Dendritic BC1 RNA in translational control mechanisms

Translational control at the synapse is thought to be a key determinant of neuronal plasticity. How is such control implemented? We report that small untranslated BC1 RNA is a specific effector of translational control both in vitro and in vivo. BC1 RNA, expressed in neurons and germ cells, inhibits a rate-limiting step in the assembly of translation initiation complexes. A translational repression element is contained within the unique 3′ domain of BC1 RNA. Interactions of this domain with eukaryotic initiation factor 4A and poly(A) binding protein mediate repression, indicating that the 3′ BC1 domain targets a functional interaction between these factors. In contrast, interactions of BC1 RNA with the fragile X mental retardation protein could not be documented. Thus, BC1 RNA modulates translation-dependent processes in neurons and germs cells by directly interacting with translation initiation factors

Investigating the modulation of viral translation by the Hepatitis C virus nonstructural protein 5A

Hepatitis C virus NS5A is a multi-functional viral protein essential for viral replication and assembly, although the exact role the protein plays in the viral lifecycle remains unclear. A vast array of functions have been attributed to NS5A in recent years, despite the lack of enzymatic activity. NS5A has been found to interact with over 130 host proteins including many which are central to cellular signaling pathways. NS5A is composed of three domains separated by regions of low complexity. All three domains perform important functions in the viral lifecycle. Domains I and II are essential for viral replication whereas domain III is required for viral assembly. However, the role that NS5A and its individual domains may play in modulating viral translation remains controversial. Previous studies have utilized translation reporter systems that do not accurately reflect the role of the viral 3´-UTR in modulating viral translation. We and others have shown that NS5A binds to the poly-U/UC region of the 3´-UTR. In addition to serving as the initiation site for negative strand synthesis the 3´-UTR functions to significantly enhance viral translation. The mechanism of translation enhancement remains unclear but may involve long range RNA-RNA interaction with the IRES, the binding of cellular proteins which stimulate translation and/or the recycling of ribosomes. Therefore, the protein-RNA interaction between NS5A and the poly-U/UC region has the potential to modulate viral translation. Therefore we set out to determine the role of NS5A and its individual domains in modulating viral translation and the role of the NS5A-poly-U/UC region interaction in this modulation. Utilizing monocistronic RNA reporters which contain the viral 5´- and 3´-UTRs and an internal Renilla luciferase reporter gene, we determined that NS5A specifically down-regulates viral translation in a dose-dependent manner through a mechanism dependent upon the presence of the poly-U/UC region in the viral 3´-UTR. Furthermore, we have re-tested the effect using full-length HCV genomic RNA reporters. These results suggest that NS5A is able to interfere with the stimulation of viral translation exerted by the 3´-UTR. This down-regulatory function of NS5A may function in mediating a switch from translation to replication, a step required in the lifecycle of a positive sensed RNA virus. Having established a role for NS5A in modulating viral translation, we then aimed to determine which region of NS5A was responsible for this effect. We found that each of NS5A domains was capable of this modulatory effect on viral translation independently. Although surprising, this finding is not entirely unexpected as each domain has been shown to retain the ability to bind to the poly-U/UC region. Within NS5A domain I we identified a 61 aa. region sufficient for translation down-regulation. Furthermore, we have identified a number of positively charged residues within this region involved in the modulation of viral translation, in particular arginine 112 (R112). This residue has previously been found to be at the domain I dimer contact interface and to form an intermolecular hydrogen bond with glutamic acid 148 (E148). We found that mutations R112A and E148A individually negate the ability of domain I to modulate viral translation and these mutations impede the formation of domain I dimers. Additionally, the R112A mutation appears to affect the ability of domain I to interact with the poly-U/UC region of the viral 3´-UTR alluding to the possible mechanism of translation modulation. Finally this mutation was lethal in an HCV subgenomic replication, confirming the link between NS5A dimerization, RNA binding and viral replication. These results collectively point to a crucial role for the NS5A arginine 112 residue in the modulation of HCV lifecycle by NS5A. Within NS5A domain II, we identified a 47 aa. region sufficient for translation modulation. Through the mutation of positively charged amino acids within this region, we found that lysine 312 (K312) was essential for this effect. The ability of this domain to modulate viral translation was completely lost when K312 was mutated within a full domain II protein fragment. The mechanism behind this modulation remains unclear but the 47 aa. region identified has been previously found to contain a region proposed to make contact with poly-U RNA and the K312 residue was suspected to interact directly with such RNA. Furthermore, this region interacts with the host protein cyclophilin A, an interaction that enhances the RNA binding ability of domain II. These findings strongly suggest that domain II modulates viral translation by binding within the poly-U/UC region. While investigating the modulation of viral translation by NS5A domain III we determined that the C-terminal 31 aa. are sufficient for the effect of this domain on viral translation. Through alanine scanning mutagenesis we identified glutamic acid 446 (E446) as playing a key role in the modulatory function of this region. Within a domain III protein fragment mutation of this E446 residue abolishes the modulatory function of this domain towards HCV translation. The mechanism behind this modulation and the role of E446 in this effect remains to be determined. These findings suggest that in addition to being essential for viral replication and assembly, NS5A has an important role in modulating viral translation through a mechanism requiring the poly-U/UC region of the viral 3´-UTR. Furthermore, each domain of NS5A appears to contribute to this effect. These results support the description of NS5A as a multi-functional protein and the further characterization of its functions may aid in the development of novel antivirals targeting the numerous functions of this complex, and at times puzzling, viral protein

