424 research outputs found

    The DNA damage response promotes Polyomavirus JC infection by nucleus to cytoplasm NF-Kappa B activation.

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    Background: Infection of glial cells by human neurotropic polyomavirus JC (JCV), the causative agent of the CNS demyelinating disease progressive multifocal leukoencephalopathy (PML), rapidly inflicts damage to cellular DNA. This activates DNA damage response (DDR) signaling including induction of expression of DNA repair factor Rad51. We previously reported that Rad51 co-operates with the transcription factor NF-κB p65 to activate JCV early transcription. Thus Rad51 induction by JCV infection may provide positive feedback for viral activation early in JCV infection. DDR is also known to stimulate NF-κB activity, a phenomenon known as nucleus to cytoplasm or “insideout” NF-κB signaling, which is initiated by Ataxia telangiectasia mutated (ATM) protein, a serine/threonine kinase recruited and activated by DNA double-strand breaks. Downstream of ATM, there occurs a series of posttranslational modifications of NF-κB essential modulator (NEMO), the γ regulatory subunit of inhibitor of NF-κB (IκB) kinase (IKK), resulting in NF-κB activation. Methods: We analyzed the effects of downstream pathways in the DDR by phosphospecific Western blots and analysis of the subcellular distribution of NEMO by cell fractionation and immunocytochemistry. The role of DDR in JCV infection was analyzed using a small molecule inhibitor of ATM (KU-55933). NEMO sumoylation was investigated by Western and association of ATM and NEMO by immunoprecipitation/Western blots. Results: We show that JCV infection caused phosphorylation and activation of ATM while KU-55933 inhibited JCV replication. JCV infection caused a redistribution of NEMO from cytoplasm to nucleus. Co-expression of JCV large Tantigen and FLAG-tagged NEMO showed the occurrence of sumoylation of NEMO, while co-expression of ATM and FLAG-NEMO demonstrated physical association between ATM and NEMO. Conclusions: We propose a model where JCV infection induces both overexpression of Rad51 protein and activation of the nucleus to cytoplasm NF-κB signaling pathway, which then act together to enhance JCV gene expression

    Comparative analyses of tumorigenic mechanisms of Merkel cell polyomavirus T antigens

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    The work described in this dissertation began in 2013 and is focused on characterizing the mechanisms by which Merkel cell polyomavirus (MCV) Large T (LT) and small T (sT) antigens induce tumorigenesis through comparative analyses with oncoproteins from other tumor viruses. A peptide motif in the C-terminal region of MCV LT that bears little sequence homology with other human polyomavirus LT proteins is shown to be critical for maintaining stability of the full-length LT protein (Chapter 3). Comparison between LT antigens of MCV and SV40 demonstrate that MCV LT in direct contrast to SV40 LT is incapable of avidly binding tumor suppressor p53 and inhibiting its transactivation capabilities (Chapter 4). Lastly, promiscuous E3 ligase targeting by MCV sT through its LT-stabilization domain (LSD) results in the formation of a genomically unstable phenotype, a known hallmark of cancer (Chapter 5). Many of the features of genomic instability induced by MCV sT such as centrosome overduplication parallel what has been observed previously for human papillomavirus 16 E7 oncoprotein. Overall, these comparative analyses have not only provided greater insight into MCV biology and how its T antigens function in causing an aggressive cancer like Merkel cell carcinoma (MCC), but they have also revealed new avenues for continued study involving MCV T antigens that will continue to move the field of tumor virology forward

    Insight into Merkel Cell Polyomavirus Replication Through the Study of the Viral Early Proteins: Large and Small Tumor Antigens

