The F-box E3 ligase protein FBXO11 regulates EBNA3C-associated degradation of BCL6.

Most mature B-cell malignancies originate from the malignant transformation of germinal center (GC) B cells. The GC reaction appears to have a role in malignant transformation, in which a major player of the GC reaction is BCL6, a key regulator of this process. We now demonstrate that BCL6 protein levels were dramatically decreased in Epstein-Barr virus (EBV)-positive lymphoblastoid cell lines and Burkitt's lymphoma cell lines. Notably, BCL6 degradation was significantly enhanced in the presence of both EBNA3C and FBXO11. Furthermore, the amino-terminal domain of EBNA3C, which contains residues 50-100, interacts directly with FBXO11. The expression of EBNA3C and FBXO11 resulted in a significant induction of cell proliferation. Furthermore, BCL6 protein expression levels were regulated by EBNA3C via the Skp Cullin Fbox (SCF)FBXO11 complex, which mediated its ubiquitylation, and knockdown of FBXO11 suppressed the transformation of lymphoblastoid cell lines. These data provide new insights into the function of EBNA3C in B-cell transformation during GC reaction and raise the possibility of developing new targeted therapies against EBV-associated cancers. IMPORTANCE: The novel revelation in our study involves the suppression of BCL6 expression by the essential Epstein-Barr virus (EBV) antigen EBNA3C, shedding new light on our current comprehension of how EBV contributes to lymphomagenesis by impeding the germinal center reaction. It is crucial to note that while several EBV latent proteins are expressed in infected cells, the collaborative mechanisms among these proteins in regulating B-cell development or inducing B-cell lymphoma require additional investigation. Nonetheless, our findings carry significance for the development of emerging strategies aimed at addressing EBV-associated cancers.

KSHV-encoded LANA bypasses transcriptional block through the stabilization of RNA Pol II in hypoxia.

IMPORTANCE: Hypoxia can induce the reactivation of Kaposi sarcoma-associated virus (KSHV), which necessitates the synthesis of critical structural proteins. Despite the unfavorable energetic conditions of hypoxia, KSHV utilizes mechanisms to prevent the degradation of essential cellular machinery required for successful reactivation. Our study provides new insights on strategies employed by KSHV-infected cells to maintain steady-state transcription by overcoming hypoxia-mediated metabolic stress to enable successful reactivation. Our discovery that the interaction of latency-associated nuclear antigen with HIF1α and NEDD4 inhibits its polyubiquitination activity, which blocks the degradation of RNA Pol II during hypoxia, is a significant contribution to our understanding of KSHV biology. This newfound knowledge provides new leads in the development of novel therapies for KSHV-associated diseases.

Insight into the Epigenetics of Kaposi's Sarcoma-Associated Herpesvirus.

Epigenetic reprogramming represents a series of essential events during many cellular processes including oncogenesis. The genome of Kaposi's sarcoma-associated herpesvirus (KSHV), an oncogenic herpesvirus, is predetermined for a well-orchestrated epigenetic reprogramming once it enters into the host cell. The initial epigenetic reprogramming of the KSHV genome allows restricted expression of encoded genes and helps to hide from host immune recognition. Infection with KSHV is associated with Kaposi's sarcoma, multicentric Castleman's disease, KSHV inflammatory cytokine syndrome, and primary effusion lymphoma. The major epigenetic modifications associated with KSHV can be labeled under three broad categories: DNA methylation, histone modifications, and the role of noncoding RNAs. These epigenetic modifications significantly contribute toward the latent-lytic switch of the KSHV lifecycle. This review gives a brief account of the major epigenetic modifications affiliated with the KSHV genome in infected cells and their impact on pathogenesis.

Targeting Kaposi's sarcoma associated herpesvirus encoded protease (ORF17) by a lysophosphatidic acid molecule for treating KSHV associated diseases.

