RNA virus persistence in lymphocytes has been studied extensively in vitro, but the influence of lymphocyte homeostatic mechanisms and antiviral immunity on persistence has not been well studied in an in vivo system. It is demonstrated here that vesicular stomatitis virus (VSV), a negative-strand RNA virus, is maintained in B lymphocytes in vivo despite the existence of homeostatic mechanisms that drive the cells to proliferate under conditions of B cell deficiency and a strong antibody response to the virus. It is also shown that antiviral antibodies inhibit VSV reactivation from persistently infected primary B cells in vitro. A model is proposed for virus persistence in vivo in which B cell homeostatic signals drive virus expression in some infected cells, resulting in an antibody response, which maintains virus persistence in B cells. In the course of conducting experiments to define the homeostatic signals that might act on persistently infected B cells in vivo, it was found that a fraction of small, resting splenic B cells proliferates after adoptive transfer into B cell deficient hosts (sublethally irradiated, xid, or SCID). This process, termed homeostatic proliferation, is driven by B cell deficiency since proliferation is limited in B cell sufficient hosts. This reveals the existence of a mechanism by which B cells sense their own numbers. The proliferation is unique in that the replicating cells do not upregulate cell surface markers, such as CD25 and B7-2, associated with antigen or mitogen induced proliferation. They do, however, show transient increases in other activation markers (CD69, CD71), demonstrating the action of an inductive signal. Homeostatic proliferation is a property of both mature and immature B cells, but in competition experiments, only mature B cells inhibit proliferation. xid B cells express a defective form of Bruton's tyrosine kinase (Btk); as a result, these cells proliferate poorly in response to stimulation through a number of cell surface receptors including the BCR, IL-5R, IL-10R, the toll-like receptor RP-105, and CD38. Homeostatic proliferation is severely reduced in xid B cells; thus, this process is regulated by a Btk-dependent inductive signal, which is counterbalanced by an inhibitory signal provided by mature B cells. B cell homeostatic proliferation does not rely on transcription factors (c-rel and p50) critical for conventional proliferation induced by antigen or mitogen (c-rel), or for peripheral B cell survival (p50), suggesting that multiple signals drive this process and that survival and proliferation signals are not identical. VSV persists in small, resting primary B cells for several weeks in vitro, and virus replication is restricted at multiple levels depending on the activation state of the cells. After adoptive transfer of infected B cells into B cell deficient (xid) recipients, viral RNA, but not infectious particles, can be detected by RT-PCR in recipient spleens for at least 72 days. RT-PCR analysis of FACS sorted donor cells stained with CFSE reveals that viral RNA is maintained in transferred B cells but can also found in recipient cells. Infected B cells can undergo homeostatic proliferation and an antibody response is generated to the virus, suggesting that homeostatic signals induce virus expression in some transferred cells. Virus persistence is maintained despite an active immune response to the virus. In fact, persistence may be maintained by antiviral antibody since in vitro treatment of infected primary B cells with anti-VSV antibody inhibits virus reactivation at multiple levels (transcription, protein synthesis, assembly/release of infectious particles). This inhibition is reversible upon antibody removal, demonstrating that functional virus is maintained in antibody treated cells. Antibody specific for a single viral protein (VSV G) is sufficient since inhibition is mediated by monoclonal antibodies specific for a VSV G; neutralizing activity is not required because inhibition occurs with non-neutralizing monoclonal antibodies to VSV G. It is proposed that antibody binding to VSV G on infected B cells generates inhibitory signal(s) that suppress signaling pathways required for virus replication in B cells. Finally, a model of RNA virus persistence in B cells is proposed in which lymphocyte homeostatic signals promote virus expression, leading to the production of antiviral antibodies, which suppress virus replication inside infected B cells and help to maintain persistence.

