Virus-induced Transformation Research Articles

Cellular SUMO-specific proteases regulate HAdV-C5 E1B-55K SUMOylation and virus-induced cell transformation.

Various viral proteins are post-translationally modified by SUMO-conjugation during the human adenovirus (HAdV) replication cycle. This modification leads to diverse consequences for target proteins as it influences their intracellular localization or cell transformation capabilities. SUMOylated HAdV proteins include the multifunctional oncoprotein E1B-55K. Our previous research, along with that of others, has demonstrated a substantial influence of yet another adenoviral oncoprotein, E4orf6, on E1B-55K SUMOylation levels. Protein SUMOylation can be reversed by cellular sentrin/SUMO-specific proteases (SENPs). In this study, we investigated the interaction of E1B-55K with cellular SENPs to understand deSUMOylation activities and their consequences for cell transformation mediated by this adenoviral oncoprotein. We show that E1B-55K interacts with and is deSUMOylated by SENP 1, independently of E4orf6. Consistent with these results, we found that SENP 1 prevents E1A/E1B-dependent focus formation in rodent cells. We anticipate these findings to be the groundwork for future studies on adenovirus-host interactions, the mechanisms that underlie E1B-55K SUMOylation, as well as the role of this major adenoviral oncoprotein in HAdV-mediated cell transformation.

The effect of virus-induced cellular transformation on oncogenesis

Aim to summarize the scientific data presented in the recent publications on tumor-associated processes induced by viruses. We analyzed 23 international publications devoted to the development and course of tumor-related processes associated with oncogenic viruses.
 The tumor-associated mechanisms are based on the processes of cell transformation, which largely depend on the state of telomeres. No less important are viral and cellular oncogenes, molecular circuits that control cell proliferation. Viral oncogenes encode proteins that increase the concentration of telomerase in the infected cells, and thereby increase the number of cell division cycles. The immune homeostasis, maintaining the integrity of body tissues, is regulated by activating and inhibiting metabolic pathways. The errors in the functioning of these signaling pathways caused by oncogenic viruses can lead to cell transformation and oncogenesis. Guaninenucleotide-binding protein RAS and protein kinase AKT are important components of signaling pathways that contribute to the production of D-type cyclins that control the cell cycle and regulate the activity of metabolic enzymes. Cyclin-dependent kinase is an important factor controlling cell cycles, damage and problems with nucleic acid replication, as well as proper assembly of the mitotic spindle. These processes can be disrupted by the transformation caused by oncogenic viruses. In most cases, viral oncogenes undergo additional changes that contribute to their transformation potential. The transformative activity of viral gene products correlates with binding to specific cellular proteins. In the immunopathogenesis of oncogenesis, an important role belongs to the inactivation of tumor suppressors, and the processes of phosphorylation.

Identification of genes for seed isoflavones based on bulk segregant analysis sequencing in soybean natural population.

We identified four hub genes for isoflavone biosynthesis based on BSA-seq and WGCNA methods and validated that GmIE3-1 positively contribute to isoflavone accumulation in soybean. Soybean isoflavones are secondary metabolites of great interest owing to their beneficial impact on human health. Herein, we profiled the seed isoflavone content by HPLC in 1551 soybean accessions grown in two locations for two years and constructed two extreme pools with high (4065.1µgg-1) and low (1427.23µgg-1) isoflavone contents to identify candidate genes involved in isoflavone biosynthesis pathways using bulk segregant analysis sequencing (BSA-seq) approach. The results showed that the average sequencing depths were 50.3× and 65.7× in high and low pools, respectively. A total of 23,626 polymorphic SNPs and 5299 InDels were detected between both pools and 1492 genes with different variations were identified. Based on differential genes in BSA-seq and weighted gene co-expression network analysis (WGCNA), four hub genes, Glyma.06G290400 (designated as GmIE3-1), Glyma.01G239200, Glyma.01G241500, Glyma.13G256100 were identified, encoding E3 ubiquitin-protein ligase, arm repeat protein interacting with ABF2, zinc metallopeptidase EGY3, and dynamin-related protein 3A, respectively. The allelic variation in GmIE3-1 showed a significant influence on isoflavone accumulation. The virus-induced gene silencing (VIGS) and RNAi hairy root transformation of GmIE3-1 revealed partial suppression of this gene could cause a significant decrease (P < 0.0001) of total isoflavone content, suggesting GmIE3-1 is a positive regulator for isoflavones. The present study demonstrated that the BSA-seq approach combined with WGCNA, VIGS and hairy root transformation can efficiently identify isoflavone candidate genes in soybean natural population.