Kaposi's sarcoma associated herpesvirus (KSHV) is causative agent of Kaposi's sarcoma, Multicentric Castleman Disease and Pleural effusion lymphoma. KSHV-encoded ORF17 encodes a protease which cleaves -Ala-Ala-, -Ala-Ser- or -Ala-Thr-bonds. The protease plays an important role in assembly and maturation of new infective virions. In the present study, we investigated expression pattern of KSHV-encoded protease during physiologically allowed as well as chemically induced reactivation condition. The results showed a direct and proportionate relationship between ORF17 expression with reactivation time. We employed virtual screening on a large database of natural products to identify an inhibitor of ORF17 for its plausible targeting and restricting Kaposi's sarcoma associated herpesvirus assembly/maturation. A library of 307,814 compounds of biological origin (A total 481,799 structures) has been used as a screen library. 1-oleoyl-2-hydroxy-sn-glycero-3-phospho-(1'-myo-inositol) was highly effective against ORF17 in in-vitro experiments. The screened compound was tested for the cytotoxic effect and potential for inhibiting Kaposi's sarcoma associated herpesvirus production upon induced reactivation by hypoxia, TPA and butyric acid. Treatment of reactivated KSHV-positive cells with 1-oleoyl-2-hydroxy-sn-glycero-3-phospho-(1'-myo-inositol) resulted in significant reduction in the production of Kaposi's sarcoma associated herpesvirus. The study identified a lysophosphatidic acid molecule for alternate strategy to inhibit KSHV-encoded protease and target Kaposi's sarcoma associated herpesvirus associated malignancies.

Epigenetic Reprogramming of Kaposi's Sarcoma-Associated Herpesvirus during Hypoxic Reactivation.

The biphasic life cycle (latent and lytic) of Kaposi's sarcoma-associated Herpesvirus (KSHV) is regulated by epigenetic modification of its genome and its associated histone proteins. The temporal events driving epigenetic reprogramming of the KSHV genome on initial infection to establish latency has been well studied, but the reversal of these epigenetic changes during lytic replication, especially under physiological conditions such as hypoxia, has not been explored. In this study, we investigated epigenetic reprogramming of the KSHV genome during hypoxic reactivation. Hypoxia induced extensive enrichment of both transcriptional activators and repressors on the KSHV genome through H3K4Me3, H3K9Me3, and H3K27Me3, as well as histone acetylation (H3Ac) modifications. In contrast to uniform quantitative enrichment with modified histones, a distinct pattern of RTA and LANA enrichment was observed on the KSHV genome. The enrichment of modified histone proteins was due to their overall higher expression levels, which was exclusively seen in KSHV-positive cells. Multiple KSHV-encoded factors such as LANA, RTA, and vGPCR are involved in the upregulation of these modified histones. Analysis of ChIP-sequencing for the initiator DNA polymerase (DNAPol1α) combined with single molecule analysis of replicated DNA (SMARD) demonstrated the involvement of specific KSHV genomic regions that initiate replication in hypoxia.

HPV18 oncoproteins driven expression of PKM2 reprograms HeLa cell metabolism to maintain aerobic glycolysis and viability.

The molecular basis of human papillomavirus (HPV)-mediated cellular immortalization and malignant transformation has illustrated an indispensable role of viral E6/E7-oncoproteins. However, the impact of viral-oncoproteins on the metabolic phenotype of cancer cells remains ambiguous. We showed silencing of HPV18-encoded E6/E7-oncoprotein significantly reduced glucose consumption, lactate production, ATP level and viability. Silencing of HPV18-encoded E6/E7 in HeLa cells significantly down-regulated expression and activity of HK1, HK2, LDHA, and LDHB. Interestingly, there was an increased pyruvate kinase activity due to switch in expression from PKM2 isoform to PKM1. The switch in favor of alternatively spliced isoform PKM1, was regulated by viral-E6/E7-oncoprotein by inhibiting the c-Myc/hnRNP-axis. Further, the near absence of the PKM1 protein despite an adequate amount of PKM1 mRNA in HeLa cells was due to its proteasomal degradation. Our results suggests HPV18-encoded E6/E7 driven preferential expression of PKM2 is essential to support aerobic glycolysis and cell proliferation. Supplementary Information: The online version contains supplementary material available at 10.1007/s13337-022-00776-w.

Co-pyrolysis of petroleum coke and banana leaves biomass: Kinetics, reaction mechanism, and thermodynamic analysis.