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    The association of Merkel cell polyomavirus (MCPyV) and Merkel cell carcinoma (MCC) established in 2008 has opened doors for the study of virus-induced oncogenesis. Much of what is known about polyomaviruses (PyVs) stems from decades of studies on SV40. However, recent research has shown differences among PyVs that might help explain the uniqueness of MCPyV that makes it the only PyV discovered to date that is associated with a human cancer. Therefore, it is important to understand the biology of this virus and its oncogenic potential. My study focuses on two of the early proteins of MCPyV, namely the large tumor antigen (LT) and the small tumor antigen (sT). Both proteins are multi-functional, contributing to viral replication and the stimulation of cellular proliferation. MCPyV LT is the viral helicase that binds to the viral origin (Ori) to initiate unwinding and the replication of the double-stranded DNA genome. Like other PyVs, MCPyV requires numerous cellular proteins to replicate its genome. In my research, I show that, in the presence of the viral Ori, the binding of LT to the Ori forms replication factories in the nucleus. A number of cellular factors involved in the host DNA damage response (DDR) re-localize to the sites of MCPyV LT-mediated replication. Inhibition of the DNA damage response by either drug treatment or siRNA knockdown decreases MCPyV replication, suggesting that an intact host DDR pathway is essential for the optimal replication of MCPyV. Previous research has shown that MCPyV sT indirectly enhances viral replication by stabilizing LT. In my study, I discover that sT is a metalloprotein that coordinates two iron-sulfur clusters. Mutations in the highly conserved cysteines found in MCPyV sTs abolishes its ability to stimulate LT-mediated viral DNA replication, and that sT can enhance LT-mediated replication in a manner that is independent of LT stabilization. Moreover, I show that sT translocates to the nuclear replication factories formed in the presence of LT and Ori, suggesting a more direct role of sT in promoting viral DNA replication. Interestingly, upon treatment with the potent antiviral agent cidofovir, sT-mediated enhancement of MCPyV replication is robustly inhibited, while replication driven by LT alone was not affected much. This finding supports the use of cidofovir in controlling PyV infection and offers MCPyV sT as a potential drug target to dampen viral growth. In summary, my work elucidates various aspects of MCPyV replication, including the involvement of the host DDR machinery and the role of sT that could potentially be targeted by drug treatment. This study contributes to the understanding of the virology of MCPyV and opens avenues for further research

    Connecting viral oncoproteins to microRNA, autophagy and metabolism in Merkel cell carcinoma

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    Merkel cell carcinoma (MCC) is an aggressive type of skin cancer. Around 80% of MCCs harbor an integrated Merkel cell polyomavirus (MCPyV) genome with a mutation in the large T antigen (LT) gene, leading to expression of truncated LT. It is evident that the viral truncated LT and small T antigen (sT) play important roles in MCC tumorigenesis. Yet, the molecular mechanisms how these viral oncoproteins contribute to MCC development remain unclear. The studies presented in this thesis aim to further our understanding of the functional role of these MCPyV oncoproteins in MCC tumorigenesis. In Paper I, we demonstrated that MCPyV T-antigens induce miR-375, miR-30a-3p and miR- 30a-5p through the DnaJ domain of the viral T-antigen. These miRNAs could target multiple autophagy genes (ATG7, SQSTM1 and BECN1) and suppress autophagy in MCC cells. Additionally, we showed that both sT and truncated LT also suppress autophagy, but not the full-length LT. Inhibition of autophagy, but not pan-caspases, could rescue cell death induced by the mTOR inhibitor Torin-1, suggesting that suppression of autophagy is crucial for cell survival in MCC. In Paper II, we found paranuclear dot-like staining of c-KIT in MCPyV positive (MCPyV+) MCC cell lines and tumor samples. Mechanistically, we showed that MCPyV truncated LT induces paranuclear retention of c-KIT through its Vam6p binding site. Our results also revealed that c-KIT interacts with BECN1. Silencing of c-KIT increased autophagy and apoptosis, and decreased LT expression. Inhibition of autophagic degradation in c-KIT depleted cells restored the LT expression, suggesting the importance of autophagy suppression to sustain the expression of viral oncoprotein and cell survival. In Paper III, we showed that overexpression of miR-375 suppressed cell growth and migration in MCPyV- MCC cell lines, while suppression of miR-375 decreased cell growth and increased apoptosis in MCPyV+ MCC cell lines. The expression of LDHB, a target of miR-375, was inversely correlated with miR-375. Silencing of LDHB decreased cell growth in MCPyV- MCC cells, but rescued cell growth suppression resulted from miR-375 inhibition in MCPyV+ MCC cells. Our findings suggest that miR-375 regulation of LDHB plays distinct roles in MCPyV+ and MCPyV- MCC. In Paper IV, we demonstrated that MCPyV T-antigens reduce LDHB expression and promote glycolysis in MCC. Overexpression of LDHB reduced cell viability and increased apoptosis in MCPyV+ MCC cells. Ectopic expression of LDHB reversed the growth- promoting effect of MCPyV oncoproteins. Inhibition of glycolysis reduced cell growth in MCPyV+ MCC cells, while inhibition of mitochondrial respiratory activity inhibited cell growth in MCPyV- MCC cells. Our findings suggest that MCPyV+ and MCPyV- MCC cells are dependent on different energy metabolism for cell growth. Overall, this thesis work highlights the diverse functions of MCPyV oncoproteins and their involvement in regulating miRNA expression, autophagy and energy metabolism