SARS-CoV2 Infection and Comorbidities, Role in Oncogenesis.

The widespread infectious disease caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) significantly impacts major human diseases. It is undoubtedly evident that cancer patients are more susceptible to the infection and at a higher risk of severe COVID-19 than the general population. Moreover, the rise in cancers incidence is waiting in the Globe as a long-term effect of post-COVID-19 complications. Multiple mostly unknown mechanisms participate and determine the oncogenic impact of virus-induced transformation. Imbalance in oncogenesis is considered critical in cancer development. Modified immunogenicity and metabolic features emerge as pivotal in COVID-19 pathogenesis and the organism system's response. The molecular mechanisms of the onset of the metabolic disorder have not yet been fully elucidated. The pathology is complicated, multifactorial, and emerging in various processes. Preventive anticancer therapy taking into account the change in metabolic processes, helps them respond better to anti-COVID-19 treatment than relying only on antiviral drugs. The modified therapeutic algorithm was provided to reduce the likelihood of post-acute complications in patients with preexisting pathologies and the onset of other chronic pathologies and cancers.

Virus-infection in cochlear supporting cells induces audiosensory receptor hair cell death by TRAIL-induced necroptosis.

Although sensorineural hearing loss (SHL) is relatively common, its cause has not been identified in most cases. Previous studies have suggested that viral infection is a major cause of SHL, especially sudden SHL, but the system that protects against pathogens in the inner ear, which is isolated by the blood-labyrinthine barrier, remains poorly understood. We recently showed that, as audiosensory receptor cells, cochlear hair cells (HCs) are protected by surrounding accessory supporting cells (SCs) and greater epithelial ridge (GER or Kölliker's organ) cells (GERCs) against viral infections. Here, we found that virus-infected SCs and GERCs induce HC death via production of the tumour necrosis factor-related apoptosis-inducing ligand (TRAIL). Notably, the HCs expressed the TRAIL death receptors (DR) DR4 and DR5, and virus-induced HC death was suppressed by TRAIL-neutralizing antibodies. TRAIL-induced HC death was not caused by apoptosis, and was inhibited by necroptosis inhibitors. Moreover, corticosteroids, the only effective drug for SHL, inhibited the virus-induced transformation of SCs and GERCs into macrophage-like cells and HC death, while macrophage depletion also inhibited virus-induced HC death. These results reveal a novel mechanism underlying virus-induced HC death in the cochlear sensory epithelium and suggest a possible target for preventing virus-induced SHL.

In Vivo Functional Verification of Four Related Genes Involved in the 1-Deoxynojirimycin Biosynthetic Pathway in Mulberry Leaves.

The alkaloid 1-deoxynojirimycin (DNJ) is one of the major bioactive compounds in mulberry leaves (Morus alba L.). Previously, we discovered four key genes involved in the pathway from lysine to piperidine in the biosynthesis of DNJ in mulberry leaves, MaLDC (MG727866), MaCAO (MH205733), MaSDR1 (MT989445), and MaSDR2 (MT989446), which encoded lysine decarboxylase, copper amine oxidase, and short-chain dehydrogenase/reductase 1 and 2, respectively. However, the in vivo functions of these four genes have not been verified yet. Here, these four genes were successfully cloned and used for the establishment of C58C1 Agrobacterium rhizogenes mediated overexpression genetic transformation systems and GV3101 Agrobacterium-mediated virus-induced gene silencing transformation systems in order to verify the influence of these four genes on the biosynthetic content of DNJ in mulberry leaves. The results showed that the content of DNJ increased after the four genes were overexpressed. When these four genes were silenced, the gene expression was blocked, which affected the biosynthesis of DNJ, and the DNJ content decreased. The above results indicated that these four genes participated in DNJ biosynthesis. This study provided a foundation for further elucidating the regulatory mechanisms of DNJ biosynthesis in mulberry leaves.

