-α-mediated p65 phosphorylation at ser536. Our findings together indicate that HBoV NS1 and NS1-70 inhibit NF-κB activation. This is the first time that HBoV has been shown to inhibit NF-κB activation, revealing a potential immune-evasion mechanism that is likely important for HBoV pathogenesis

The RIG-I agonist M8 triggers cell death and natural killer cell activation in human papillomavirus-associated cancer and potentiates cisplatin cytotoxicity

Although the activation of innate immunity to treat a wide variety of cancers is gaining increasing attention, it has been poorly investigated in human papillomavirus (HPV)-associated malignancies. Because these tumors harbor a severely impaired cGAS-STING axis, but they still retain a largely functional RIG-I pathway, another critical mediator of adaptive and innate immune responses, we asked whether RIG-I activation by the 5'ppp-RNA RIG-I agonist M8 would represent a therapeutically viable option to treat HPV+ cancers. Here, we show that M8 transfection of two cervical carcinoma-derived cell lines, CaSki and HeLa, both expressing a functional RIG-I, triggers intrinsic apoptotic cell death, which is significantly reduced in RIG-I KO cells. We also demonstrate that M8 stimulation potentiates cisplatin-mediated cell killing of HPV+ cells in a RIG-I dependent manner. This combination treatment is equally effective in reducing tumor growth in a syngeneic pre-clinical mouse model of HPV16-driven cancer, where enhanced expression of lymphocyte-recruiting chemokines and cytokines correlated with an increased number of activated natural killer (NK) cells in the tumor microenvironment. Consistent with a role of RIG-I signaling in immunogenic cell killing, stimulation of NK cells with conditioned medium from M8-transfected CaSki boosted NK cell proliferation, activation, and migration in a RIG-I-dependent tumor cell-intrinsic manner. Given the highly conserved molecular mechanisms of carcinogenesis and genomic features of HPV-driven cancers and the remarkably improved prognosis for HPV+ oropharyngeal cancer, targeting RIG-I may represent an effective immunotherapeutic strategy in this setting, favoring the development of de-escalating strategies

Global analyses revealed age-related alterations in innate immune responses after stimulation of pathogen recognition receptors

Aging leads to dysregulation of multiple components of the immune system that results in increased susceptibility to infections and poor response to vaccines in the aging population. The dysfunctions of adaptive B and T cells are well documented, but the effect of aging on innate immunity remains incompletely understood. Using a heterogeneous population of peripheral blood mononuclear cells (PBMCs), we first undertook transcriptional profiling and found that PBMCs isolated from old individuals (≥ 65 years) exhibited a delayed and altered response to stimulation with TLR4, TLR7/8, and RIG-I agonists compared to cells obtained from adults (≤ 40 years). This delayed response to innate immune agonists resulted in the reduced production of pro-inflammatory and antiviral cytokines and chemokines including TNFα, IL-6, IL-1β, IFNα, IFNγ, CCL2, and CCL7. While the major monocyte and dendritic cell subsets did not change numerically with aging, activation of specific cell types was altered. PBMCs from old subjects also had a lower frequency of CD40+ monocytes, impaired up-regulation of PD-L1 on monocytes and T cells, and increased expression of PD-L2 and B7-H4 on B cells. The defective immune response to innate agonists adversely affected adaptive immunity as TLR-stimulated PBMCs (minus CD3 T cells) from old subjects elicited significantly lower levels of adult T-cell proliferation than those from adult subjects in an allogeneic mixed lymphocyte reaction (MLR). Collectively, these age-associated changes in cytokine, chemokine and interferon production, as well as co-stimulatory protein expression could contribute to the blunted memory B- and T-cell immune responses to vaccines and infections

Distinct Mechanisms for Induction and Tolerance Regulate the Immediate Early Genes Encoding Interleukin 1β and Tumor Necrosis Factor α