Enveloped RNA viruses like human immunodeficiency virus type-1 (HIV-1) and SARS-CoV-2 enter host cells through fusion with the plasma membrane, a process facilitated by specific viral envelope proteins that recognize and bind to receptors expressed on the host cell surface. These receptors can diverge based on the type of cell and virus. For HIV-1, the primary receptors on myeloid cells are CD4 and CCR5 or CXCR4. For SARS-CoV-2, although the primary receptor is ACE2, other myeloid-cell specific sialic acid binding lectins can also facilitate entry. Following cellular invasion, different viral RNA species can be detected by distinct host nucleic acid sensors, resulting in type I interferons and pro-inflammatory cytokine induction. While these innate immune responses are essential for controlling viral infections, overactivation can lead to chronic inflammation, tissue damage, and disease pathogenesis. Herein, I examine the contribution of HIV-1 and SARS-CoV-2 de-novo RNA expression and the molecular mechanisms that contribute to innate immune activation in myeloid cells. Despite advancements in combination antiretroviral therapy (ART) in suppressing systemic viral replication in individuals infected with HIV, residual viral RNA expression in tissue reservoirs remains a significant hindrance to curative efforts. I hypothesized that persistent expression of viral RNAs in myeloid cells triggers dysregulated innate immune activation, and inflammasomes activation. This study centers on the long-lived tissue-resident innate immune cells - macrophages and microglia, which, owing to their self-renewing nature, operate as reservoirs of viral RNA production, and are thought to lead to chronic immune activation even in the absence of productive replication. Our previous studies suggest that de novo expression of unspliced intron-containing HIV-1 RNA (herein referred to as icRNA) triggers activation of pro-inflammatory cytokines in myeloid cells. Here, I demonstrate that cytosolic expression of HIV-1 icRNA, but not multiply-spliced viral RNAs induces inflammasome activation, LDH release and IL-1β secretion in productively infected monocyte-derived macrophages (MDM) and induced pluripotent stem cell (iPSC)-derived microglia. Interestingly, knockdown of RLRs, RIG-I and MDA5 or endosomal TLRs failed to abrogate HIV-1 icRNA-induced IL-1β secretion. Rather, knockdown of NLRP1, but not NLRP3, inflammasome resulted in a significant reduction in IL-1β secretion, underscoring NLRP1's pivotal role in the HIV-1 icRNA-induced IL-1β secretion. Furthermore, Rev-Crm1-dependent nucleocytoplasmic export of HIV-1 icRNA was required for NLRP1-mediated Caspase-1 activation, IL-1β secretion, LDH release and cell death. Similarly, SARS-CoV-2, while not establishing productive infection in macrophages, can activate these cells, contributing to a hyper-inflammatory response marked by the heightened expression of pro-inflammatory cytokines, which is understood to be a principal driver of COVID-19 pathology. SARS-CoV-2 established an abortive infection in macrophages. CD169, a macrophage-specific sialic-acid binding lectin, mediated ACE2-independent SARS-CoV-2 entry in human macrophages and establishment of restricted infection. Interestingly, CD169-mediated SARS-CoV-2 entry in macrophages led to the expression of viral genomic and subgenomic RNAs, with negligible viral protein expression and no release of infectious virus particles, implying a post-entry restriction to SARS-CoV-2 replication in macrophages that was curbed by exogenous ACE2 expression. Despite restricted viral RNA expression, cytoplasmic RLRs, RIG-I and MDA5, sensed abortive viral transcripts, and induced pro-inflammatory responses in a MAVS dependent manner. This dissertation reveals striking parallels between the role of viral RNAs in driving pro-inflammatory responses in HIV-1 and SARS-CoV-2 infections. These findings collectively underscore the central role of cytoplasmic sensing of viral RNAs and their contribution to chronic inflammation in virus-infected myeloid cells. Elucidating these molecular mechanisms further may pave the way for novel therapeutic interventions to mitigate the persistent innate immune activation and immunopathology detected in HIV-1 and SARS-CoV-2 infected individuals.