Differential Regulation of Cellular FAM111B by Human Adenovirus C Type 5 E1 Oncogenes

The adenovirus type 5 (HAdV-C5) E1 transcription unit encodes regulatory proteins that are essential for viral replication and transformation. Among these, E1A and E1B-55K act as key multifunctional HAdV-C5 proteins involved in various steps of the viral replication cycle and in virus-induced cell transformation. In this context, HAdV-C5-mediated dysregulations of cellular factors such as the tumor suppressors p53 and pRB have been intensively investigated. However, cellular components of downstream events that could affect infection and viral transformation are widely unknown. We recently observed that cellular FAM111B is highly regulated in an E1A-dependent fashion. Intriguingly, previous reports suggest that FAM111B might play roles in tumorigenesis, but its exact functions are not known to date. Here, we set out to investigate the role of FAM111B in HAdV-C5 infections. We found that (i) FAM111B levels are upregulated early and downregulated late during infection, that (ii) FAM111B expression is differentially regulated, that (iii) FAM111B expression levels depend on the presence of E1B-55K and E4orf6 and that (iv) a FAM111B knockdown increases HAdV-C5 replication. Our data indicate that FAM111B acts as an anti-adenoviral host factor that is involved in host cell defense mechanisms in productive HAdV-C5 infection. Moreover, these findings suggest that FAM111B might play an important role in the host antiviral immune response that is counteracted by HAdV-C5 E1B-55K and E4orf6 oncoproteins.

Screening and functional characterization of candidate resistance genes to powdery mildew from Dasypyrum villosum#4 in a wheat line Pm97033.

Three genes designated DvLox, Pm21#4, and Pm21#4-H identified in a wheat-Dasypyrum villosum#4 T6V#4S·6DL translocation line Pm97033 conferred wheat for powdery mildew resistance. Powdery mildew (PM) caused by Blumeria graminis f. sp. tritici (Bgt) is one of the most devastating diseases in wheat. To date, only a few genes conferring resistance to wheat PM are cloned. Dasypyrum villosum is a wild relative of wheat, which provides Pm21 conferring wheat immunity to PM. In this study, we obtained many differentially expressed genes (DEGs) from a wheat-D. villosum#4 T6V#4S·6DL translocation line Pm97033 using RNA-sequencing. Among them, 7 DEGs associated with pathogen resistance were up-regulated in front of Bgt infection. Virus-induced gene silencing and transformation assays demonstrated that two of them, DvLox and Pm21#4 encoding a lipoxygenase and a encoding coiled-coil/nucleotide-binding site/leucine-rich repeat resistance protein, conferred wheat PM resistance. The transgenic wheat plants expressing DvLox enhanced PM resistance, and the transgenic wheat plants expressing Pm21#4 showed PM immunity. The Pm21#4-silenced Pm97033 plants by the cluster regularly interspaced short palindromic repeats-associated endonuclease (CRISPR/Cas9) system were susceptible to PM. Thus, Pm21#4 is a key gene contributing PM immune resistance in Pm97033. Constitutively expression of Pm21#4-H, which is silenced in Pm97033 and D. villosum#4, endowed a PM-susceptible wheat variety Fielder with PM immune resistance.

Marek's Disease Virus Infection Induced Mitochondria Changes in Chickens.

Mitochondria are crucial cellular organelles in eukaryotes and participate in many cell processes including immune response, growth development, and tumorigenesis. Marek’s disease (MD), caused by an avian alpha-herpesvirus Marek’s disease virus (MDV), is characterized with lymphomas and immunosuppression. In this research, we hypothesize that mitochondria may play roles in response to MDV infection. To test it, mitochondrial DNA (mtDNA) abundance and gene expression in immune organs were examined in two well-defined and highly inbred lines of chickens, the MD-susceptible line 72 and the MD-resistant line 63. We found that mitochondrial DNA contents decreased significantly at the transformation phase in spleen of the MD-susceptible line 72 birds in contrast to the MD-resistant line 63. The mtDNA-genes and the nucleus-genes relevant to mtDNA maintenance and transcription, however, were significantly up-regulated. Interestingly, we found that POLG2 might play a potential role that led to the imbalance of mtDNA copy number and gene expression alteration. MDV infection induced imbalance of mitochondrial contents and gene expression, demonstrating the indispensability of mitochondria in virus-induced cell transformation and subsequent lymphoma formation, such as MD development in chicken. This is the first report on relationship between virus infection and mitochondria in chicken, which provides important insights into the understanding on pathogenesis and tumorigenesis due to viral infection.