A +RNA virus diptych : Chikungunya virus-host interactions and arteriviral programmed ribosomal frameshifting

In part 1 of the thesis quantitative proteomics was used to determine changes in abundance and phosphorylation status of host proteins during infection with the human pathogen chikungunya virus (CHIKV). Several proteins were identified that may be specifically downregulated during CHIKV infection to create a suitable environment for viral replication. eEF2 was identified as a factor that becomes strongly phosphorylated during infection with several viruses which may be a mechanism to stall translation in response to viral infection. In part 2 of the thesis the discovery of a novel and unusual -2/-1 programmed ribosomal frameshifting (PRF) mechanism is described that is used during translation of the nonstructural open reading frame of the economically important porcine reproductive and respiratory syndrome virus (PRRSV). This mechanism relies on a slippery site and stimulatory RNA signal in the PRRSV genome and is stimulated by the viral protein nsp1beta and host poly (C) binding proteins 1 and 2. Frameshifting results in the synthesis of two previously unidentified proteins, nsp2 TF (-2 PRF) and nsp2N (-1 PRF). Virus mutants that can no longer make the frameshift products are attenuated and may be used for vaccine development.UBL - phd migration 201

The DEAD-box helicase DDX3 supports the assembly of functional 80S ribosomes

The DEAD-box helicase DDX3 has suggested functions in innate immunity, mRNA translocation and translation, and it participates in the propagation of assorted viruses. Exploring initially the role of DDX3 in the life cycle of hepatitis C virus, we observed the protein to be involved in translation directed by different viral internal ribosomal entry sites. Extension of these studies revealed a general supportive role of DDX3 in translation initiation. DDX3 was found to interact in an RNA-independent manner with defined components of the translational pre-initiation complex and to specifically associate with newly assembling 80S ribosomes. DDX3 knock down and in vitro reconstitution experiments revealed a significant function of the protein in the formation of 80S translation initiation complexes. Our study implies that DDX3 assists the 60S subunit joining process to assemble functional 80S ribosomes

