The E3/E4 ubiquitin conjugation factor UBE4B interacts with and ubiquitinates the HTLV-1 Tax oncoprotein to promote NF-κB activation.

Human T-cell leukemia virus type 1 (HTLV-1) is the etiological agent of adult T-cell leukemia/lymphoma (ATLL), and the neurological disease HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). The HTLV-1 Tax protein persistently activates the NF-κB pathway to enhance the proliferation and survival of HTLV-1 infected T cells. Lysine 63 (K63)-linked polyubiquitination of Tax provides an important regulatory mechanism that promotes Tax-mediated interaction with the IKK complex and activation of NF-κB; however, the host proteins regulating Tax ubiquitination are largely unknown. To identify new Tax interacting proteins that may regulate its ubiquitination we conducted a yeast two-hybrid screen using Tax as bait. This screen yielded the E3/E4 ubiquitin conjugation factor UBE4B as a novel binding partner for Tax. Here, we confirmed the interaction between Tax and UBE4B in mammalian cells by co-immunoprecipitation assays and demonstrated colocalization by proximity ligation assay and confocal microscopy. Overexpression of UBE4B specifically enhanced Tax-induced NF-κB activation, whereas knockdown of UBE4B impaired Tax-induced NF-κB activation and the induction of NF-κB target genes in T cells and ATLL cell lines. Furthermore, depletion of UBE4B with shRNA resulted in apoptotic cell death and diminished the proliferation of ATLL cell lines. Finally, overexpression of UBE4B enhanced Tax polyubiquitination, and knockdown or CRISPR/Cas9-mediated knockout of UBE4B attenuated both K48- and K63-linked polyubiquitination of Tax. Collectively, these results implicate UBE4B in HTLV-1 Tax polyubiquitination and downstream NF-κB activation.

Multi-targeted therapy of everolimus in Kaposi's sarcoma associated herpes virus infected primary effusion lymphoma.

Kaposi's sarcoma associated herpes virus (KSHV) infected primary effusion lymphoma (PEL) is a rare aggressive form of non-Hodgkin's lymphoma of B cells. KSHV latent and lytic antigens modulate several host cellular signalling pathways especially mammalian target of rapamycin (mTOR), STAT-3 and nuclear factor-kappa B (NF-κB) for rapid tumor progression and immune evasion. Current chemotherapeutic strategies are becoming ineffective as they kill only dividing cells and inefficient to target molecular pathways crucial for active virus replication and its survival. In this study, we evaluated the efficacy of everolimus, an mTOR inhibitor in inducing apoptosis of PEL cells. Dose-dependent treatment of everolimus triggered mitochondria-mediated caspase-dependent apoptosis in PEL cells. Everolimus downregulated KSHV latent antigen expression with concurrent blocking of lytic reactivation for active virus replication. Everolimus also inhibited latent antigen mediated constitutively active STAT-3 and NF-κB signalling. We co-cultured everolimus treated PEL cells with immature dendritic cells and found activation of dendritic cells with increase in surface expression of CD86 and HLA-DR. As everolimus targets and disrupts KSHV antigens as well as antigen facilitated multiple signalling pathways necessary for KSHV survival and maintenance of infection with synchronised boosting of immune system against viral infection, it can be a better therapeutic approach towards treatment of PEL.

The tyrosine kinase KDR is essential for the survival of HTLV-1-infected T cells by stabilizing the Tax oncoprotein.

Human T-cell leukemia virus type 1 (HTLV-1) infection is linked to the development of adult T-cell leukemia/lymphoma (ATLL) and the neuroinflammatory disease, HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). The HTLV-1 Tax oncoprotein regulates viral gene expression and persistently activates NF-κB to maintain the viability of HTLV-1-infected T cells. Here, we utilize a kinome-wide shRNA screen to identify the tyrosine kinase KDR as an essential survival factor of HTLV-1-transformed cells. Inhibition of KDR specifically induces apoptosis of Tax expressing HTLV-1-transformed cell lines and CD4 + T cells from HAM/TSP patients. Furthermore, inhibition of KDR triggers the autophagic degradation of Tax resulting in impaired NF-κB activation and diminished viral transmission in co-culture assays. Tax induces the expression of KDR, forms a complex with KDR, and is phosphorylated by KDR. These findings suggest that Tax stability is dependent on KDR activity which could be exploited as a strategy to target Tax in HTLV-1-associated diseases.

Mechanisms of Innate Immune Sensing of HTLV-1 and Viral Immune Evasion.