DNA Tumor Viruses and Cell Metabolism.

Viruses play an important role in cancerogenesis. It is estimated that approximately 20% of all cancers are linked to infectious agents. The viral genes modulate the physiological machinery of infected cells that lead to cell transformation and development of cancer. One of the important adoptive responses by the cancer cells is their metabolic change to cope up with continuous requirement of cell survival and proliferation. In this review we will focus on how DNA viruses alter the glucose metabolism of transformed cells. Tumor DNA viruses enhance “aerobic” glycolysis upon virus-induced cell transformation, supporting rapid cell proliferation and showing the Warburg effect. Moreover, viral proteins enhance glucose uptake and controls tumor microenvironment, promoting metastasizing of the tumor cells.

Oxidative stress enables Epstein-Barr virus-induced B-cell transformation by posttranscriptional regulation of viral and cellular growth-promoting factors.

Infection of human B lymphocytes by Epstein-Barr virus (EBV) leads to the establishment of immortalized lymphoblastoid cell lines (LCLs) that are widely used as a model of viral oncogenesis. An early consequence of infection is the induction of DNA damage and activation of the DNA damage response, which limits the efficiency of growth transformation. The cause of the DNA damage remains poorly understood. We have addressed this question by comparing the response of B lymphocytes infected with EBV or stimulated with a potent B-cell mitogen. We found that although the two stimuli induce comparable proliferation during the first 10 days of culture, the EBV-infected blasts showed significantly higher levels of DNA damage, which correlated with stronger and sustained accumulation of reactive oxygen species (ROS). Treatment with ROS scavengers decreased DNA damage in both mitogen-stimulated and EBV-infected cells. However, while mitogen-induced proliferation was slightly improved, the proliferation of EBV-infected cells and the establishment of LCLs were severely impaired. Quenching of ROS did not affect the kinetics and magnitude of viral gene expression but was associated with selective downregulation of the viral LMP1 and phosphorylated cellular transcription factor STAT3 that have key roles in transformation. Analysis of the mechanism by which high levels of ROS support LMP1 expression revealed selective inhibition of viral microRNAs that target the LMP1 transcript. Our study provides novel insights into the role of EBV-induced oxidative stress in promoting B-cell immortalization and malignant transformation.

The mycotoxin aflatoxin B1 stimulates Epstein-Barr virus-induced B-cell transformation in in vitro and in vivo experimental models.

Although Epstein-Barr virus (EBV) infection is widely distributed, certain EBV-driven malignancies are geographically restricted. EBV-associated Burkitt's lymphoma (eBL) is endemic in children living in sub-Saharan Africa. This population is heavily exposed to food contaminated with the mycotoxin aflatoxin B1 (AFB1). Here, we show that exposure to AFB1 in in vitro and in vivo models induces activation of the EBV lytic cycle and increases EBV load, two events that are associated with an increased risk of eBL in vivo. AFB1 treatment leads to the alteration of cellular gene expression, with consequent activations of signaling pathways, e.g. PI3K, that in turn mediate reactivation of the EBV life cycle. Finally, we show that AFB1 triggers EBV-driven cellular transformation both in primary human B cells and in a humanized animal model. In summary, our data provide evidence for a role of AFB1 as a cofactor in EBV-mediated carcinogenesis.

Up-regulation of human cervical cancer proto-oncogene contributes to hepatitis B virus-induced malignant transformation of hepatocyte by down-regulating E-cadherin.