Interaction of hepatitis C virus polymerase with host cell proteins

Hepatitis C virus (HCV) interacts with host cell proteins to modify cellular pathways creating a favourable environment that facilitates its replication and persistence. The purpose of the work presented in this thesis was to identify cellular proteins that can interact with NS5B, the virus's RNA-dependent RNA polymerase, that may contribute to the virus's biology. A number of cellular proteins were found to interact with NS5B using the yeast two-hybrid system. These proteins were involved in cellular pathways such as interferon signalling, lipid transport and metabolism, protein trafficking, cell proliferation and apoptosis. Of these, phospholipid scramblase 1 (PLSCR1) and zinc finger protein 143 (ZNF143) were selected for further investigation. The interactions were confirmed in vitro, and, for PLSCR1, the region that interacted with NS5B was determined to be within the amino-terminal region of the protein (61-137 a.a.). NS5B interacted with PLSCR1 and ZNF143 via a single interacting region localized in its N-terminus (1-153 a.a.).Expression of PLSCR1 or ZNF143 enhanced the ability of interferon to stimulate transcription from an interferon-stimulated response element (ISRE) reporter construct. Co-expression with NS5B was found to down-regulate this activity. Expression of a number of interferon-stimulated genes was investigated in the presence of NS5B, PLSCR1 or ZNF143 but no significant effect was observed. Overexpression of PLSCR1 had no effect on HCV sub-genomic replicon replication, while reduction of its expression by short hairpin RNA (shRNA) enhanced replication. Overexpression of ZNF143 was found to have a suppressive effect on replication but downregulating its expression did not enhance replication. In addition to using the yeast two-hybrid system to identify NS5B- interacting proteins, an in vitro pulldown assay coupled with mass spectrometry identified α- and β -tubulin associated with NS5B in vitro and in vivo. Subsequently this association was demonstrated to be an indirect interaction but the intermediatory partner was not identified. The domain that mediated the association with α- and β-tubulin was determined to be within the N-terminus of NS5B (1-153 a.a.). Nocodazole, an inhibitor of tubulin polymerization, had a marked effect on the association of α -tubulin with NS5B displacing it from the complex but had no effect on β -tubulin's association. Utilizing an HCV sub- genomic replicon, nocodazole was shown to have a significant inhibitory effect on replication. Taken together the data presented in this thesis showed that NS5B had a multitude of potential interactions with a variety of cellular proteins. The biological significance of some of these interactions on the cellular response to IFN and replicon replication was investigated. This work has generated a number of novel observations on the interaction between the virus and the cell that warrant future investigatio

Papel de la vía endocítica en la infección del virus de la peste porcina africana