    Merkel cell polyomavirus: molecular insights into the most recently discovered human tumour virus.

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    A fifth of worldwide cancer cases have an infectious origin, with viral infection being the foremost. One such cancer is Merkel cell carcinoma (MCC), a rare but aggressive skin malignancy. In 2008, Merkel cell polyomavirus (MCPyV) was discovered as the causative agent of MCC. It is found clonally integrated into the majority of MCC tumours, which require MCPyV oncoproteins to survive. Since its discovery, research has begun to reveal the molecular virology of MCPyV, as well as how it induces tumourigenesis. It is thought to be a common skin commensal, found at low levels in healthy individuals. Upon loss of immunosurveillance, MCPyV reactivates, and a heavy viral load is associated with MCC pathogenesis. Although MCPyV is in many ways similar to classical oncogenic polyomaviruses, such as SV40, subtle differences are beginning to emerge. These unique features highlight the singular position MCPyV has as the only human oncogenic polyomavirus, and open up new avenues for therapies against MCC

    Novel Regulatory Mechanisms by Which Large T Antigen Coordinates the Merkel Cell Polyomavirus Life Cycle

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    Due to its association with Merkel cell carcinoma (MCC), a substantial effort has been made to better understand how Merkel cell polyomavirus (MCPyV) proteins drive oncogenesis; however, our understanding of the early steps of MCPyV infection remains poor. The polyomavirus Large Tumor antigen (LT) is a highly multi-functional protein with a wide range of activities, including: stimulation of cellular proliferation through its interaction with retinoblastoma protein and DnaJ heatshock protein family members; arrest of the cell cycle through a poorly understood activity localized to the C-terminal region; and regulation of the initiation of viral DNA replication. LT proteins also play important roles in regulating viral transcription. How these various functions are regulated to ensure an orderly progression of events conducive for the viral life cycle has not been well established. In this study, I show how phosphorylation of MCPyV LT plays an important role in regulating its many functions. I identify threonines 297 and 299 as key phospho-sites which regulate LT\u27s ability to initiate replication. T297 phosphorylation inhibits LT binding to the viral origin of replication and acts as an off switch, while phosphorylation of T299 is required to stimulate LT-mediated replication of viral genomes. This study was the first to identify phosphorylation sites of LT and link them to important protein functions. Cross-reactivity to a phospho-specific antibody revealed yet another phosphorylation site on MCPyV LT as S816. We discovered that this phosphorylation event is mediated by ATM kinase, and may play a role in the MCPyV LT C-terminal domain\u27s ability to arrest the cell cycle. This study helps to further elucidate MCPyV\u27s association with the host DNA Damage Response (DDR) and provides some rational for the recruitment of these factors to viral replication centers. Finally, studies of the viral non-coding control region (NCCR) reveal a surprising interaction between LT and sT on the late promoter. MCPyV LT is able to robustly stimulate the late promoter only in the context of an intact Ori and sT co-expression. Using phosphomutant LTs and mutant Ori sequences, I highlight the importance of LT binding to the Ori and stimulation of replication as key factors in LT-mediated activation of the late promoter in the context of sT co-expression. LT alone actually represses the late promoter and requires sT coexpression to efficiently stimulate the late promoter after replication. This study therefore reveals an important dependence on sT expression for the regulation of transcription that has not yet been reported with other polyomaviruses. In sum, this study demonstrates multiple mechanisms of regulation including protein phosphorylation, protein-DNA interactions, and co-expression of key viral proteins as regulators of LT function. These studies may help elucidate critical factors required for establishing a robust cellular infection system which is greatly needed to further our understanding of the basic virology of this important human tumor virus