Insights into thermal degradation behaviour, kinetics, reaction mechanism, possible synergism, and thermodynamic analysis of co-pyrolysis of carbonaceous materials are crucial for efficient design of co-pyrolysis reactor systems. Present study deals with comprehensive kinetics and thermodynamic investigation of co-pyrolysis of petroleum coke (PC) and banana leaves biomass (BLB) for realizing the co-pyrolysis potential. Thermogravimetric non-isothermal studies have been performed at 10, 20, and 30 °C/min heating rates. Synergistic effect between PC and BLB was determined by Devolatilization index (Di) and mass loss method. Kinetic parameters were estimated using seven model-free methods. Standard activation energy for PC + BLB blend from FWO, KAS, Starink, and Vyazovkin methods was ≈165 kJ/mol and that from Friedman and Vyazovkin advanced isoconversional methods was ≈171 kJ/mol. The frequency factor calculated for the blend from Kissinger method was found to be in the range of 106-1016s-1. Devolatilization index (Di) showed synergistic effect of blending. The data pertaining to co-pyrolysis was found to fit well with R2 (second order) and D3 (three dimensional) from Z(α) master plot. Thermodynamic parameters, viz. ΔH ≈ 163 kJ/mol and ΔG ≈ 151 kJ/mol were calculated to determine the feasibility and reactivity of the co-pyrolysis process. The results are expected to be useful in the design of petcoke and banana leaves biomass co-pyrolysis systems.

HIF1α-Regulated Expression of the Fatty Acid Binding Protein Family Is Important for Hypoxic Reactivation of Kaposi's Sarcoma-Associated Herpesvirus.

The hypoxic microenvironment and metabolic reprogramming are two major contributors to the phenotype of oncogenic virus-infected cells. Infection by Kaposi's sarcoma-associated herpesvirus (KSHV) stabilizes hypoxia-inducible factor 1α (HIF1α) and reprograms cellular metabolism. We investigated the comparative transcriptional regulation of all major genes involved in fatty acid and amino acid metabolism in KSHV-positive and -negative cells grown under normoxic or hypoxic conditions. We show a distinct regulation of genes involved in both fatty acid and amino acid metabolism in KSHV-positive cells grown in either normoxic or hypoxic conditions, with a particular focus on genes involved in the acetyl coenzyme A (acetyl-CoA) pathway. The fatty acid binding protein (FABP) family of genes, specifically FABP1, FABP4, and FABP7, was also observed to be synergistically upregulated in hypoxia by KSHV. This pattern of FABP gene expression was also seen in naturally infected KSHV BC3 or BCBL1 cells when compared to KSHV-negative DG75 or BL41 cells. Two KSHV-encoded antigens, which positively regulate HIF1α, the viral G-protein coupled receptor (vGPCR), and the latency-associated nuclear antigen (LANA) were shown to drive upregulation of the FABP gene transcripts. Suppression of FABPs by RNA interference resulted in an adverse effect on hypoxia-dependent viral reactivation. Overall, this study provides new evidence, which supports a rationale for the inhibition of FABPs in KSHV-positive cells as potential strategies, for the development of therapeutic approaches targeting KSHV-associated malignancies.IMPORTANCE Hypoxia is a detrimental stress to eukaryotes and inhibits several cellular processes, such as DNA replication, transcription, translation, and metabolism. Interestingly, the genome of Kaposi's sarcoma-associated herpesvirus (KSHV) is known to undergo productive replication in hypoxia. We investigated the comparative transcriptional regulation of all major genes involved in fatty acid and amino acid metabolism in KSHV-positive and -negative cells grown under normoxic or hypoxic conditions. Several metabolic pathways were observed differentially regulated by KSHV in hypoxia, specifically, the fatty acid binding protein (FABP) family genes (FABP1, FABP4, and FABP7). KSHV-encoded antigens, vGPCR and LANA, were shown to drive upregulation of the FABP transcripts. Suppression of FABPs by RNA interference resulted in an adverse effect on hypoxia-dependent viral reactivation. Overall, this study provides new evidence, which supports a rationale for the inhibition of FABPs in KSHV-positive cells as potential strategies, for the development of therapeutic approaches targeting KSHV-associated malignancies.

