Identification of FAM111A as an SV40 Host Range Restriction and Adenovirus Helper Factor

The small genome of polyomaviruses encodes a limited number of proteins that are highly dependent on interactions with host cell proteins for efficient viral replication. The SV40 large T antigen (LT) contains several discrete functional domains including the LXCXE or RB-binding motif, the DNA binding and helicase domains that contribute to the viral life cycle. In addition, the LT C-terminal region contains the host range and adenovirus helper functions required for lytic infection in certain restrictive cell types. To understand how LT affects the host cell to facilitate viral replication, we expressed full-length or functional domains of LT in cells, identified interacting host proteins and carried out expression profiling. LT perturbed the expression of p53 target genes and subsets of cell-cycle dependent genes regulated by the DREAM and the B-Myb-MuvB complexes. Affinity purification of LT followed by mass spectrometry revealed a specific interaction between the LT C-terminal region and FAM111A, a previously uncharacterized protein. Depletion of FAM111A recapitulated the effects of heterologous expression of the LT C-terminal region, including increased viral gene expression and lytic infection of SV40 host range mutants and adenovirus replication in restrictive cells. FAM111A functions as a host range restriction factor that is specifically targeted by SV40 LT

Proteomic Analysis of Merkel Cell Polyomavirus

Over the past 8 years, the discovery of 11 new human polyomaviruses (HPyVs) has revived interest in this DNA tumor virus family. Although HPyV infection is widespread and largely asymptomatic, one of these HPyVs, Merkel cell polyomavirus (MCPyV), is a human tumor virus. JC virus (JCPyV), BK virus (BKPyV), HPyV7, and trichodysplasia-spinulosa virus (TSPyV) can cause non-neoplastic diseases in the setting of immunosuppression. Probably, one of the most common themes among the oncogenic viruses rests in the ability of one or more of the viral proteins to deregulate pathways involved in the control of cell proliferation. The focus of this work is on MCPyV, which is a human pathogen. In addition to its importance in human health, there is growing interest in adapting MCPyV for drug delivery and other biotechnology applications, several viral coat proteins can spontaneously assemble into capsids in vitro with morphologies identical to the native virions and virion assembly is a powerful model system for studying protein complex formation. The protein capsid of the virion is a non-covalent association of protein subunits that is responsible for an array of functions, including cell attachment, cell entry, and DNA release. Even for the best studied family members, the mechanism of assembly is still poorly understood. Studies on the proteins of MCPyV provide information about the composition of the virus, as well as individual virus-virus protein and virus-host protein interactions. Mass spectrometry offers a unique perspective on the properties of viruses. Its broad application to viral structure provides unique insights into many biological processes, including viral–antibody binding, protein–protein interactions and protein dynamics. Electrospray ionization (ESI) mass spectrometry is a powerful approach for analyzing biomolecules and biomolecular complexes. Previous studies have provided evidence that non-covalent biomolecular complexes can be observed by ESI mass spectrometry. Mass spectrometry of viral proteins is now routine and since viruses are typically well characterized, in that the capsid protein and genome sequences are known, identifying a virus based on the mass of the protein or based on enzymatic digestion is relatively straightforward. This thesis describes the use of mass spectrometry for the identification of proteins associated with the MCPy virion and for the analysis of the post-translational modifications of the capsid proteins, such as ubiquitinylation, phosphorylation and acetylation

Comparative analyses of tumorigenic mechanisms of Merkel cell polyomavirus T antigens

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

A Novel Human DnaJ Protein, hTid-1, a Homolog of the Drosophila Tumor Suppressor Protein Tid56, Can Interact with the Human Papillomavirus Type 16 E7 Oncoprotein