    Merkel cell Polyomavirus (MCPyV) in non-Merkel Cell Carcinoma (non-MCC)

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    Merkel cell polyomavirus (MCPyV) has been detected in respiratory specimens including those from Cystic Fibrosis (CF) patients, raising questions about its immunological and clinical relevance in the respiratory tract. MCPyV is considered the etiological agent of Merkel cell carcinoma (MCC) and, considering the widespread prevalence of the virus across the body, the involvement of MCPyV in tumours other than MCC cannot be excluded. It has been proposed that MCPyV could be associated with non-MCC cancers such as non-small-cell lung carcinoma (NSCLC). A potential association between MCPyV infection and EGFR expression it has also been suggested since BRAF gene, a downstream target of EGFR pathway, was found to be higher expressed in MCPyV positive samples than negative ones. In this study, as objective one, the prevalence of MCPyV-DNA in respiratory samples of a large cohort of CF patients was estimated analyzing viral load and sequencing the LT, NCCR and VP1 regions. In addition, in order to shed light on the potential pathogenic role of MCPyV in CF, demographic, microbiological and clinical data collected from MCPyV-DNA positive and negative patients were compared. Moreover, the transcript expression of TLR9 and distinct IFN-I genes (IFNα, IFNβ and IFNε) were also examined in respiratory samples of MCPyV positive CF patients according to their bacteriological and clinical status. Respiratory samples (n = 1138) were randomly collected from respiratory tract of CF patients (n = 539). MCPyV-DNA was detected in 268 out of 1138 respiratory specimens (23.5%), without any difference in the prevalence of MCPyV-DNA, according to age, gender or bacteriological status of CF individuals. Thirteen out of 137 CF patients remained positive for MCPyV-DNA over the time. Detection of MCPyV-DNA in respiratory specimens was not associated with the occurrence of exacerbation events. Both MCPyV positive adolescents (11–24 years) and adults (≥25 years) had lower mRNA levels of TLR9, IFNβ, IFNε and IFNα than the negative patients of the same age group, while MCPyV positive children produced increased levels of TLR9 and IFN-I genes (p < 0.05 for TLR9, IFNβ, IFNε) with respect to the negative ones. There were significant differences in TLR9 levels (p < 0.01), but not in those of IFNs, between MCPyV-DNA positive and negative patients with S. aureus, P. aeruginosa or both. Overall, these results indicate that MCPyV-DNA is frequently detected in the respiratory samples of CF patients and might influence the expression levels of IFN-related genes in an age dependent manner. The concomitant detection of MCPyV together with S. aureus and/or P. aeruginosa correlated with alterations in TLR9 levels suggesting that virus-bacteria coinfections might contribute to affect antiviral immunity in CF patients. The second aim of this study was to investigate the role of MCPyV as etiological viral agent of NSCLCs by examining a series of NSCLC-patients for both the presence of specific MCPyV DNA and the expressions of viral RNA transcripts (LT and VP1). The integrated form of the MCPyV-positive NSCLCs was also examinate. Formalin-fixed paraffin-embedded tissue (FFPE) of NSCLCs and corresponding nonmalignant lung tissue were obtained from 112 Italian patients undergoing surgery. PCR revealed that 9 out of 32 squamous cell carcinomas (SCCs), 9 out of 45 adenocarcinomas, 1 out of 32 large-cell carcinomas, and 1 out of 3 pleomorphic carcinomas were positive for MCPyV DNA. Some MCPyV DNA-positive cancers expressed LT RNA transcripts. The viral integration sites were identified in one SCC. The other samples didn’t show an integrated MCPyV genome but frameshift mutations in the LT gene. Analysis of EGFR showed that the infection rate of MCPyV was higher in NSCLCs with EGFR mutations than without EGFR mutations; however, this difference was not statistically significant. The obtained results showed the expression of a viral oncoprotein, the presence of integrated MCPyV, and a truncated LT gene with a preserved retinoblastoma tumor-suppressor protein-binding domain in NSCLCs. Although the viral prevalence was low, the tumor-specific molecular signatures support the possibility that MCPyV could be associated with the pathogenesis of NSCLC in a subset of patients. To conclude, since MCPyV infection is observed in occurrence of EGFR mutation, our results could indicate that MCPyV could be considered an EGFR mutagen and might spark interests to deepen the prognostic value of EGFR in NSCLCs