Introduction: RNA viruses are economically important pathogens of fish, and among these viruses, infectious pancreatic necrosis virus (IPNV) is of particular concern for the aquaculture industry, especially for farmed rainbow trout (Oncorhynchus mykiss) and Atlantic salmon (Salmo salar). This non-enveloped aquatic virus, which was first isolated in the UK in 1971, belongs to the family of Birnaviridae and has a bi-segmented dsRNA genome of about 6kb. IPNV is classified in 6 genogroups with correspondence to 10 known serotypes and an additional proposed genogroup of marine aquabirnaviruses (MABV). IPNV causes high mortality in fry and a reduced mortality in adult fish, respectively. Fish, which survive, can become carriers and this can lead to a clinical outbreak by releasing infective material into water or by vertical transmission via oocytes, milt and seminal fluids. Methods: This project aimed at determining the phylogeny and genomic changes of IPNV in Scotland by whole genome sequence analysis of IPNV isolates (diagnostic TCID50 supernatants) spanning 3 decades since 1982, using next generation sequencing technology. Viral RNA of IPNV culture supernatant (CHSE-214 and TO cell culture) was processed for next generation sequencing on an Illumina MiSeq platform. Library preparation was performed using the Nextera XT DNA Library Kit, prior to sequencing according to the manufacturer's MiSeq Reagent Kit v3 (150cycles) protocol. To optimize whole genome next generation sequencing for IPNV, we compared two RNA processing protocols, the Glasgow (GLAP) and the Goettingen protocol (GOEP) with focus on missing terminal nucleotides after a de novo genome assembly. Sequences were used to determine the phylogeny and selection pressure on the genome as well as a possible virus-host adaptation. Results: The results showed that both protocols were able to give full length genomes as well as genomes with missing terminal nucleotides. The phylogenetic analysis of 57 sequenced IPVN isolates shows that 78.95 % of the isolates group within genogroup V, which includes serogroup Sp and 5.26 % within genogroup I which includes serogroup Ja. Segment A of 15.79 % of the isolate grouped within genogroup III, which includes serotype Ca1 and Te but only 7.02 % of the segment B isolates grouped in the genogroup III. The remaining 8.77 % of segment B groups within genogroup II, containing the Ab serotype. Previous research has shown that residue substitutions at positions 217 and 221 in the major capsid protein VP2 have an impact on the virulence of the virus, leading to different virulence types: virulent (T217, A221), low virulence (P217, A221), avirulent (T217, T221) and persistent (P217, T221). Whole genome sequence results show that 58.93 % of the sequenced isolates belong to the persistent, 32.14 % to the low virulent type, only one isolate was of a virulent type and 7.15 % had not virulence assigned amino acid compositions in positions 217 and 221. The selection pressure analysis showed that especially VP2 is experiencing selection pressure in the variable region. In the VP1 protein we see two sites under positive selection pressure within specific motifs. VP5 showed positive selected sites mostly within the truncated region of the protein. Other proteins showed no particular interesting sites of selection. The codon adaptation analysis showed highest adaptation index for VP2. Besides VP5, which had an CAI index below one, therefore showing negative adaptation, other IPNV proteins had an CAI of barely above the value of 1. The dinucleotide abundance, focussing on CpG, showed that CpG is underrepresented in segment A and B. Discussion Phylogenetic analysis of the sequenced IPNV strains shows separate clustering of different genogroups. Genetic reassortment is observed in segment B showing a grouping within genogroup III and II although the segment A of these isolates was grouping exclusively within III. We found that over 50 % of the isolates belong to the persistent and over 30 % to the low virulent type, assuming that due to not sterilising vaccination these types were selected in the vaccinated population. The results from the CAI calculations indicate an adaptation of IPNV to its host. Together with the findings that CpG is underrepresented in IPNV it suggests that this leads to an immune escape. Especially since the selection pressure analysis showed positive selection in VP2 within the virulence determination sites of the protein, indicating that IPNV "tries" to downregulate immune recognition. The prevalence of mostly persistent type of isolates indicates together with the assumption of adaptation and immune escape that IPNV is evolving with the host in order to ensure survival.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 4 uppsatser.