AbstractWe have cloned hTid-1, a human homolog of the Drosophila tumor suppressor protein Tid56, by virtue of its ability to form complexes with the human papillomavirus E7 oncoprotein. The carboxyl terminal cysteine-rich metal binding domain of E7 is the major determinant for interaction with hTid-1. The carboxyl terminus of E7 is essential for the functional and structural integrity of E7 and has previously been shown to function as a multimerization domain. The hTid-1 protein is a member of the DnaJ-family of chaperones. Its mRNA is widely expressed in human tissues, including the HPV-18-positive cervical carcinoma cell line HeLa and human genital keratinocytes, the normal host cells of the HPVs. The hTid-1 gene has been mapped to the short arm of chromosome 16. The large tumor antigens of polyomaviruses encode functional J-domains that are important for viral replication as well as cellular transformation. The ability of HPV E7 to interact with a cellular DnaJ protein suggests that these two viral oncoproteins may target common regulatory pathways through J-domains

pRb Inactivation in Mammary Cells Reveals Common Mechanisms for Tumor Initiation and Progression in Divergent Epithelia

Retinoblastoma 1 (pRb) and the related pocket proteins, retinoblastoma-like 1 (p107) and retinoblastoma-like 2 (p130) (pRb(f), collectively), play a pivotal role in regulating eukaryotic cell cycle progression, apoptosis, and terminal differentiation. While aberrations in the pRb-signaling pathway are common in human cancers, the consequence of pRb(f) loss in the mammary gland has not been directly assayed in vivo. We reported previously that inactivating these critical cell cycle regulators in divergent cell types, either brain epithelium or astrocytes, abrogates the cell cycle restriction point, leading to increased cell proliferation and apoptosis, and predisposing to cancer. Here we report that mouse mammary epithelium is similar in its requirements for pRb(f) function; Rb(f) inactivation by T(121), a fragment of SV40 T antigen that binds to and inactivates pRb(f) proteins, increases proliferation and apoptosis. Mammary adenocarcinomas form within 16 mo. Most apoptosis is regulated by p53, which has no impact on proliferation, and heterozygosity for a p53 null allele significantly shortens tumor latency. Most tumors in p53 heterozygous mice undergo loss of the wild-type p53 allele. We show that the mechanism of p53 loss of heterozygosity is not simply the consequence of Chromosome 11 aneuploidy and further that chromosomal instability subsequent to p53 loss is minimal. The mechanisms for pRb and p53 tumor suppression in the epithelia of two distinct tissues, mammary gland and brain, are indistinguishable. Further, this study has produced a highly penetrant breast cancer model based on aberrations commonly observed in the human disease

Understanding the role of Merkel cell polyomavirus oncoproteins in the cellular transformation of mammalian Merkel cells

Merkel cell carcinoma (MCC) is a highly aggressive skin cancer of neuroendocrine origin with a high propensity for metastasis through the dermal lymphatic system. In 2008, Merkel cell polyomavirus (MCPyV) was discovered monoclonally integrated within the host genome of more than 80% of MCC tumours. MCPyV is known to contribute to the transformation and maintenance of MCC tumour cells through the expression of the Large and Small Tumour (LT and ST) antigens. To date, a number of mechanisms by which MCPyV T antigen expression promotes cell transformation and viral replication have been elucidated. Although, no studies have addressed the molecular mechanisms associated with MCPyV T antigen expression and the highly aggressive, metastatic nature of MCC. Herein, a quantitative proteomic approach has been used to identify cellular proteins and gene ontology pathways that are differentially expressed upon MCPyV ST expression. This highlighted that MCPyV ST expression promotes the upregulation of cellular proteins involved in microtubule-associated cytoskeletal organisation and dynamics. Further analysis, has demonstrated that MCPyV ST promotes cell motility, migration and invasion and that MCPyV ST-induced microtubule destabilisation is fundamental for this phenotype. Bioinformatical analysis highlighted the upregulation of stathmin, a microtubule destabilising protein, which is required for MCPyV ST-induced cell motility. Furthermore, through protein interaction studies and cell motility assays, the cellular phosphatase catalytic subunit PP4C has been implicated in the regulation of this process. Herein, these findings present the first study into the metastatic nature of MCC and the underlying mechanisms by which MCPyV T antigen expression can promote cell migration. This in turn should allow for enhanced therapeutic studies and chemotherapy regimes, in order to improve current treatments of MCC