Vascular endothelial growth factor encoded by Parapoxviruses can regulate metabolism and survival of triple negative breast cancer cells.

Dysbiotic microbiomes are linked to many pathological outcomes including different metabolic disorders like diabetes, atherosclerosis and even cancer. Breast cancer is the second leading cause of cancer associated death in women, and triple negative breast cancer (TNBC) is the most aggressive type with major challenges for intervention. Previous reports suggested that Parapoxvirus signatures are one of the predominant dysbiotic viral signatures in TNBC. These viruses encode several genes that are homologs of human genes. In this study, we show that the VEGF homolog encoded by Parapoxviruses, can induce cell proliferation, and alter metabolism of breast cancer and normal breast cells, through alteration of MAPK-ERK and PI3K-AKT signaling. In addition, the activity of the transcription factor FoxO1 was altered by viral-encoded VEGF through activation of the PI3K-AKT pathway, leading to reprogramming of cellular metabolic gene expression. Therefore, this study provides new insights into the function of viral-encoded VEGFs, which promoted the growth of the breast cancer cells and imparted proliferative phenotype with altered metabolism in normal breast cells.

Pyrolysis of banana leaves biomass: Physico-chemical characterization, thermal decomposition behavior, kinetic and thermodynamic analyses.

In the present work, non-isothermal thermogravimetric experiments were conducted at three heating rates, viz. 10, 20, and 30 °C/min to study the thermal degradation of banana leaves biomass, where the key objective was to determine the kinetic triplet (activation energy, pre-exponential variable, and reaction model) and thermodynamic parameters. The kinetic study was carried out using five model-free isoconversional methods, viz. Flynn-Wall-Ozawa, Kissinger-Akahira-Sunose, Starink, Friedman, and Kissinger. Results showed that average activation energy ranges between 70.75 and 92.12 kJ/mol for the studied isoconversional model-free methods. The average activation energy obtained by KAS (79.36 kJ/mol) was found to be in the proximity of that obtained by FWO (84.02 kJ/mol). Pre-exponential factor obtained from Kissinger method was found to vary from 107 to 1033 s-1. Master plot showed that data fits well with second order reaction model till 0.2 conversion and then follows third order reaction model from 0.2 to 0.5 conversion.

Quassinoid analogs with enhanced efficacy for treatment of hematologic malignancies target the PI3Kγ isoform.

Development of novel PI3K inhibitors is an important strategy to overcome their resistance and poor tolerability in clinical trials. The quassinoid family member Brusatol shows specific inhibitory activity against hematologic malignancies. However, the mechanism of its anti-cancer activity is unknown. We investigated the anti-cancer activity of Brusatol on multiple hematologic malignancies derived cell lines. The results demonstrated that the PI3Kγ isoform was identified as a direct target of Brusatol, and inhibition was dramatically reduced on cells with lower PI3Kγ levels. Novel synthetic analogs were also developed and tested in vitro and in vivo. They shared comparable or superior potency in their ability to inhibit malignant hematologic cell lines, and in a xenograft transplant mouse model. One unique analog had minimal toxicity to normal human cells and in a mouse model. These new analogs have enhanced potential for development as a new class of PI3K inhibitors for treatment of hematologic malignancies.

KSHV-encoded LANA protects the cellular replication machinery from hypoxia induced degradation.

Kaposi's sarcoma associated herpesvirus (KSHV), like all herpesviruses maintains lifelong persistence with its host genome in latently infected cells with only a small fraction of cells showing signatures of productive lytic replication. Modulation of cellular signaling pathways by KSHV-encoded latent antigens, and microRNAs, as well as some level of spontaneous reactivation are important requirements for establishment of viral-associated diseases. Hypoxia, a prominent characteristic of the microenvironment of cancers, can exert specific effects on cell cycle control, and DNA replication through HIF1α-dependent pathways. Furthermore, hypoxia can induce lytic replication of KSHV. The mechanism by which KSHV-encoded RNAs and antigens regulate cellular and viral replication in the hypoxic microenvironment has yet to be fully elucidated. We investigated replication-associated events in the isogenic background of KSHV positive and negative cells grown under normoxic or hypoxic conditions and discovered an indispensable role of KSHV for sustained cellular and viral replication, through protection of critical components of the replication machinery from degradation at different stages of the process. These include proteins involved in origin recognition, pre-initiation, initiation and elongation of replicating genomes. Our results demonstrate that KSHV-encoded LANA inhibits hypoxia-mediated degradation of these proteins to sustain continued replication of both host and KSHV DNA. The present study provides a new dimension to our understanding of the role of KSHV in survival and growth of viral infected cells growing under hypoxic conditions and suggests potential new strategies for targeted treatment of KSHV-associated cancer.