Tesis Doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 17-10-2013En este trabajo de tesis hemos caracterizado que la infección del virus de la peste porcina africana (VPPA) es dependiente de la vía endocítica. Una vez el VPPA se internaliza en las células, se incorpora inmediatamente a la vía endocítica. Esta vía comprende una serie de vesículas que sufren cambios madurativos altamente regulados por las Rab GTPasas y los fosfoinosítidos (FIs). Estos cambios modifican las características fisiológicas de las vesículas endocíticas a lo largo del proceso de maduración. La infección, comienza con el paso del VPPA por los endosomas tempranos (EE), dada la elevada colocalización entre el virus con estas vesículas desde los primeros minutos de la infección. Posteriormente, el VPPA alcanzaba los endosomas tardíos (LE), donde se producía la desencapsidación de un modo dependiente de pH ácido antes de la primera hora post infección. La señalización relacionada con la vía endocítica era crucial para el éxito de la infección, en concreto una diana molecular de gran relevancia, la GTPasa Rab7, reguladora de la dinámica del LE. La infectividad del VPPA resultaba severamente afectada cuando se anulaba la función de la GTPasa Rab7. Otra señalización relevante para el LE es dependiente del fosfatidil inositol 3,5 bifosfato. El bloqueo de la síntesis de este FI, especialmente antes de la infección, producía una disminución de la expresión de proteínas virales y de la producción viral. Una de las etapas cruciales de la infección, correspondió a los cuerpos multivesiculares (MBVs), un estadío madurativo intermedio, previo al LE. Los MBVs se caracterizan por presentar numerosas vesículas intraluminales (ILVs) en su interior. Las ILVs están enriquecidas por un lípido no convencional, el ácido lisobisfosfatídico (LBPA). Al anular su acción mediante un anticuerpo bloqueante, se afectaba la capacidad infectiva del virus. Asimismo, se demostró que la infección por VPPA en sus primeras etapas era altamente dependiente del flujo de colesterol libre desde los endosomas (LE) hacia sus destinos celulares. Las mencionadas moléculas de señalización de la vía endocítica son dianas moleculares para el virus y las membranas endosomales podrían jugar un papel como plataformas para la formación de la organela de replicación viral (ORV) o factoría vírica. Hasta ahora, se consideraba que las ORVs eran acúmulos de proteínas y DNA viral en los que no podía detectarse ninguna organela a excepción de ribosomas. Sin embargo, hemos descrito por primera vez, que las membranas endosomales se encuentran entremezcladas con los focos de acúmulo de proteínas víricas en las fases iniciales de su formación. Además, la formación de la factoría del VPPA conlleva una redistribución de todos los compartimentos endosomales hacia el área donde se constituye el complejo de ensamblaje del virus, resultando fundamental en las etapas iniciales de la infección por VPPA.In this thesis we have characterized the dependence of the African swine fever virus (ASFV) infection on the endocytic pathway. Once ASFV is internalized into cells, it is immediately incorporated to the endocytic pathway. This pathway comprises a number of vesicles undergoing maturational changes highly regulated by Rab GTPases and phosphoinositides (PIs). These changes modify the physiological characteristics of the endocytic vesicles along the maturation process. Infection begins with the passage of ASFV through early endosomes (EE), given the high colocalization between virus proteins and these vesicles found from the first 30 minutes of infection. Subsequently, according to our results, ASFV reaches the late endosomes (LE) where desencapsidation occurs in an acid pH-dependent manner before the first hour post infection. The endocytic pathway related signaling is crucial for a successful ASFV infection, specifically a highly relevant molecular target is the GTPase Rab7, regulating dynamics of LE. ASFV infectivity was severely affected when impairing the Rab7 function. Another relevant LE signaling was phosphatidylinositol 3,5 bisphosphate dependent. Blocking the synthesis of this PI, especially starting before infection, caused a severe decrease in viral protein expression and virus production. A crucial endosomal stage for ASFV infection corresponded to multivesicular bodies (MBV), an intermediate maturation stage prior to LE. MBVs are characterized by numerous intraluminal vesicles (ILVs). ILVs are enriched by an unconventional lipid, the lysobisphosphatidic acid (LBPA) which directly correlates with intracellular cholesterol levels. By withdrawing its action with a blocking antibody, virus infectivity was also affected. Furthermore, it was demonstrated that ASFV infection at its early stages was highly dependent on the flow of free cholesterol from LE to their cell destinations. The mentioned signaling molecules of the endocytic pathway might are molecular targets for the virus. Also, endosomal membranes might play a role as a scaffold for the formation of viral replication organelle or viral factory. Until now, it was assumed that viral factories were composed by proteins and viral DNA in bulk accumulations, in which no organelle could be detected, exception made for ribosomes. However, we have described for first time that endosomal membranes were found interspersed between early aggregates of viral proteins accumulation at the initial stages of the viral replication site formation, which is microtubule dependent. Furthermore, the formation of ASFV factory entailed redistribution of all endosomal compartments to the area where the assembly complex of the virus is constituted, resulting in a key process for ASFV infection

Target identification and molecular characterization of the RNA-binding protein XSeb4R in Xenopus laevis