A Systems Biology Analysis of PP2A Functions in Cancer Cells

Cancer is characterized by aberrant activation of phosphorylation signalling cascades. However, despite the critical role of phosphatases in protein phosphorylation, their contribution to cancer cell signalling is only emerging. Notably, Protein phosphatase 2A (PP2A) has a well-established tumor suppressor function but it is poorly understood which of its many targets are relevant for this function. This is partly due to the wide range of activities that PP2A participates in and partly due to the fact that PP2A activity regulation, as well as the deregulation in cancer, occurs via many auxiliary subunits and endogenous inhibitor proteins. In this MD-PhD thesis, we have used various systems biology approaches, including phosphoproteomics, high throughput drug sensitivity screening, and transcriptomics to study the functions of the most frequently mutated PP2A subunit, PPP2R1A, as well as three of its endogenous inhibitor proteins, CIP2A, PME1, and SET in cancer cells. This study demonstrates that PP2A reactivation is poorly tolerated by several types of cancer cells and results in downregulation of multiple oncogenic pathways, as well as induction of senescence. Specifically, CIP2A is a regulator of MYC transactivation in basal type breast cancers and our results indicate multiple cooperative mechanisms by which PP2A regulates MYC. Analysis of PP2A dephosphorylome also provided novel insights into general organization of phosphorylation signalling and emphasized the role of PP2A inhibition in the nucleus. By combining the phosphoprotemics data with cancer cell responses to over 300 drugs, we have identified mechanistically distinct types of interactions between drug sensitivity and PP2A activity. We further validated that inhibition of PP2A in KRAS mutant lung cancers confers resistance to MAPK pathway inhibitors including the combination of Raf and MEK inhibitors. Together, these findings provide new evidence to support PP2A reactivation as cancer therapeutic strategy and to support evaluating PP2A activity as a predicitive marker for cancer therapy responses.Syövälle ominainen piirre on fosforylaation perustuvien signaalipolkujen poikkeava aktivoituminen. Huolimatta fosfataasien keskeisestä tehtävästä proteiinien fosforylaatiossa, niiden merkityksestä syöpäsolujen signlaalinvälityksessä on vasta vähän tietoa. Erityisesti Proteiinifosfataasi 2A:lla (PP2A) on selkeästi osoitettu olevan kasvunestäjproteiini-ominaisuuksia, mutta se mitkä PP2A:n monista kohdeproteiineista ovat tärkeitä syövän kannalta tunnetaan huonosti. Osittain tämä johtuu PP2A:n toimintojen moninaisuudesta ja osittain siitä, että PP2A:n säätely, sekä syövissä esiintyvät säätelyn häiriöt, tapahtuvat ylimääräisten alayksiköiden ja inhibiiittoriproteiinien kautta Tässä väitöskirjatutkimuksessa olemme tutkineet PP2A:n yleisimmin mutatoituneen alayksikön, PPP2R1A:n, sekä kolmen inhibiittoriproteiinin, CIP2A:n, PME-1:n ja SETin, toimintoja syöpäsoluissa käyttäen erilaisia systeemibilogisia lähestymistapoja, mukaan lukien fosfoproteomiikkaa, lääkeherkkyysseulontaa ja transkriptomi-analyysiä. Tämä tutkimus osoittaa, että monet syöpäsolut sietävät huonosti PP2A:n uudelleen aktivoimista, joka johtaa useiden onkogeenisten signaalipolkujen estymiseen ja senesenssin käynnistymiseen. Tuloksemme viittaavat siihen, että PP2A säätelee MYC-onkogeeniä useilla toisiaan tukevilla mekanismeilla, ja CIP2A:lla on merkitystä MYCin transaktivaation säätelyssä basaalityypin rintasyövässä. PP2A:n defosforylomista saatu tieto auttaa myös ymmärtämään yleisellä tasolla fosforylaatiosignaloinnin järjestäytymistä soluissa ja osoittaa, että PP2A:n inhibitiolla on keskeinen merkitys tumassa. Yhdistämällä fosfoproteomiikan ja syöpäsolujen vasteet yli 300 lääkkeelle olemme tunnistaneet useita mekanismeiltaan erilaisia yhteisvaikutuksia PP2A:n aktiivisuuden ja lääkeherkkyyksien välillä. PP2A:n estämisestä aiheutuva resistenssin MAPK-signaalipolun inhbiittoreille KRAS-mutaatioita kantasvissa syöpäsoluissa vahvistettiin lisätutukimuksilla. PP2A:n estäminen teki solut resistenteiksi myös MEK ja RAF inhibiittorien yhdistelmälle. Yhdessä nämä tulokset puoltavat PP2A:n reaktivaatiota syövän hoitostrategiana ja PP2A:n aktiivisuuden määrittämistä syöpähoitojen ennustekijänä.Siirretty Doriast