La génétique inverse est devenue une méthode clé pour la production de virus à ARN génétiquement modifiés et pour comprendre les propriétés cellulaires et biologiques des virus. Cependant les méthodes les plus fréquemment utilisées, basées sur le clonage de génomes viraux complets dans des plasmides, sont laborieuses et imprévisibles. La première partie de cette thèse présente des études sur la mise au point d'un nouveau système de génétique inverse, appelé méthode ISA (Amplicons-Sous génomique-Infectieux), qui permet la génération, en quelques jours, de virus infectieux sauvages et génétiquement modifiés appartenant à trois familles différentes de virus à ARN simple brin de polarité positive, avec une grande maîtrise des séquences virales. Dans la deuxième partie de cette thèse, nous avons appliqué pour la première fois à un arbovirus (CHIKV), le ré-encodage des codons - une méthode développée récemment et très excitante pour le développement de vaccins vivants atténués. En utilisant une approche aléatoire de ré-encodage des codons qui attribue au hasard des codons sur la base de la séquence en acides aminés correspondante, nous avons mis en évidence des pertes importantes de fitness réplicatif sur des cellules de primates et d'arthropodes. La diminution du fitness réplicatif est en corrélation avec le degré de ré-encodage, une observation qui peut aider à la modulation de l'atténuation virale. En utilisant l'expérience acquise avec le CHIKV, nous avons transposé avec succès ce mécanisme d'atténuation au JEV et amélioré notre maîtrise du processus d'atténuation en utilisant une combinaison de la synthèse de novo et de la méthode ISA. / Reverse genetics has become a key methodology for producing genetically modified RNA viruses and deciphering cellular and viral biological properties, but the most commonly used methods, based on the preparation of plasmid-based complete viral genomes, are laborious and unpredictable. The first part of this thesis presents studies relating to the development of a new reverse genetics system, designated the ISA (Infectious-Subgenomic-Amplicons) method, which enabled the generation of both wild-type and genetically modified infectious viruses belonging to three different families of positive, single stranded RNA viruses within days with great control of the viral sequences. In the second part of this thesis, we applied for the first time to an arbovirus (CHIKV), codon re-encoding - a recently developed and very exciting method for the development of live attenuated vaccines. Using a random codon re-encoding approach which randomly attributed nucleotide codons based on their corresponding amino acid sequence, we identified major fitness losses of CHIKV in both primate and arthropod cells. The decrease of replicative fitness correlated with the extent of re-encoding, an observation that may assist in the modulation of viral attenuation. Detailed analysis of these observed replicative fitness losses indicated that they are the consequence of several independent re-encoding induced events. Using the experience acquired on the CHIKV, we successfully transposed this attenuation mechanism to JEV and improved our control of the attenuation process by using a combination of de novo synthesis and the ISA method.

No description available.

No description available.

Japanese encephalitis virus (JEV) is one of the major causes of encephalitis in Asia. JEV causes serious inflammation of the brain, which may lead to permanent brain damage and has a high mortality rate. Almost 3 billion people live in JE endemic areas and JEV causes an estimated 20,000 cases of disease and 6000 deaths per year. JEV is a positive stranded RNA virus belonging to the Flavivirus genus of the family Flaviviridae. The genome of JEV is about 11 kb long and codes for a polyprotein which is cleaved by both host and viral encoded proteases to form 3 structural and 7 non-structural proteins. JEV transmission occurs through a zoonotic cycle involving mosquitoes and vertebrate amplifying hosts, chiefly pigs and ardeid birds. Humans are infected when bitten by an infected mosquito and are dead end hosts. The role of humoral and cell mediated immune responses during JEV infection have been studied by several groups. While the humoral responses play a central role in protection against JEV, the cell mediated immune responses contributing to this end are not fully understood. The MHC molecules have been known to play predominant roles in host responses to viral infections and the consequences of virus infection on the expression of MHC molecules are varied. The expression of MHC-I molecules is known to decrease upon infection with many viruses such as HIV, MCMV, HCMV, Adv, and EBV. In contrast, infection with flavivirus such as West Nile Virus (WNV) has been shown to increase the cell surface expression of both MHC-I and MHC-II molecules. It has been reported previously that WNV infection increases the cell surface expression of adhesion molecules such as ICAM-1, VCAM-1 as well as E-Selectin and these changes were mediated directly by WNV and not by soluble cytokines. In contrast to classical MHC-I molecules, the nonclassical MHC-I molecules do not belong to a single group of structurally and functionally homologous proteins and normally have lower cell surface expression. Earlier studies have shown that the expression of nonclassical MHC-I molecules were induced during infection with JHM strain of mouse hepatitis virus (MHV). However, the functional significance of this induction is unclear. Expression of nonclassical MHC-I molecules upon flaviviral infection is not very well understood. In this thesis, evidence is presented that JEV infection induces the expression of both classical and nonclassical MHC-I molecules on primary mouse brain astrocytes, mouse embryonic fibroblasts (MEFs) and H6 (hepatoma cell). The levels