Hepatocellular carcinoma (HCC) is one of the most fatal human malignancies, Human cervical cancer proto-oncogene (HCCR) aberrantly expressed in a number of malignant tumors, including HCC. HCC is associated with Hepatitis B virus (HBV) infection in a large percentage of cases. To explore the regulation and function of HCCR expression in the development of HCC, we detected HCCR expression in HBV expressing hepatocytes. Results showed that the expression of HCCR was higher in HBV-expressing hepatocytes than that in control cells. Examining different components of HBV revealed that the HBx promotes HCCR expression in hepatocytes via the T-cell factor (TCF)/β-catenin pathway. HCCR expression in HBx transgenic mice increased with as the mice aged and developed tumors. We also found that overexpression of HCCR in hepatocytes promoted cell proliferation, migration, and invasion and reduced cell adhesion. Suppressing HCCR expression abolished the effect of HBx-induced hepatocyte growth. In addition, HCCR represses the expression of E-cadherin by inhibition its promoter activity, which might correlate with the effects of HCCR in hepatocytes. Taken together, these results demonstrate that HBx-HCCR-E-cadherin regulation pathway might play an important role in HBV-induced hepatocarcinogenesis. They also imply that HCCR is a potential risk marker for HCC and/or a potential therapeutic target.

Role of the microenvironment in tumourigenesis: focus on virus-induced tumors.

Tumor microenvironment can differ considerably in various types of tumors in terms of cellular and cytokine networks and molecular drivers. The well known link between inflammation and cancer has recently found a number of genetic and molecular confirmations. In this respect, numerous reports have revealed that infection and chronic inflammation can contribute to cancer development, progression and control. Adhesion molecules, chemokines and proinflammatory cytokines, that enroll leukocytes, are persistently present in cancer microenvironment, thus increasing the risk for developing tumors. In this respect, cancer-derived microvescicles, in particular exosomes, exert an important role in the recruitment and reprogramming of components of tumor microenvironment. The relationship between cancer and virus infection has generated, in recent years, a great interest for studies aiming to better understand the role of the immune system in the control of these infections and of the immune cofactors in the promotion of the virus-induced neoplastic transformation. This suggests that virus-induced immune alterations may play a role to create an immunotolerogenic microenvironment during the carcinogenesis process.

Higher incidence of Epstein-Barr virus-induced lymphocyte transformation in multiple sclerosis

Epstein-Barr virus (EBV) infection is associated with multiple sclerosis (MS), and EBV may transform lymphoblastoid cell lines more frequently in MS patients than controls, but it is not clear whether this reflects a higher viral load or an enhanced ability to reactivate EBV. MS patients and controls were examined for their B-cell subsets and during 16 weeks for spontaneous lymphocyte transforming events. MS patients had normal distribution of B-cell subsets, but a significantly higher incidence of B-cell transforming events, which occurred with kinetics similar to controls. The higher incidence suggests an increased frequency of latent EBV-infected B cells in MS.

Polyomavirus BK large tumor antigen exerts tolerogenic signatures with immunodominant p53-binding regions in prostate cancer