Elicitation and enhancement of T and B cell responses

The Major histocompatibility complex class I (MHC-I) has been characterized in such great depth that a number of its key properties are well understood and part of its behavior can even be predicted. It is therefore intriguing that the impact of small epitope modifications on immunogenicity and the elicitation of T-cell repertoires remain often unpredictable and full of surprises. Substitution of the secondary anchor residue at peptide position 3 from a serine to a proline (p3P) significantly increased the stabilization capacity and immunogenicity of the melanoma-associated H-2Db (Db)-restricted epitope gp100 (EGS). Despite this strong enhancement the conformation of the modified epitope (EGP) was not altered and vaccination with EGP generated T-cell responses that recognized cells expressing EGS with high functional avidity. Based on these promising results, the p3P modification was applied to the highly immunodominant Lymphocytic Choriomeningitis Virus epitope gp33 and the associated escape variants Y4F and Y4A. As for gp100, p3P was found to increase the MHC stabilization capacity and immunogenicity of the modified epitopes V3P, PA and PF, while not altering their structures. Accordingly, T-cell responses were cross-reactive between native and p3P enhanced epitopes and, when used for vaccination of C57BL/6 mice, PF elicited a focused T-cell response against Db/Y4F. In parallel, surface plasmon resonance (SPR) measurements revealed that p3P did not only enhance MHC stabilization capacity but also directly increased the affinity of the cognate T-cell receptors (TCRs). To fully characterize the molecular details underlying these two enhancing effects, the thermostability, TCR binding and molecular dynamics (MD) of Db/EGP were measured in comparison with Db/EGS. Furthermore, the contribution of Y159, a highly conserved tyrosine that is structurally juxtaposed to p3P, was assessed using a set of soluble Db-Y159 variants. In conclusion, these measurements clearly demonstrated that specific interactions of p3P with the aromatic ring of Y159 are responsible for the significantly increased MHC stabilization capacity. Surprisingly, the enhanced TCR binding was found to be entirely independent of Y159, suggesting a direct contribution of the buried proline residue to TCR binding. These findings underscore the potential to enhance MHC-I-restricted epitopes at secondary anchor residues, while specifically indicating that proline can directly increase TCR affinity, which could not have been anticipated from our current understanding of the factors shaping TCR recognition. Not entirely different from T-cell elicitation, the induction of broadly neutralizing antibodies against the Human Immunodeficiency Virus type 1 (HIV-1) is to date still an elusive goal despite extensive characterization of the respective antibody-epitope interactions. One of the central challenges is that the virus is highly adapted to immune pressure and the most relevant antibody epitopes on the HIV envelope proteins (Env) are the least immunogenic. Therefore, a highly heterologous prime-boost vaccination strategy was designed in which priming of rabbits with HIV-1 env plasmids was followed by a recombinant Simian Immunodeficiency Virus (SIV) Env boost. While the SIV Env trimers were inherently favorable because of their higher stability, the approach was specifically chosen to preferentially boost antibody responses against the few sites that are conserved in HIV and SIV Env. The described approach was generally validated and warrants future investigations as it lead to the elicitation of potent neutralizing antibodies even though it remains to be fully established if the highly heterologous nature of the prime boost strategy was solely responsible. In summary, the studies presented in this thesis provide the structural and functional platform for a novel and intriguing MHC-I peptide enhancement. Additionally, heterologous immunizations of rabbits offer a promising addition to existing vaccination strategies against HIV