of adhesion molecules as well as molecules involved in antigen processing and presentation were also analyzed and our results clearly demonstrate that JEV infection induces their expression on astrocytes, MEFs and H6. The role of NF-κB and type-I IFNs in the induction of classical and nonclassical MHC-I molecules as well as molecules involved in antigen processing and presentation were also analyzed and our results demonstrated that type-I IFN mediated signaling is responsible for the induction of these molecules during JEV infection. Chapter 1 discusses the innate and adaptive immune system, the role of classical and nonclassical MHC molecules in the initiation of immune response and diverse strategies adapted by different viruses to evade the immune response. It also includes a detailed discussion about the IFN and NF-κB signaling pathways and their modulation by viral infection. Finally, the genome organization, epidemiology, transmission cycle, pathogenesis and pathology, clinical features, humoral as well as cell mediated immune response to JEV infection and the current vaccine status to JEV infection are briefly discussed. Chapter 2 describes the general materials and methods used in this study. It includes the details of the reagents and cell lines used in the experiments. It also discusses the various techniques such as RT-PCR, FACS analysis, EMSA and ELISA. Chapter 3 focusses on the validation of different knockout MEFs used in the study as well as confirming the purity of primary astrocyte cultures established from pub brains. The susceptibility of various cells to JEV infection has also been investigated. Our results confirmed the authenticity of all the cells and the purity of primary astrocyte cultures used in the study. Our results also indicated that all the cells used in the study are susceptible to JEV infection. Chapter 4 discusses the expression of MHC and related genes involved in immune response upon JEV infection of primary mouse brain astrocytes, MEFs and H6. Chapter 4 demonstrates for the first time that JEV infection induces the expression of nonclassical MHC-I or class Ib molecules namely Qa-1, Qb1 and T10 in addition to the induction of classical MHC-I molecules. In contrast to WNV, there was no increase in the cell surface expression of MHC-II molecules upon JEV infection of primary mouse brain astrocytes. JEV infection also induces the expression of adhesion molecules as well as molecules involved in antigen processing and presentation namely Tap1, Tap2, Tapasin, Lmp2, Lmp7 and Lmp10. Chapter 5 demonstrates that JEV infection induces NF-κB activation in astrocytes and MEFs. Studies using MEFs deficient in classical and alternate pathways of NF-κB activation indicate that JEV activates the classical pathway of NF-κB activation and is dependent on canonical lKKβ/IKK2 activity. JEV infection of astrocytes, MEFs and H6 induces the production of type-I IFNs. To determine the mechanism of type-I IFN induction during JEV infection, MEFs deficient in NF-κB signaling and IFN signaling were used. Results indicate that type-I IFN production in MEFs occurs by both NF-κB dependent and independent mechanisms. In contrast, the production of IFN-α was completely abrogated in IFNAR-\- MEFs whereas IFN-β production was greatly reduced. Production of type-I IFNs in IFNGR-\- MEFs is also reduced upon JEV infection but the reason for this is unclear. Chapter 6 demonstrates that JEV induced expression of classical MHC-I molecules occurs by type-I IFN mediated signaling. This result is in contrast to WNV infection, in which both NF-κB and type-I IFNs are involved in the induction of classical MHC-I molecules. Type-I IFNs were also shown to be involved in the induction of nonclassical MHC molecules namely, Qa-1 and Qb1 during JEV infection. In contrast, the expression of T10, another nonclassical MHC molecule occurs independent of type-I IFN signaling. The expression of molecules involved in antigen processing and presentation namely, Tap1, Tap2, Lmp2 and Lmp7 was type-I IFN-mediated, whereas the expression of Tapasin and Lmp10 was mediated by both type-I IFN dependent and independent mechanisms. The expression of VCAM-1 was dependent on NF-κB mediated signaling. Chapter 7 precisely describes the underlying mechanism of induction of MHC and various other related molecules and their significance during JEV infection. In addition, it also includes a working model for the induction of these molecules during JEV infection. In summary, this is the first study in which the mechanism of JEV mediated induction of classical as well as nonclassical MHC molecules has been studied in detail. This study clearly demonstrated that type-I IFNs are involved in the induction of classical and nonclassical MHC-I molecules during JEV infection. The functional significance of this JEV mediated induction of classical MHC-I molecules is unclear, but it has been proposed that this is to escape from the action of NK cells. The absence of MHC-II induction during JEV infection could be important because it may lead to the initiation of an immune response which is different from that induced during other viral infections which induce the expression of MHC-II molecules. In contrast to classical MHC-I molecules, the functional and biological significance of nonclassical MHC-I molecules are poorly studied. Nonclassical MHC-I molecules play an important role in bridging adaptive and innate immune response. So the nonclassical MHC molecules induced during JEV infection may play an important role in the initiation of immune response during JEV infection. The role these nonclassical MHC-I molecules in antigen presentation during JEV infection is not known. These nonclassical antigens are also recognized by NK and γδT cells, thus the expression of nonclassical MHC-I molecules during JEV infection might also confer a protective role.