    Regulation of cellular degradation pathways by viral oncoproteins and microRNAs

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    The cellular degradation system actively participates in cell homeostasis. Despite ongoing efforts and much progress in recent years, the underlying mechanisms of these pathways are not entirely understood. This thesis aims to contribute further insights how microRNAs and viral oncoproteins regulate key genes involved in cellular degradation pathways. In Paper I, we found overexpression of miR-223-3p in testicular germ cell tumors (TGCTs), in which its expression was negatively correlated with the mRNA level of the FBXW7 ubiquitin E3 ligase. Overexpression of miR-223-3p suppressed, while its inhibition increased, FBXW7 protein level in TGCT cell lines, suggesting FBXW7 as a target of miR-223-3p. Using both gain- and loss-of-function experiments, we showed that miR-223-3p induced cell growth and reduced apoptosis. Ectopic expression of the FBXW7 open reading frame could reverse the effect of miR-223-3p. In conclusion, we suggest the oncogenic role of miR-223-3p – FBXW7 regulation in TGCT. In Paper II, we demonstrated that miR-375, miR-30a-3p and miR-30a-5p are regulated by Merkel cell polyomavirus (MCPyV) T-antigens through the DnaJ domain. These miRNAs could suppress autophagy and target ATG7, SQSTM1 (p62) and BECN1. Lower protein levels of ATG7 and p62 are associated with MCPyV-positive MCC tumors. Additionally, we showed that ectopic expression of MCPyV oncoproteins could suppress autophagy and blockage of autophagy rescued Torin-1 mediated cytotoxicity in MCC cells. This study provides evidence that MCPyV oncoproteins induce microRNAs and suppress autophagy in MCC, suggesting the importance of autophagy suppression in protecting MCC cell survival. In Paper III, we uncover a function and mechanism of MCPyV oncoprotein in autophagy evasion through c-KIT receptor tyrosine kinase. We show that the viral oncoprotein promotes c-KIT retention in late endosomes through its Vam6p binding site, which promotes c-KIT binding to Beclin-1 and enhances Beclin-1-BCL2 interaction. Silencing of c-KIT induces autophagy, which leads to degradation of the viral oncoprotein. Thus, MCPyV has developed a strategy to hijack cellular degradation system to sustain the viral oncoprotein expression. In Paper IV, we identify CK20 paranuclear dot as a part of aggressome in MCC, in which these structures are associated with MCPyV status, localized at microtubule-organizing center and dependent on BAG3 expression and dynein-mediated microtubule transport. Additionally, we show that the MCPyV truncated large T-antigen promotes aggresome formation through its Vam6p binding site. This study suggests a model of BAG3-dependent aggresome formation contributed by viral oncoprotein in MCC. Overall, this thesis work has demonstrated the involvement of microRNAs and viral oncoproteins in regulation of cellular degradation pathways, which contributes to the understanding of the molecular interplays between cellular degradation system and cancer development in TGCT and MCC (as illustrated in the Graphical Abstract)