Während entwicklungsbiologische Prozesse im Kontext der transkriptionalen Regulation ausführlich untersucht wurden, ist weniger über die Regulation durch translationale Regulation bekannt. RNA-Bindeproteine sind in alle Schritte des RNA-Metabolismus involviert und können dabei mehrere und unterschiedliche Funktionen erfüllen. Das RNA-Bindeprotein XSeb4R wurde als positiver Regulator der primären Neurogenese in Xenopus laevis identifiziert (Boy et al., 2004). Allerdings sind weder Zieltranskripte von XSeb4R, noch die Funktion im Kontext des RNA-Lebenszyklus bekannt. Im Rahmen dieser Arbeit wurde gezeigt, dass XSeb4R an den 3 UTR seiner Ziele bindet, die Stabilität dieser Transkripte erhöht und deren Translation aktiviert. Die translationale Aktivierung wird durch Interaktion von XSeb4R mit kappenassoziierten Proteinen ermöglicht. Als Zieltranskripte von XSeb4R konnten wichtige Regulatoren der Emryogenese, wie den neuralen Determinierungsfaktor Xngnr-1 und den Hauptregulator der Keimblattformation, VegT, identifiziert werden. Mit Hilfe einer RNA-Immuno-Präzipitation wurde ein weiteres XSeb4R-Zieltranskript identifiziert, Xl G14, dessen Funktion während der Embryogenese weiterer Studien bedarf. Zusammengefasst betonen diese Erkenntnisse die Signifikanz der Genexpression durch spezifische Translationsregulatoren wie XSeb4R und den Bedarf an weiteren Studien solcher Faktoren, um die komplexen Mechanismen der Embryonalentwicklung entwirren zu können

Characterisation of the translation efficiency and quasispecies composition of the 5'untranslated region of Hepatitis C virus in genotype 1 and 3 infected patients

Hepatitis C virus (HCV) infects over 170 million people worldwide. Chronic infection occurs in 50-80% of cases and eventually leads to cirrhosis and hepatocellular carcinoma. HCV can be classified into six genotypes. Genotypes 1, 2 and 3 have a world-wide distribution but their prevalence differs from one geographical area to another. In Scotland there is an approximate 50/50 split between patients infected with HCV genotype 1 and genotype 3. One difference which has been consistently demonstrated is the better response of patients infected with genotypes 2 and 3 to interferon treatment than those infected with genotype 1. The HCV lifecycle is only partly understood owing to the lack of a productive cell culture system. There is no vaccine to prevent infection by HCV. Given the predicted future impact of the disease, there is a great need to understand the molecular basis of the HCV life cycle. Protein translation is one of the important processes in HCV replication. It involves an internal ribosome entry site (IRES) in the 5'untranslated region (5'UTR). Comparison of the sequence and function of the 5'UTR from different genotypes might differentiate features essential to the virus life cycle in all genotypes from those relevant only to individual genotypes. In this study, the 5'UTR region of genotype 3 was compared with that of genotype 1 with respect to translation initiation and quasispecies composition. The association between translation efficiencies, serum viral loads and the histology of the liver was also investigated. (Abstract shortened by ProQuest.)

Analysis of RNA-Protein interactions involved in calicivirus translation and replication

The interaction of host-cell nucleic acid-binding proteins with the genomes of positive-stranded RNA viruses is known to play a role in the translation and replication of many viruses. To date, however, the characterisation of similar interactions with the genomes of members of the Caliciviridae family has been limited to in vitro binding analysis. In this study, feline calicivirus (FCV) and murine norovirus (MNV) have been used as model systems to identify and characterise the role of host-cell factors that interact with the viral RNA and RNA structures that regulate virus replication. It was demonstrated that RNA-binding proteins such as polypyrimidine tract-binding protein (PTB), poly(C)-binding proteins (PCBPs) and La protein interact with the extremities of MNV and FCV genomic and subgenomic RNAs. PTB acted as a negative-regulator in FCV translation and is possibly involved in the switch between translation and replication during the late stages of the infection, as PTB is exported from the nucleus to the cytoplasm, where calicivirus replication takes place. Furthermore, using the MNV reverse-genetics system, disruption of 5' end stem-loops reduced infectivity ~15-20 fold, while disruption of an RNA structure that is suspected to be part of the subgenomic RNA synthesis promoter and an RNA structure at the 3' end completely inhibited virus replication. Restoration of infectivity by repair mutations in the subgenomic promoter region and the recovery of viruses that contained repressor mutations within the disrupted structures, in both the subgenomic promoter region and the 3’ end, confirmed a functional role for these RNA secondary structures. Overall this study has yielded new insights into the role of RNA structures and RNA-protein interactions in the calicivirus life cycle

Molecular Biology and pathogenesis of coronavirus: Virus and host factors involved in viral RNA transcription

Ph.DDOCTOR OF PHILOSOPH