Tumor suppressor p53, the guardian of the genome, has been intensely studied molecule owing to its central role in maintaining cellular integrity. While the level of p53 protein is maintained low in unstressed conditions, there is a rapid increase in the functional p53 protein levels during stress conditions. It is now well documented in literature that p53 protein accumulates in the cells following DNA damage by posttranslational modifications leading to increased stability and half life of protein. Additionally, recent studies have also highlighted the significance of increased p53 translation during stress conditions. Interestingly, an alternative initiation codon has been shown to be present within the coding region of p53 mRNA. Translation initiation from this internal AUG results in an N-terminally truncated p53 isoform, described as ΔN-p53. However, the mechanisms underlying co-translational regulation of p53 and ΔN-p53 are still poorly understood. Studies have suggested that synthesis of both p53 and its ΔN-p53 isoform is regulated during cell cycle and also stress and cell-type specific manner. Interestingly, reports also demonstrate continued synthesis of both p53 isoforms during stress conditions. In contrast, global rates of cap-dependent translation initiation are shown to be reduced during stress conditions. This translation attenuation is observed mainly due to restricted availability of critical initiation factors. Interestingly, preferential synthesis of a vital pool of survival factors persists even during these circumstances. Studies have suggested that this selective translation is mediated via alternative mechanisms of translation initiation. One of the important mechanisms used for protein synthesis during these conditions is internal initiation. In this mechanism, the ribosomes are recruited to a complex RNA structural element known as ‘Internal Ribosome Entry Site (IRES)’, generally present in the 5’ untranslated region (UTR) of mRNA. Therefore, it is possible that the translation of p53 and ΔN-p53 could also be regulated by IRES mediated translation, especially during stress conditions. In this thesis the role of internal initiation in translational control of p53 and ΔN-p53 has been investigated. Additionally, the putative secondary structure of p53 IRES RNA has been determined. Further, it has been shown that polypyrimidine tract binding (PTB) protein acts as an important regulator of p53 IRES activities. The probable mechanism of action of PTB protein has also been investigated. The results suggest that interaction with PTB alters the p53 IRES conformation which could facilitate translation initiation. Finally, the possible physiological significance of existence of p53 IRES elements has been addressed. In the first part of the thesis, the presence of internal ribosome entry site within p53 mRNA has been investigated. As a first step, the 5’UTRs mediating the translation of both p53 and ΔN-p53 were cloned in the intercistronic regions of bicistronic constructs. Results of in vivo transfection of these bicistronic constructs suggested the presence of two IRES elements within p53 mRNA, with activities comparable to known viral and cellular IRESs. The IRES directing the translation of p53 is in the 5'-untranslated region of the mRNA, whereas the IRES mediating the translation of ΔN-p53 extends further into the protein-coding region. To further validate, stringent assays were performed to rule out the possibility of any cryptic promoter activity, re-initiation/scanning or alternative splicing in the p53 mRNA. Transfection of in vitro synthesized bicistronic RNAs confirmed the presence of IRES elements within p53 mRNA. Incidentally, this constitutes the first report on translational control of p53 by internal initiation. In the second part of the thesis, the secondary structure of p53 IRES RNA has been investigated. Structural analysis of p53 RNA was performed using structure-specific nucleases and modifying chemicals. The results obtained from chemical modification and nuclease probing experiments were used to constrain Mfold predicted structures. Based on this, a putative secondary structure model for p53 IRES RNA has been derived. Sequence alignment suggested that the p53 IRES RNA showed significant sequence conservation across mammalian species. To study the effect of mutations on the IRES structure, mutant p53 IRESs were used that harbor silent mutations at critical locations within the p53 IRES element. Incidentally, one of the mutant constructs used in the study was observed to be a naturally occurring mutation in a chronic lymphocyte leukemia patient. RNA structure analyses of these two mutant p53 IRES RNAs were performed. The nuclease mapping data suggested conformational alteration in these mutant RNAs with respect to wild type. Consistently, a comparative Circular-Dichroism spectroscopy of the Wt and mutant RNAs