Event Abstract Back to Event Polyomavirus BK large tumor antigen exerts tolerogenic signatures with immunodominant p53-binding regions in prostate cancer Maurizio Provenzano1* 1 Division of Urology, University Hospital of Zurich, Oncology Unit, Switzerland Polyomavirus BK (BKV) large tumor antigen (L-Tag) has been recently identified as a potential co-factor in the development of prostate cancer (PCa) but its role as target of immune responses in this malignancy remains unexplored. A regulatory profiling elicited by L-Tag peptide-pool stimulation has been observed in BKV seropositive PCa patients bearing BKV positive lesions and evidence of biochemical recurrence. Differently, T cells against p53-binding regions of L-Tag exquisitely belong to effector/memory CD8+ T cells populations in BKV seropositive healthy donors. The ambiguous activity of L-Tag prompted us to investigate the role of functional regions within L-Tag in eliciting peculiar immune responses in PCa. Two peptides, L-Tag406-414 and L-Tag579-587, nested in the p53-binding regions of L-Tag and previously documented to induce pro-inflammatory responses in healthy donors, triggered an immune regulatory response (IL-10- and TGF-β-producing CD4+CD25+CD127- T cells) with suppressive properties in 61% and 54% of PCa patients studied (n=20), respectively. In contrast, four peptides identified within L-Tag regions non specifically required for virus-induced malignant transformation, recalled IFN-γ-producing effector/memory CD8+ and CD4+ T cells with cytotoxic (CD107+) and no-exhausted (PD-1-) phenotype, able to boost immunogenic activities in PCa patients with a BKV-driven tolerogenic signatures. These findings suggest that strategic regions of L-Tag appointed to carry out oncogenic activities might orchestrate tumor-promoting environment. However, it also gives evidence that a systemic boosting of BKV seropositive PCa patients with immunogenic portions of BKV L-Tag would generate potent antigen-specific immune responses and thereby break the tolerogenic potential governed by BKV L-Tag in this disease. References G. Sais, S. Wyler, T. Hudolin, I. Banzola, C. Mengus, L. Bubendorf, P. J. Wild, H. H. Hirsch, T. Sulser, G. C. Spagnoli and M. Provenzano: Differential patterns of large tumor antigen-specific immune responsiveness in patients with BK polyomavirus-positive prostate cancer or benign prostatic hyperplasia. J Virol, 86(16), 8461-71 (2012) M. Provenzano, L. Bracci, S. Wyler, T. Hudolin, G. Sais, R. Gosert, P. Zajac, G. Palu, M. Heberer, H. H. Hirsch and G. C. Spagnoli: Characterization of highly frequent epitope-specific CD45RA+/CCR7+/- T lymphocyte responses against p53-binding domains of the human polyomavirus BK large tumor antigen in HLA-A*0201+ BKV-seropositive donors. J Transl Med, 4, 47 (2006) Keywords: virus-induced oncogenesis, breaking immunological tolerance, regulatory T cells, prostate cancer, polyomavirus BK Large tumor antigen Conference: 15th International Congress of Immunology (ICI), Milan, Italy, 22 Aug - 27 Aug, 2013. Presentation Type: Abstract Topic: Host-pathogen interactions Citation: Provenzano M (2013). Polyomavirus BK large tumor antigen exerts tolerogenic signatures with immunodominant p53-binding regions in prostate cancer. Front. Immunol. Conference Abstract: 15th International Congress of Immunology (ICI). doi: 10.3389/conf.fimmu.2013.02.00055 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 08 Mar 2013; Published Online: 22 Aug 2013. * Correspondence: Dr. Maurizio Provenzano, Division of Urology, University Hospital of Zurich, Oncology Unit, Zurich, Zurich, 8091, Switzerland, maurizio.provenzano@gmail.com Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Maurizio Provenzano Google Maurizio Provenzano Google Scholar Maurizio Provenzano PubMed Maurizio Provenzano Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Design stars: how Small DNA Viruses Remodel the Host Nucleus

Numerous host components are encountered by viruses during the infection process. While some of these host structures are left unchanged, others may go through dramatic remodeling processes. In this review, we summarize these host changes that occur during small DNA virus infections, with a focus on host nuclear components and pathways. Although these viruses differ significantly in their genome structures and infectious pathways, there are common nuclear targets that are altered by various viral factors. Accumulating evidence suggests that these nuclear remodeling processes are often essential for productive viral infections and/or viral-induced transformation. Understanding the complex interactions between viruses and these host structures and pathways will help to build a more integrated network of how the virus completes its life cycle and point toward the design of novel therapeutic regimens that either prevent harmful viral infections or employ viruses as nontraditional treatment options or molecular tools.

KSHV infects a subset of human tonsillar B cells, driving proliferation and plasmablast differentiation