Human T lymphotropic virus-1 (HTLV-1) was the first identified oncoretrovirus, which infects and establishes a persistent infection in approximately 10-20 million people worldwide. Although only ~5% of infected individuals develop pathologies such as adult T-cell leukemia/lymphoma (ATLL) or a neuroinflammatory disorder termed HTLV-1-asssociated myelopathy/tropical spastic paraparesis (HAM/TSP), asymptomatic carriers are more susceptible to opportunistic infections. Furthermore, ATLL patients are severely immunosuppressed and prone to other malignancies and other infections. The HTLV-1 replication cycle provides ligands, mainly nucleic acids (RNA, RNA/DNA intermediates, ssDNA intermediates, and dsDNA), that are sensed by different pattern recognition receptors (PRRs) to trigger immune responses. However, the mechanisms of innate immune detection and immune responses to HTLV-1 infection are not well understood. In this review, we highlight the functional roles of different immune sensors in recognizing HTLV-1 infection in multiple cell types and the antiviral roles of host restriction factors in limiting persistent infection of HTLV-1. We also provide a comprehensive overview of intricate strategies employed by HTLV-1 to subvert the host innate immune response that may contribute to the development of HTLV-1-associated diseases. A more detailed understanding of HTLV-1-host pathogen interactions may inform novel strategies for HTLV-1 antivirals, vaccines, and treatments for ATLL or HAM/TSP.

Quinacrine has anticancer activity in breast cancer cells through inhibition of topoisomerase activity.

The small molecule Quinacrine (QC, a derivative of 9-aminoacridine), an anti-malaria drug, displays activity against cancer cell lines and can simultaneously suppress nuclear factor-κB (NF-κB) and activate p53 signaling. In this study, we investigated the anticancer mechanism underlying these drug activities in breast cancer cell lines. QC caused a dose-dependent decrease of both anchorage dependent and independent growth of breast cancer cells (MCF-7 and MDA-MB-231) without affecting normal breast epithelial cells (MCF-10A), as evident from clonogenic cell survival, [3-(4,5-dimethylthiazol-2yl-)-2,5-diphenyl tetrazolium bromide] viability, wound healing and soft agar growth. QC activated the proapoptotic marker Bax, PARP cleavage, p53 and its downstream target, p21 (Cip1/Waf1) and downregulated the antiapoptotic marker Bcl-xL and relative luciferase activity of NF-κB in MCF-7 cells. Results of DAPI nuclear staining and FACS analysis show that QC increased apoptosis in a dose-dependent manner. QC caused apoptosis by increasing the cell population in S-phase and simultaneously decreasing the G1 and G2/M populations. A dose-dependent increase of DNA damage as measured by the comet assay was seen in MCF-7 cells after exposure to QC. With regards to the mechanism of DNA damage, we found that QC inhibited topoisomerase activity in MCF-7 cells by increasing the unwinding of supercoiled DNA. Collectively, the results demonstrate that QC has efficient anticancer potential against breast cancer cells via not only an induction of p53 and p21 but also an induction of S phase arrest, DNA damage and inhibition of topoisomerase activity.

RETRA induces necroptosis in cervical cancer cells through RIPK1, RIPK3, MLKL and increased ROS production.

Cervical cancer is the fourth most prevalent cancer in women worldwide, predominantly infected with human papillomavirus (HPV). The current chemo and radiotherapies are mostly futile due to acquired resistance to apoptosis and warrant new therapeutic approaches targeting potent non-apoptotic cell death pathways to eliminate cervical cancer cells. Induction of necroptosis by pharmaceutical interventions is emerging as a promising tool in multiple apoptotic resistant cancer cells. RETRA (REactivation of Transcriptional Reporter Activity) is a small molecule known to induce expression of p53 regulated genes in mutant (mt) p53 cells but, detailed mechanisms of its anticancer effects are poorly known. The present study investigated the potentials of RETRA as an anticancer agent and found that it induces necroptosis selectively in cervical cancer cells irrespective of p53 status through the phosphorylation of receptor-interacting protein kinase 1,3 (RIPK1, RIPK3) and mixed lineage kinase domain-like protein (MLKL) with no cytotoxic effects in normal human peripheral blood mononuclear cells (PBMCs). RETRA-treated cells also displayed necroptotic morphology of disintegrated plasma membranes with intact nuclei and also showed cell cycle arrest at the S phase with the upregulation of p21 and downregulation of cyclin-D3. Intriguingly, the combinatorial approach of using RETRA with Necrostain-1, a known inhibitor of necroptosis, reversed the effect of RETRA and rescued cell death. Moreover, induction of necroptosis by RETRA is associated with mitochondrial hyperpolarization and elevated ROS production. Collectively, these findings suggest that RETRA induces cell death via necroptosis with increased production of ROS, accentuating the therapeutic implication of RETRA in cervical cancer cells.

