Orientador: Paula Rahal / Coorientador: Yury Khudyakov / Banca: Ana Carolina Gomes Jardim / Banca: Isabel Maria Vicente Guedes de Carvalho Mello Membro / Banca: Camila Malta Romano / Banca: Paola Jocelan Scarin Provazzi Trabulsi / Banca: Cintia Bittar / Resumo: Embora novos medicamentos de ação direta anti-HCV tenham sido recentemente aprovados no mercado, com exceção do genótipo 1, a terapia mais utilizada ainda é baseada em Interferon (IFN) e Ribavirina. Desta forma, o genótipo 3 é o que apresenta mais baixa taxa de sucesso no tratamento, no atual cenário. Um dos fatores determinantes do insucesso deste tratamento no paciente é a variabilidade viral. O HCV apresenta alta taxa de mutação durante a replicação, implicando na produção de variantes intra-hospedeiro, denominadas quasispecies. A região hipervariável 1 (HVR1) da proteína do envelope é uma região de alta variabilidade do genoma viral. A detecção das quasispecies minoritárias pode ser realizada de forma confiável e eficiente por pirossequenciamento de alta cobertura (UPDS), técnica capaz de detectar variantes presentes em frequência <1% na população. Com base neste contexto, foram determinados quasispecies da HVR1-HCV de 14 pacientes infectados com o genótipo 3 do vírus, utilizando a técnica de UPDS. No total, 64.400 sequencias da HVR1 foram obtidas de amostras pré-tratamento. Destas, 27.398 sequências de alta qualidade foram filtradas e corrigidas. Valores de distância genética e entropia de Shannon não foram correlacionados a resposta ao tratamento. Estas sequências foram posteriormente submetidas a construção de redes median-joining e análise Bayesiana de estrutura populacional (BAPS). A análise identificou amostras com diferentes estruturas, desde altamente conservadas (apenas uma população de quasispecies) até altamente estratificadas (6 subpopulações). Este resultado foi condizente com a estrutura das redes median-joining. Várias mutações ao longo da HVR1 do mesmo paciente e algumas repetiram em pacientes do mesmo grupo de resposta. Quanto a avaliação de análise das sequências de aminoácidos, embora haja grande variação quanto a sequência e propriedades físico-químicas, pode-se ... / Abstract: Hepatitis C is a major public health problem. New HCV antiviral drugs were released on market on 2010; however, excluding for genotype 1, the most used therapy used currently is still based on Interferon (IFN) and Ribavirin. Nowadays, genotype 3 is the one with the highest rate of treatment failure. Viral genome variability is one of the factors that lead in therapy failure. HCV presents a high mutability during replication course, implicating in arising of intra-host variants called quasispecies. The hypervariable region 1 (HVR1) from envelope protein presents as quasispecies and may be related to IFN therapy resistance. Resistant quasispecies may not represent majority of variants population in the host, therefore, in these cases traditional sequencing techniques are unable to detect. For detection of minority quasispecies, ultra-deep pyrosequencing (UPDS) is a reliable and efficient tool, being able to detect even variants with frequency <1% in the population. Regarding this issue, we determined HVR1 quasispecies from 14 patients infected with HCV genotype 3 using the UPDS approach. In total, 64,400 HVR1 sequences were obtained from pre-therapy sample. From these sequences, 27,398 ones with high quality were filtered. Genetic distance and Shannon entropy values were not related to therapy outcome. These sequences were analyzed using median-joining networks and Bayesian population structural analysis. These analysis identified samples with different structures, from high conserved (one sub-population) to high stratified ones (6 sub-populations). Networks analysis also confirmed this result. Mutations exclusive for a type of response of therapy were identified along HVR1. Amino acid sequences indicated that this region presents conserved structure, even if sequence and physical and chemicals properties seem flexible. Especially in turns and coils positions, this conservation seems notable. Potential epitopes positions are concentrated ... / Doutor