Interleukin-1β and Tumor Necrosis Factor α play related, but distinct, roles in immunity and disease. Our study revealed major mechanistic distinctions in the Toll-like receptor (TLR) signaling-dependent induction for the rapidly expressed genes (IL1B and TNF) coding for these two cytokines. Prior to induction, TNF exhibited pre-bound TATA Binding Protein (TBP) and paused RNA Polymerase II (Pol II), hallmarks of poised immediate-early (IE) genes. In contrast, unstimulated IL1B displayed very low levels of both TBP and paused Pol II, requiring the lineage-specific Spi-1/PU.1 (Spi1) transcription factor as an anchor for induction-dependent interaction with two TLR-activated transcription factors, C/EBPβ and NF-κB. Activation and DNA binding of these two pre-expressed factors resulted in de novo recruitment of TBP and Pol II to IL1B in concert with a permissive state for elongation mediated by the recruitment of elongation factor P-TEFb. This Spi1-dependent mechanism for IL1B transcription, which is unique for a rapidly-induced/poised IE gene, was more dependent upon P-TEFb than was the case for the TNF gene. Furthermore, the dependence on phosphoinositide 3-kinase for P-TEFb recruitment to IL1B paralleled a greater sensitivity to the metabolic state of the cell and a lower sensitivity to the phenomenon of endotoxin tolerance than was evident for TNF. Such differences in induction mechanisms argue against the prevailing paradigm that all IE genes possess paused Pol II and may further delineate the specific roles played by each of these rapidly expressed immune modulators. © 2013 Adamik et al

Immunometabolism pathways as the basis for innovative anti-viral strategies (INITIATE): a Marie Sklodowska-Curie innovative training network

The past century has witnessed major advances in the control of many infectious diseases, yet outbreaks and epidemics caused by (re-) emerging RNA viruses continue to pose a global threat to human health. As illustrated by the global COVID19 pandemic, high healthcare costs, economic disruption and loss of productivity reinforce the unmet medical need to develop new antiviral strategies to combat not only the current pandemic but also future viral outbreaks. Pivotal for effective anti-viral defense is the innate immune system, a first line host response that senses and responds to virus infection. While molecular details of the innate immune response are well characterized, this research field is now being revolutionized with the recognition that cell metabolism has a major impact on the antiviral and inflammatory responses to virus infections. A detailed understanding of the role of metabolic regulation with respect to antiviral and inflammatory responses, together with knowledge of the strategies used by viruses to exploit immunometabolic pathways, will ultimately change our understanding and treatment of pathogenic viral diseases. INITIATE is a Marie Sklodowska-Curie Actions Innovative Training Network (MSCA-ITN), with the goal to train 15 early stage PhD researchers (ESRs) to become experts in antiviral immunometabolism (https://initiate-itn.eu/). To this end, INITIATE brings together a highly complementary international team of academic and corporate leaders from 7 European countries, with outstanding track records in the historically distinct research fields of virology, immunology and metabolism. The ESRs of INITIATE are trained in these interdisciplinary research fields through individual investigator-driven research projects, specialized scientific training events, workshops on academia-industry interactions, outreach & communication. INITIATE will deliver a new generation of creative and entrepreneurial researchers who will be able to face the inevitable future challenges in combating viral diseases

Froude supercritical flow processes and sedimentary structures: new insights from experiments with a wide range of grain sizes

Recognition of Froude supercritical flow deposits in environments that range from rivers to the ocean floor has triggered a surge of interest in their flow processes, bedforms and sedimentary structures. Interpreting these supercritical flow deposits is especially important because they often represent the most powerful flows in the geological record. Insights from experiments are key to reconstruct palaeo‐flow processes from the sedimentary record. So far, all experimentally produced supercritical flow deposits are of a narrow grain‐size range (fine to medium sand), while deposits in the rock record often consist of a much wider grain‐size distribution. This paper presents results of supercritical‐flow experiments with a grain‐size distribution from clay to gravel. These experiments show that cyclic step instabilities can produce more complex and a larger variety of sedimentary structures than the previously suggested backsets and ‘scour and fill’ structures. The sedimentary structures are composed of irregular lenses, mounds and wedges with backsets and foresets, as well as undulating planar to low‐angle upstream and downstream dipping laminae. The experiments also demonstrate that the Froude number is not the only control on the sedimentary structures formed by supercritical‐flow processes. Additional controls include the size and migration rate of the hydraulic jump and the substrate cohesion. This study further demonstrates that Froude supercritical flow promotes suspension transport of all grain sizes, including gravels. Surprisingly, it was observed that all grain sizes were rapidly deposited just downstream of hydraulic jumps, including silt and clay. These results expand the range of dynamic mud deposition into supercritical‐flow conditions, where local transient shear stress reduction rather than overall flow waning conditions allow for deposition of fines. Comparison of the experimental deposits with outcrop datasets composed of conglomerates to mudstones, shows significant similarities and highlights the role of hydraulic jumps, rather than overall flow condition changes, in producing lithologically and geometrically complex stratigraphy