Nm23-H1 induces apoptosis in primary effusion lymphoma cells via inhibition of NF-κB signaling through interaction with oncogenic latent protein vFLIP K13 of Kaposi's sarcoma-associated herpes virus.

BACKGROUND: Primary effusion lymphoma (PEL) is an aggressive form of non-Hodgkin lymphoma of B cells caused by Kaposi's Sarcoma-associated Herpes Virus (KSHV). KSHV encoded latent and lytic antigens promote oncogenic transformation and evade apoptosis through the modulation of various host cellular signaling pathways. Nm23-H1 is a known metastatic suppressor whose expression inversely correlates with the metastatic potential of different cancers. Here, we set out to assess the role of Nm23-H1 in PEL development. METHODS: Flow cytometry and real-time PCR assays were performed for Nm23-H1 expression analysis. Induction of apoptosis was assessed using Western blotting and flow cytometry-based assays in Nm23-H1 overexpressing cells. Co-immunoprecipitation assays, confocal microscopy and imaging flow cytometry were performed to determine Nm23-H1 and vFLIP K13 protein-protein interaction. A PEL cell-induced xenograft model was established in non-obese diabetic/severely combined immunodeficient (NOD/SCID) mice to validate the effect of Nm23-H1 overexpression. RESULTS: We found that Nm23-H1 expression was significantly downregulated both at transcriptional and protein levels in PEL cell lines and that its overexpression triggered mitochondrial-mediated caspase-dependent apoptosis. We revealed Nm23-H1 interacts with the latent protein vFLIP K13 and that Nm23-H1 overexpression leads to inhibition of vFLIP K13 driven nuclear factor-kappa B (NF-κB) signaling with concurrent inhibition of autocrine and paracrine growth factors and downregulation of latent KSHV antigens without induction of active lytic reactivation. We also confirmed the effects of Nm23-H1 overexpression in a PEL cell-induced xenograft model in NOD/SCID mice. CONCLUSION: Downregulation of Nm23-H1 expression in KSHV-infected PEL cells and its overexpression trigger apoptosis by impairing vFLIP K13-driven NF-κB signaling, suggesting therapeutic implications of Nm23-H1 for primary effusion lymphomas.

Abatement of gas-phase VOCs via dielectric barrier discharge plasmas.

Non-thermal plasma (NTP) technology is regarded as promising method for abatement of volatile organic compounds (VOCs) and has gained substantial interests in the fields of air purification. In this present work at atmospheric pressure, dielectric barrier discharge (DBD) plasma has been employed and utilized to evaluate feasibility of toluene, benzene, and m-xylene degradation in a parallel plate type DBD reactor taking argon as a carrier gas. The composition of post-plasma-treated by-products is studied by various spectroscopic techniques such as GC, GCMS, and FTIR, and the corresponding computational work is carried out by Gaussian software. The by-products obtained are analyzed in order to ascertain their structure and stability. The degradation of the VOCs to CO and CO2 has been affirmed from the GC and GCMS analyses. Furthermore, the probable degradation pathway for the degradation of the aforementioned VOCs has been deciphered and the most feasible path is suggested. Additionally, DFT calculations have been performed to have an idea about the energies of the pathways involved. This research work has been carried out without use of any catalyst. Surprisingly, the degradation observed is much higher than the reported values. These findings would be helpful towards the abatement of VOCs by the use of non-thermal plasma.

Mechanisms of Oncogenesis by HTLV-1 Tax.

The human T-cell lymphotropic virus type 1 (HTLV-1) is the etiological agent of adult T-cell leukemia/lymphoma (ATLL), a neoplasm of CD4+CD25+ T cells that occurs in 2-5% of infected individuals after decades of asymptomatic latent infection. Multiple HTLV-1-encoded regulatory proteins, including Tax and HTLV-1 basic leucine zipper factor (HBZ), play key roles in viral persistence and latency. The HTLV-1 Tax oncoprotein interacts with a plethora of host cellular proteins to regulate viral gene expression and also promote the aberrant activation of signaling pathways such as NF-κB to drive clonal proliferation and survival of T cells bearing the HTLV-1 provirus. Tax undergoes various post-translational modifications such as phosphorylation and ubiquitination that regulate its function and subcellular localization. Tax shuttles in different subcellular compartments for the activation of anti-apoptotic genes and deregulates the cell cycle with the induction of DNA damage for the accumulation of genomic instability that can result in cellular immortalization and malignant transformation. However, Tax is highly immunogenic and therefore HTLV-1 has evolved numerous strategies to tightly regulate Tax expression while maintaining the pool of anti-apoptotic genes through HBZ. In this review, we summarize the key findings on the oncogenic mechanisms used by Tax that set the stage for the development of ATLL, and the strategies used by HTLV-1 to tightly regulate Tax expression for immune evasion and viral persistence.