Kaposi sarcoma-associated herpesvirus (KSHV; also known as HHV8) is the causative agent of two B cell tumors, multicentric Castleman disease (MCD) and primary effusion lymphoma (PEL). However, little is known about the nature of the specific B cell subtype(s) most susceptible to infection. Identifying these cells would provide direct insight into KSHV transmission and virus-induced transformation. To identify this subset and to determine whether infection alters its cellular phenotype, we exposed human tonsillar cells to KSHV and characterized infected cells using high-throughput multispectral imaging flow cytometry (MIFC). Stable expression of the virally encoded latency-associated nuclear antigen (LANA), a marker of latent KSHV infection, was observed predominantly in cells expressing the l light chain of the B cell receptor. These LANA+ B cells proliferated and exhibited similarities to the cells characteristic of MCD (IgMl-expressing plasmablasts), including blasting morphology with elevated expression of Ki67, variable expression of CD27, and high levels of IgM and IL-6 receptor. Furthermore, the proportion of infected cells showing a blasting phenotype increased upon addition of exogenous IL-6. Our data lead us to propose that oral transmission of KSHV involves the latent infection of a subset of tonsillar IgMl-expressing B cells, which then proliferate as they acquire the plasmablast phenotype characteristic of MCD.

Herpesvirus Telomerase RNA(vTR)-Dependent Lymphoma Formation Does Not Require Interaction of vTR with Telomerase Reverse Transcriptase (TERT)

Telomerase is a ribonucleoprotein complex involved in the maintenance of telomeres, a protective structure at the distal ends of chromosomes. The enzyme complex contains two main components, telomerase reverse transcriptase (TERT), the catalytic subunit, and telomerase RNA (TR), which serves as a template for the addition of telomeric repeats (TTAGGG)n. Marek's disease virus (MDV), an oncogenic herpesvirus inducing fatal lymphoma in chickens, encodes a TR homologue, viral TR (vTR), which significantly contributes to MDV-induced lymphomagenesis. As recent studies have suggested that TRs possess functions independently of telomerase activity, we investigated if the tumor-promoting properties of MDV vTR are dependent on formation of a functional telomerase complex. The P6.1 stem-loop of TR is known to mediate TR-TERT complex formation and we show here that interaction of vTR with TERT and, consequently, telomerase activity was efficiently abrogated by the disruption of the vTR P6.1 stem-loop (P6.1mut). Recombinant MDV carrying the P6.1mut stem-loop mutation were generated and tested for their behavior in the natural host in vivo. In contrast to viruses lacking vTR, all animals infected with the P6.1mut viruses developed MDV-induced lymphomas, but onset of tumor formation was significantly delayed. P6.1mut viruses induced enhanced metastasis, indicating functionality of non-complexed vTR in tumor dissemination. We discovered that RPL22, a cellular factor involved in T-cell development and virus-induced transformation, directly interacts with wild-type and mutant vTR and is, consequently, relocalized to the nucleoplasm. Our study provides the first evidence that expression of TR, in this case encoded by a herpesvirus, is pro-oncogenic in the absence of telomerase activity.

THIS ARTICLE HAS BEEN RETRACTED Epstein–Barr virus‐encoded latent membrane protein 1 activates β‐catenin signaling in B lymphocytes

The Epstein-Barr virus-encoded latent membrane protein 1 is considered the Epstein-Barr virus oncogene based on its importance in Epstein-Barr virus-induced B-lymphocyte transformation. Beta-catenin is a potential oncogene, and its accumulation has been implicated in a variety of human cancers. Here, we found that beta-catenin protein was highly expressed in Epstein-Barr virus-immortalized B-cell lines compared with peripheral blood mononuclear cells from healthy donors. Beta-catenin expression in Epstein-Barr virus-immortalized B-cell line decreased following treatment with LY294002, an inhibitor of phosphatidylinositol 3-kinase. Treatment with LY294002 or knockdown of beta-catenin by small interfering RNA reduced the growth of Epstein-Barr virus-immortalized B-cell line. Transient transfection of latent membrane protein 1 expression plasmid increased beta-catenin protein expression and beta-catenin-dependent transcription. Latent membrane protein 1 deletions mutants lacking the carboxyl-terminal activating region 1 domain failed to enhance beta-catenin protein expression and beta-catenin-dependent transcriptional activity. They also failed to increase phosphorylated AKT expression. Dominant-negative AKT suppressed latent membrane protein 1-induced beta-catenin-dependent transcriptional activity. These results suggest that latent membrane protein 1 activates beta-catenin through the phosphatidylinositol 3-kinase/AKT signaling pathway. Activation of the beta-catenin pathway by Epstein-Barr virus may contribute to the lymphoproliferation characteristic of Epstein-Barr virus-infected B-cells.