Role of ectopically expressed mtDNA encoded cytochrome c oxidase subunit I (MT-COI) in tumorigenesis.

Somatic mutations within mitochondrial DNA (mtDNA) encoded cytochrome c oxidase subunit I (MT-CO1 or MT-COI) are frequent in various cancer types. In addition, perturbation from orchestrated expression of mitochondrial DNA encoded genes is also associated with complex disorders, including cancer. Since codon bias and the mitochondrial translation system restricts functional characterization of over-expressed wild type or mutant mitochondrial DNA encoded genes, the codon optimization and artificial synthesis of entire MT-CO1 allowed us to over-express the wild type and one of its deleterious mutants into the mitochondria of the transfected cells. Ectopically expressed MT-CO1 was observed to efficiently express and localized to mitochondria but showed high level of aggregation under denaturing condition. Over-expression of wild type or mutant variant of MT-CO1 promoted anchorage dependent and independent proliferation potential in in-vitro experiments and introduced the cancer cell metabolic phenotype of high glucose uptake and lactate release. Reactive oxygen species generated in cells over-expressing MT-CO1 variants acted as key effectors mediating differential expression of apoptosis and DNA damage pathway related genes. High ROS generated also down-regulated the expression of global regulators of gene expression, DNMT3A and DNMT3B. The down-regulated expression of DNMTs co-related with differential methylation of the CpG islands in the promoter region of a select set of studied genes, in a manner to promote pro-cancerous phenotype. Apart from assigning the mechanistic role to the MT-CO1 variants and their perturbed expression in cancer development, the present study provides novel insights into the functional role of somatic mutations within MT-CO1 promoting cancer phenotype.

EBV epitranscriptome reprogramming by METTL14 is critical for viral-associated tumorigenesis.

Epstein-Barr virus (EBV) is a ubiquitous oncogenic virus that induces many cancers. N6-Methyladenosine (m6A) modification regulates many cellular processes. We explored the role of m6A in EBV gene regulation and associated cancers. We have comprehensively defined m6A modification of EBV latent and lytic transcripts. Furthermore, m6A modification demonstrated a functional role in regulation of the stability of viral transcripts. The methyltransferase METTL14 was induced at the transcript and protein levels, and knock-down of METTL14 led to decreased expression of latent EBV transcripts. METTL14 was also significantly induced in EBV-positive tumors, promoted growth of EBV-transformed cells and tumors in Xenograft animal models. Mechanistically, the viral-encoded latent oncoprotein EBNA3C activated transcription of METTL14, and directly interacted with METTL14 to promote its stability. This demonstrated that EBV hijacks METTL14 to drive EBV-mediated tumorigenesis. METTL14 is now a new target for development of therapeutics for treatment of EBV-associated cancers.

Correction: Shugoshin 1 is dislocated by KSHV-encoded LANA inducing aneuploidy.

[This corrects the article DOI: 10.1371/journal.ppat.1007253.].

EBNA3C facilitates RASSF1A downregulation through ubiquitin-mediated degradation and promoter hypermethylation to drive B-cell proliferation.