Orientador: Paulo Inácio da Costa / Banca: Miriane da Costa Gileno / Banca: Vanderlei Rodrigues / Banca: Daniele Cardoso Geraldo Maia / Banca: Elisabeth Loshchagin Pizzolitto / Resumo: O vírus da hepatite C (VHC) é um vírus envelopado com cerca de 50 a 70 nm de diâmetro, fita positiva de RNA e pertence ao gênero do Hepacivirus e à família Flaviridae. A detecção e quantificação do antígeno do core, proteína do nucleocapsídeo do VHC tem obtido sucesso em muitos ensaios, sendo considerado um marcador da replicação viral, por apresentar uma sequencia de aminoácidos altamente conservada, o que lhe confere alta sensibilidade e especificidade. A proteína E2 é uma glicoproteína de envelope do VHC com 11 sítios de glicosilação e a maioria deles estão bem conservados, tornando-a um alvo antigênico. O objetivo deste estudo foi o de desenvolver métodos diagnósticos para o VHC de alta sensibilidade, baixo custo e que pudessem ser utilizados na triagem sorológica. As regiões genômicas que codificam as proteínas core (parcial 136 aa) e E2 do VHC foram expressas em E. coli da linhagem Rosetta e clonadas no vetor de expressão pET-42a e induzidas por IPTG 0,4mM, produzindo proteínas recombinantes fusionadas a proteína GST (glutationa S-transferase), que foram então purificadas por cromatografia de afinidade. A imunorreatividade foi avaliada por Western Blot, Slot Blot e os métodos diagnósticos (ensaio imunoenzimático (ELISA) de captura, indireto, imunoblotting e imunocromatográfico) desenvolvidos e aprimorados. Os métodos desenvolvidos foram mais sensíveis e específicos utilizando a mistura das proteínas recombinantes fusionadas a GST (core + E2) / Abstract: The hepatitis C virus (HCV) is an enveloped virus of about 50 to 70 nm in diameter, positive strand RNA, and belongs to the genus Hepacivirus and the family Flaviridae. The detection and quantification of the core antigen, HCV nucleocapsid protein, has been successful in many trials and is considered a marker of viral replication, since it presents a sequence of highly conserved amino acids, giving it high sensitivity and specificity. The E2 protein is an envelope glycoprotein of HCV with 11 glycosylation sites, most of these well-conserved, making it a target antigen. The aim of this study was to develop high sensitivity, low cost diagnostic methods for HCV which could be used for serological screening. The genomic regions encoding the core (part 136 aa) and E2 proteins of HCV were expressed in E. coli Rosetta strain, cloned in expression vector pET-42a, and induced with 0.4 mM IPTG, producing recombinant proteins fused to GST protein (glutathione S-transferase), which were then purified by affinity chromatography. The immunoreactivity was assessed by Western blot, Slot Blot and the developed and improved diagnostic methods (enzyme-linked immunosorbent assay (ELISA) capture and indirect, immunoblotting and immunochromatographic). The methods developed were more sensitive and specific using the mixture of the recombinant proteins fused to GST (core + E2) / Doutor

Los virus de RNA de cadena positiva conforman el grupo más numeroso y diverso de los patógenos virales de las plantas. Se replican mediante un mecanismo en el que la RNA polimerasa RNA dependiente viral, junto con otras proteínas virales y del huésped, sintetizan un intermediario de polaridad complementaria (o negativa), que a la vez sirve de molde para la síntesis del RNA genómico de polaridad positiva. Este mecanismo puede seguir un modelo geométrico o de stamping machine (Sardanyés et al. 2009). Por otro lado, las rutas de silenciamiento de RNA en plantas desempeñan un papel muy importante en la regulación de los procesos de desarrollo, mantenimiento de la estructura cromosómica y mecanismos de defensa contra virus patógenos (Matzke y Matzke, 2004). Los microRNA (miRNAs) son una clase de pequeños RNAs (sRNAs) endógenos que se producen en el núcleo a partir de transcritos precursores (pre-miRNA) en forma de horquilla, por la acción, en plantas, de Dicer 1 (DCL1). Los miRNAs maduros (21 nt) son cargados por RISC y dirigen la degradación de mRNAs endógenos que contienen una secuencia