Role of Interleukin 28B Polymorphisms in Response to Interferon Based Therapy for Hepatitis C Virus Clearance.

BACKGROUND: Interleukin-28B (IL28B) locus on a human chromosomal region mapped to 19q13 execute immune defense against viruses. During Hepatitis C Virus (HCV) infection the IL28B has a promising role in deciding the consequence of infection for spontaneous clearance of viruses or causing chronic liver infection. Treatment of chronic hepatitis C includes use of direct acting antivirals, Pegylated-Interferon (PEG-IFN) and Ribavirin (RBV) therapy. Also, interferon free regimens are suggested to be useful in resistant patients. Numerous reports including Genome-Wide Association Studies (GWAS), comprehensive meta-analysis and independent case-control studies in different population have revealed the association between certain Il-28B polymorphisms and response to the PEGIFN- RBV therapy in patients infected with HCV. METHOD: We searched all peer-reviewed relevant and recent literature manually for the present review. CONCLUSION: The GWAS studies have revealed an important role of IL28B in HCV infection, which was supported by many independent studies and meta-analysis by different groups in different ethnicities. IL28B genotyping may be use as predictors of response for IFN-based therapy and personalized treatment of hepatitis C patient.

1, 25(OH)2 D3 Induces Reactivation and Death of Kaposi's Sarcoma-Associated Herpesvirus of Primary Effusion Lymphoma cells.

Kaposi's sarcoma associated herpesvirus (KSHV) a gammaherpesvirus establishes perennial latency in the host with periodic reactivation. Occasionally change in the physiological condition like hypoxia, host cell differentiation can trigger the lytic switch and reactivation of the virus. The biologically active form of 1, 25(OH)2 D3 plays a critical role in the regulation of various physiological processes (e.g. regulation of mineral homeostasis and control of bone metabolism). Apart from its role in host physiology, 1, 25(OH)2 D3 has been implicated as a potential agent for the prevention and/or treatment of many a tumors. Here we show that 1, 25(OH)2 D3 induces both death of Kaposi sarcoma associated herpesvirus infected PEL cells and KSHV replication. 1, 25(OH)2 D3 mediated inhibition of proliferation was associated with apoptosis of the PEL cells, and virus reactivation. In addition, p38 signalling is required for KSHV reactivation. Furthermore, treatment of PEL cells with p38 inhibitor abrogated the expression of ORF57, thus blocking lytic switch. Furthermore, silencing of VDR resulted in reduced ORF57 expression compared to the control cells, signifying the potential role of 1, 25(OH)2 D3 in KSHV reactivation. Thus, our studies have revealed a novel role of 1, 25(OH)2 D3 in the regulation of KSHV reactivation and PEL cell death.

Regulators of Tfh Cell Differentiation.

The follicular helper T (Tfh) cells help is critical for activation of B cells, antibody class switching, and germinal center (GC) formation. The Tfh cells are characterized by the expression of CXC chemokine receptor 5 (CXCR5), ICOS, programed death 1 (PD-1), B cell lymphoma 6 (BCL-6), and IL-21. They are involved in clearing infections and are adversely linked with autoimmune diseases and also have a role in viral replication as well as clearance. On the one hand, Tfh cells are generated from naive CD4+ T cells with sequential steps involving cytokine signaling (IL-21, IL-6, IL-12, activin A), migration, and positioning in the GC by CXCR5, surface receptors (ICOS/ICOSL, signaling lymphocyte activation molecule-associated protein/signaling lymphocyte activation molecule) as well as transcription factor (BCL-6, c-Maf, and signal transducer and activator of transcription 3) signaling and repressor miR155. On the other hand, Tfh generation is negatively regulated at specific steps of Tfh generation by specific cytokine (IL-2, IL-7), surface receptor (PD-1, CTLA-4), transcription factors B lymphocyte maturation protein 1, signal transducer and activator of transcription 5, T-bet, KLF-2 signaling, and repressor miR 146a. Interestingly, miR-17-92 and FOXO1 act as a positive as well as a negative regulator of Tfh differentiation depending on the time of expression and disease specificity. Tfh cells are also generated from the conversion of other effector T cells as exemplified by Th1 cells converting into Tfh during viral infection. The mechanistic details of effector T cells conversion into Tfh are yet to be clear. To manipulate Tfh cells for therapeutic implication and or for effective vaccination strategies, it is important to know positive and negative regulators of Tfh generation. Hence, in this review, we have highlighted and interlinked molecular signaling from cytokines, surface receptors, transcription factors, ubiquitin ligase, and microRNA as positive and negative regulators for Tfh differentiation.