EBV latent antigen 3C (EBNA3C) is essential for EBV-induced primary B-cell transformation. Infection by EBV induces hypermethylation of a number of tumor suppressor genes, which contributes to the development of human cancers. The Ras association domain family isoform 1A (RASSF1A) is a cellular tumor suppressor, which regulates a broad range of cellular functions, including apoptosis, cell-cycle arrest, mitotic arrest, and migration. However, the expression of RASSF1A is lost in many human cancers by epigenetic silencing. In the present study, we showed that EBNA3C promoted B-cell transformation by specifically suppressing the expression of RASSF1A. EBNA3C directly interacted with RASSF1A and induced RASSF1A degradation via the ubiquitin-proteasome-dependent pathway. SCFSkp2, an E3-ubiquitin ligase, was recruited by EBNA3C to enhance RASSF1A degradation. Moreover, EBNA3C decreased the transcriptional activity of RASSF1A promoter by enhancing its methylation through EBNA3C-mediated modulation of DNMTs expression. EBNA3C also inhibited RASSF1A-mediated cell apoptosis, disrupted RASSF1A-mediated microtubule and chromosomal stability, and promoted cell proliferation by upregulating Cyclin D1 and Cyclin E expression. Our data provides new details, which sheds light on additional mechanisms by which EBNA3C can induce B-cell transformation. This will also facilitate the development of novel therapeutic approaches through targeting of the RASSF1A pathway.

Shugoshin 1 is dislocated by KSHV-encoded LANA inducing aneuploidy.

Shugoshin-1 (Sgo1) protects the integrity of the centromeres, and H2A phosphorylation is critical for this process. The mitotic checkpoint kinase Bub1, phosphorylates H2A and ensures fidelity of chromosome segregation and chromosome number. Oncogenic KSHV induces genetic alterations through chromosomal instability (CIN), and its essential antigen LANA regulates Bub1. We show that LANA inhibits Bub1 phosphorylation of H2A and Cdc20, important for chromosome segregation and mitotic signaling. Inhibition of H2A phosphorylation at residue T120 by LANA resulted in dislocation of Sgo1, and cohesin from the centromeres. Arrest of Cdc20 phosphorylation also rescued degradation of Securin and Cyclin B1 at mitotic exit, and interaction of H2A, and Cdc20 with Bub1 was inhibited by LANA. The N-terminal nuclear localization sequence domain of LANA was essential for LANA and Bub1 interaction, reversed LANA inhibited phosphorylation of H2A and Cdc20, and attenuated LANA-induced aneuploidy and cell proliferation. This molecular mechanism whereby KSHV-induced CIN, demonstrated that the NNLS of LANA is a promising target for development of anti-viral therapies targeting KSHV associated cancers.

Epstein-Barr Virus Nuclear Antigen 3C Facilitates Cell Proliferation by Regulating Cyclin D2.

Cell cycle regulation is one of the hallmarks of virus-mediated oncogenesis. Epstein-Barr virus (EBV)-induced lymphomas express a repertoire of essential viral latent proteins that regulate expression of cell cycle-related proteins to dysregulate this process, thereby facilitating the proliferation of infected cells. We now demonstrate that the essential EBV latent protein 3C (EBNA3C) stabilizes cyclin D2 to regulate cell cycle progression. More specifically, EBNA3C directly binds to cyclin D2 and they colocalize together in nuclear compartments. We show that EBNA3C regulates the promoter of cyclin D2 through cooperation with master transcription factor Bcl6 and enhances its stability by inhibiting its ubiquitin-dependent degradation. EBNA3C also promoted cell proliferation in the presence of cyclin D2, suggesting that cyclin D2 contributes to EBNA3C-mediated cell cycle progression. These results provide new clues as to the role of this essential viral latent protein and its ability to regulate expression of cellular factors, which drives the oncogenic process.IMPORTANCE Epstein-Barr virus (EBV) is the first identified human tumor virus and is associated with a range of human cancers. During EBV-induced lymphomas, the essential viral latent proteins modify the expression of cell cycle-related proteins to disturb the cell cycle process, thereby facilitating the proliferative process. The essential EBV nuclear antigen 3C (EBNA3C) plays an important role in EBV-mediated B-cell transformation. Here we show that EBNA3C stabilizes cyclin D2 to regulate cell cycle progression. More specifically, EBNA3C directly binds to cyclin D2, and they colocalize together in nuclear compartments. EBNA3C enhances cyclin D2 stability by inhibiting its ubiquitin-dependent degradation and significantly promotes cell proliferation in the presence of cyclin D2. Our results provide novel insights into the function of EBNA3C on cell progression by regulating the cyclin D2 protein and raise the possibility of the development of new anticancer therapies against EBV-associated cancers.