also validated the conformational alteration of the mutant RNAs. This also suggested that the presence of mutations in p53 IRES might result in decreased induction of p53 protein following DNA damage due to altered RNA structure. This might constitute as one of the mechanisms leading to tumor development in some types of cancers. In the third part of the thesis, the role of important cellular proteins that might modulate p53 IRES mediated translation has been studied. These cellular proteins act as IRES interacting trans-acting factors (ITAFs). Polypyrimidine tract binding (PTB) protein is an important ITAF implicated in regulating IRES mediated gene expression during apoptosis. It was observed that PTB protein specifically interacts with both the IRES elements within p53 mRNA. Interestingly, the affinity of interaction of PTB protein with both p53 IRES RNAs was observed to be significantly different. In order to determine the contact points of PTB on p53 IRES, a foot-printing assay using structure specific nuclease and recombinant-PTB protein was performed on p53 RNA. The data from foot-printing as well as primer extension inhibition assay (toe-printing analysis) suggested the presence of multiple PTB binding sites on p53 IRES RNA. Based on these results, a deletion mutant was generated that showed reduced PTB binding and also reduced IRES activity as compared to wild type. Further, to study the role of PTB in mediating p53 translation, the expression of PTB gene was partially silenced by using PTB specific siRNA. Partial depletion of endogenous PTB protein showed a significant decrease in the p53 IRES activities. These results suggest that PTB protein is essential for the p53 IRES activities. To understand the probable mechanism by which PTB regulates p53 IRES mediated translation, CD spectroscopy analysis of p53 IRES RNA was performed in the absence and presence of PTB protein. Interestingly, CD spectra analysis of the p53 RNA in the presence of PTB suggested a specific conformational change in p53 IRES, which might probably facilitate ribosome loading during internal initiation. This also suggests that abnormal expression of p53 ITAFs might lead to reduced p53 induction following DNA damage conditions. It could also be another event leading to malignant transformation of cells bearing wild type p53. It is highly tempting to speculate that the levels of p53 ITAFs could also be used as tumor biomarkers. In the fourth part of the thesis, the physiological relevance of existence of IRES elements within p53 mRNA has been investigated. The levels of p53 and ΔN-p53 proteins are known to be regulated in a cell cycle phase-dependent manner. The IRES activities of both p53 IRES elements were investigated at different phases of cell cycle. The activity of the IRES responsible for translation of p53 protein was found to be highest at G2-M transition and the maximum IRES activity corresponding to ΔN-p53 synthesis was observed at G1-S transition. These results suggested that the p53 IRES activities are regulated in a cell-cycle phase-dependent manner. Next, the regulation of p53 IRES mediated translation during stress conditions was studied. Human lung carcinoma cell line, A549 cells (that endogenously express both the p53 isoforms), were exposed to DNA damaging drug, doxorubicin. The level of p53 protein was observed to increase in a time-dependent manner. Interestingly, PTB protein, which is predominantly nuclear, was found to translocate to the cytoplasm during stress condition in a time-dependent manner. Under similar conditions, p53 protein was observed to reverse translocate from the cytoplasm to nucleus, probably to function as a transcription factor. Next, the influence of partial PTB silencing on p53 isoforms in the presence of cell stress (mediated by doxorubicin) was investigated. The data indicated reduced levels of both p53 and ΔN-p53 when PTB gene expression was partially silenced. These observations constitute “the proof of concept” that relative abundance of an ITAF, such as PTB protein, might contribute to regulating the coordinated expression of the p53 isoforms. The thesis reveals the presence as well as the physiological relevance of existence of IRES elements within p53 mRNA. The novel discovery of p53 IRES elements may provide new insights into the underlying mechanism of translational regulation. The modulation of the p53 IRES activities by PTB protein suggests that the regulated expression of p53 isoforms depends on the integrity of IRES elements and availability of cellular proteins that can serve as p53 ITAFs. Thus, studies pertaining to the identification of mutations within p53 IRES region as well as abnormal expression of p53 ITAFs such as PTB in cancer cells may have far reaching implications. These studies might lead to further advances in the field of cancer detection, prognosis and design of novel therapeutic strategies.