    Immunobiology of Emerging Human DNA Viruses

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    Many unknown species of human DNA viruses have recently (2005-2013) been discovered by using modern molecular and bioinformatic tools. The clinical and pathogenic roles of these viruses are presently known only fragmentarily; however they were found in symptomatic patients, and some have been shown to cause severe infectious illness, or cancer. Some of these emerging DNA viruses are examined in this thesis: Human Bocavirus 1 (HBoV1), Merkel cell polyomavirus (MCV or MCPyV) and Trichodysplasia spinulosa-associated polyomavirus (TSV or TSPyV). Viruses like these are of fundamental importance in the genesis of not only of acute but also of chronic or late-onset illness. The immunobiology and pathogenesis of these new viruses along with the already known DNA virus (parvovirus B19 or B19) can be found by the immunological and molecular methods. For years it was thought that parvovirus B19, was the sole human-pathogen among its family members. In 2005 a new pathogenic species, HBoV1 (previously denoted HBoV), was discovered by random-PCR from a nasopharyngeal aspirate. The existing data strongly suggest that HBoV1 causes a respiratory illness in young children. The aim of our study was to increase our knowledge on HBoV1-specific Th-cell immunity by examining T-cell proliferation and cytokine responses in asymptomatic adults. HBoV1-specific response was compared to those elicited by B19. B19-specific Th-cell immunity appears to be more divergent (in terms of cytokine response patterns) than the HBoV1-specific one. The present study also suggests that interleukin-13 (IL-13) response induced by HBoV1 may contribute to the airway pathology like asthma or bronchiolitis. A novel concept of CD4+ T-cells with cytolytic potential (CD4+ CTL) is emerging. Very recently, CD4+ CTL have been implicated in the control of persistent viral infections, e.g., Epistein-Barr virus (EBV), hepatitis C virus (HCV) and HIV-1. While human parvovirus B19 can establish persistence, yet no data exist on the presence of B19-specific CD4+ CTLs. Detection of vigorous B19-specific granzyme B (GrB) and perforin responses in seropositive individuals points to a role of CD4+ CTL also in B19 immunity. Such cells could function within immune regulation and in the triggering of autoimmune phenomena such as Systemic Lupus Erythematosus (SLE) or rheumatoid arthritis (RA). The newly discovered MCV resides in approximately 80% of Merkel cell carcinomas (MCC). The integration of MCV genome in-to the genome of host cell has been suggested to be the primary reason for this rare and aggressive skin cancer. Here we studied the T-cell immunity against this carcinogenic virus. We found that interferon-γ (IFN-γ) is the dominant cytokine among MCV-seropositive individuals and suggest that IFN-γ plays an important role in surveillance against MCV-induced disease. Our studies also suggested a role for IL-13 and IL-10 in anti-tumor immunity and immune regulation, respectively. TSV, while exhibiting high seroprevalence in general population, has been detected in trichodysplasia spinulosa (TS) skin lesions, suggesting an etiological role in this disease. In order to characterize Th-cell immunity against TSV, and to permit its comparisons with MCV-specific Th-cell immunity, we studied TSV and MCV-specific proliferation and cytokine responses in healthy volunteers and in one MCC patient. While an association between humoral and cellular responses was detectable with MCV, it was found to be weaker than the humoral and cellular responses detectable with TSV. Despite the significant homology in amino acid sequences of VP1, Th-cell crossreactivity was not evident between these viruses. As CD8+ T-cells specific for MCV LT-Ag oncoprotein clearly provide an important defence mechanism against MCC, the MCV VP1-specific Th-cells may also be important in preventing the oncogenic process, by suppressing MCV replication with antiviral cytokines such as IFN-γ. Parvoviruses (HBoV1 and B19) and polyomaviruses (MCV and TSV) induce effector CD4+ T-cell responses that are best known for their ability to protect against viral infections. Besides helper functions, CD4+ T-cell contribute to viral control and elimination by CD4-mediated cytotoxic effector functions. Thus, understanding of the CD4+ T-cell immunity is of key importance in the development of vaccines and therapeutic agents against life threatening infectious pathogens.Not availabl
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