Epstein-Barr virus nuclear antigen 3C interact with p73: Interplay between a viral oncoprotein and cellular tumor suppressor.

The p73 protein has structural and functional homology with the tumor suppressor p53, which plays an important role in cell cycle regulation, apoptosis, and DNA repair. The p73 locus encodes both a tumor suppressor (TAp73) and a putative oncogene (ΔNp73). p73 May play a significant role in p53-deficient lymphomas infected with Epstein-Barr virus (EBV). EBV produces an asymptomatic infection in the majority of the global population, but it is associated with several human B-cell malignancies. The EBV-encoded Epstein-Barr virus nuclear antigen 3C (EBNA3C) is thought to disrupt the cell cycle checkpoint by interacting directly with p53 family proteins. Doxorubicin, a commonly used chemotherapeutic agent, induces apoptosis through p53 and p73 signaling such that the lowΔNp73 level promotes the p73-mediated intrinsic pathway of apoptosis. In this report, we investigated the mechanism by which EBV infection counters p73α-induced apoptosis through EBNA3C.

Kaposi sarcoma herpes virus latency associated nuclear antigen protein release the G2/M cell cycle blocks by modulating ATM/ATR mediated checkpoint pathway.

The Kaposi's sarcoma-associated herpesvirus infects the human population and maintains latency stage of viral life cycle in a variety of cell types including cells of epithelial, mesenchymal and endothelial origin. The establishment of latent infection by KSHV requires the expression of an unique repertoire of genes among which latency associated nuclear antigen (LANA) plays a critical role in the replication of the viral genome. LANA regulates the transcription of a number of viral and cellular genes essential for the survival of the virus in the host cell. The present study demonstrates the disruption of the host G2/M cell cycle checkpoint regulation as an associated function of LANA. DNA profile of LANA expressing human B-cells demonstrated the ability of this nuclear antigen in relieving the drug (Nocodazole) induced G2/M checkpoint arrest. Caffeine suppressed nocodazole induced G2/M arrest indicating involvement of the ATM/ATR. Notably, we have also shown the direct interaction of LANA with Chk2, the ATM/ATR signalling effector and is responsible for the release of the G2/M cell cycle block.

Nm23-H1 can induce cell cycle arrest and apoptosis in B cells.

Nm23-H1 is a well-known tumor metastasis suppressor, which functions as a nucleoside-diphosphate kinase converting nucleoside diphosphates to nucleoside triphosphates with an expense of ATP. It regulates a variety of cellular activities, including proliferation, development, migration and differentiation known to be modulated by a series of complex signaling pathway. Few studies have addressed the mechanistic action of Nm23-H1 in the context of these cellular processes. To determine the downstream pathways modulated by Nm23-H1, we expressed Nm23-H1 in a Burkitt lymphoma derived B-cell line BJAB and performed pathway specific microarray analysis. The genes with significant changes in expression patterns were clustered in groups which are responsible for regulating cell cycle, p53 activities and apoptosis. We found a general reduction of cell cycle regulatory proteins including cyclins and cyclin dependent kinase inhibitors (anti proliferation), and upregulation of apoptotic genes which included caspase 3, 9 and Bcl-x. Nm23-H1 was also found to upregulate p53 and downregulate p21 expression. A number of these genes were validated by real time PCR and results from promoter assays indicated that Nm23-H1 expression downregulated cyclin D1 in a dose responsive manner. Further, we show that Nm23-H1 forms a complex with the cellular transcription factor AP1 to modulate cyclin D1 expression levels. BJAB cells expressing Nm23-H1 showed reduced proliferation rate and were susceptible to increased apoptosis which may in part be due to a direct interaction between Nm23-H1 and p53. These results suggest that Nm23-H1 may have a role in the regulation of cell cycle and apoptosis in human B-cells.