Wild-type viruses with intrinsic oncolytic capacity in human includes DNA viruses like some autonomous parvoviruses and many RNA viruses. Recent advances in molecular biology have allowed the design of several genetically modified viruses, such as adenovirus and herpes simplex virus that specifically replicate in, and kill tumor cells. However, still several hurdles regarding clinical limitations and safety issues should be overcome before this mode of therapy can become of clinical relevance. It includes limited virus spread in tumor masses, stability of virus in the blood, trapping within the liver sinusoids, transendothelial transfer, and/or vector diffusion of viral particles to tumor cells, limited tumor transduction, immune-mediated inactivation or destruction of the virus. For replication-competent vectors without approved antiviral agents, suicide genes might be used as fail-safe mechanism. Cancer stem cells are a minor population of tumor cells that possess the stem cell property of self-renewal. Therefore, viruses that target the defective self-renewal pathways in cancer cells might lead to improved outcomes.<p>In this thesis, data we generated in the field of oncolytic autonomous parvoviruses are presented.<p>We replaced capsid genes by reporter genes and assessed expression in different types of human cancer cells and their normal counterparts, either at the level of whole cell population, (CAT ELISA) or at the single cell level, (FACS analysis of Green Fluorescent Protein). Cat expression was substantial (up to 10000 times background) in all infected tumor cells, despite variations according to the cell types. In contrast, no gene expression was detected in similarly infected normal cells, (with the exception of an expression slightly above background in fibroblasts.). FACS analysis of GFP expression revealed that most tumor cells expressed high level of GFP while no GFP positive normal cells could be detected with the exception of very few (less than 0.1%) human fibroblast cells expressing high level of GFP. We also replace capsid genes by genes coding for the costimulatory molecules B7-1 and B7-2 and show that, upon infection with B7 recombinant virions, only tumor cells display the costimulatory molecules and their immunogenicity was increased without any effect on normal cells. Using a recombinant MVM containig the Herpes Simplex thymidine kinase gene, we could get efficient killing of most tumor cell types in the presence of ganciclovir, whithout affecting normal proliferating cells. We also produced tetracycline inducible packaging cell lines in order to improve recombinant vectors yields. The prospects and limitations of these different strategies will be discussed.<p>An overview is given of the general mechanisms and genetic modifications by which oncolytic viruses achieve tumor cell-specific replication and antitumor efficacy. However, as their therapeutic efficacy in clinical trials is still not optimal, strategies are evaluated that could further enhance the oncolytic potential of conditionally replicating viruses in conjunction with other standard therapies. <p>Another exciting new area of research has been the harnessing of naturally tumor-homing cells as carrier cells to deliver oncolytic viruses to tumors. The trafficking of these tumor-homing cells (stem cells, immune cells and cancer cells), which support proliferation of the viruses, is mediated by specific chemokines and cell adhesion molecules and we are just beginning to understand the roles of these molecules. Finally, we will explore some ways deserving further study in order to be able to utilize various oncolytic viruses for effective cancer treatment. <p><p> / Doctorat en sciences, Spécialisation biologie moléculaire / info:eu-repo/semantics/nonPublished