diana complementaria, regulando así su concentración. Para oponerse al silenciamiento de RNA antiviral, en la mayoría de virus de plantas y algunos virus animales han evolucionado proteínas supresoras del silenciamiento que interfieren en pasos clave de la ruta de los pequeños RNAs interferentes (siRNA): directamente secuestrando los siRNAs, inhibiendo su producción o previniendo su diseminación por el huésped a corta o larga distancia (Li y Ding, 2006). Recientemente se ha desarrollado una nueva estrategia para producir plantas resistentes a virus basada en las rutas de silenciamiento de RNA. Consiste en expresar en plantas transgénicas miRNAs artificiales (amiRNAs) dirigidos contra secuencias específicas de los virus sobre los que se desea resistencia (Niu et al., 2006). Plantas de Arabidopsis thaliana transgénicas que expresan amiRNAs en los que se ha sustituido en el pre-amiRNA la secuencia de 21 nt del miR159 maduro, por diferentes secuencias complementarias a distintas regiones del genoma de un potyvirus (Riechmann et al., 1992. Urcuqui-Inchima et al., 2001) como el virus del mosaico del nabo (TuMV) han resultado ser resistentes a este virus en ensayos de laboratorio (Niu et al., 2006). El estudio de los aspectos moleculares de la resistencia mediada por microRNAs artificiales durante la complejidad de la infección viral puede permitir la mejora en el diseño de esta estrategia de resistencia desde el punto de vista de robustez, especificidad, durabilidad y seguridad medioambiental. Sobre todo porque los virus de RNA presentan una elevada tasa de mutación (Duffy et al., 2008) y linajes evolutivos independientes de TuMV son capaces de romper la resistencia mediada por un amiRNA, debido a la aparición de alelos mutantes que evaden el reconocimiento del miRNA artificial (Lafforgue et al., 2011). / Martínez García, F. (2014). Análisis de la dinámica de replicación de un virus de RNA de plantas y del uso de microRNAs artificiales como estrategia antiviral [Tesis doctoral]. Editorial Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/35447 / TESIS / Premios Extraordinarios de tesis doctorales

Thesis (Ph. D.)--University of Alabama at Birmingham, 2006. / Title from first page of PDF file (viewed Feb. 14, 2008). Includes bibliographical references.

Host-pathogen interactions in Hepatitis C Virus infection: Deciphering the role of host proteins and microRNAs Hepatitis C virus (HCV) is a positive sense single stranded RNA virus belonging to the Hepacivirus genus of the Flaviviridae family. HCV genome consists of a single open reading frame flanked by highly structured 5‟ and 3‟ untranslated regions (UTRs) at both ends. Unlike cellular mRNAs, HCV RNA translation is independent of the cap structure and is mediated by an internal ribosomal entry site (IRES) present in the 5‟UTR. HCV replication begins with the synthesis of a complementary negative-strand RNA using the positive strand RNA genome as a template catalyzed by the NS5B RNA dependent RNA polymerase (RdRp). The de novo priming of HCV RNA synthesis by NS5B occurs at the very end of the 3‟UTR. The 3‟UTR is organized into highly structured regions namely the variable region, poly U/UC region and the 3‟X region. These regions contain cis-acting elements that determine the efficiency of viral replication. In addition, the interaction of trans-acting factors with the 3‟ UTR is also important for regulation of HCV replication. HCV 3‟UTR interacts with several cellular proteins such as the human La protein, polypyrimdine tract binding protein (PTB), poly (rC)-binding protein 2 (PCBP2) and Human antigen R (HuR). However, the molecular basis of regulation of viral replication by these proteins is not well understood. Many proteins that are hijacked by HCV as well as other cytoplasmic RNA viruses, such as La, PCBP2, HuR and PTB are RNA binding proteins (RBPs). They are involved in post transcriptional regulation of cellular gene expression. Thus the subversion of these proteins by the virus can affect their normal physiological functions. In addition to proteins, recent reports also describe the involvement of non-coding RNAs including microRNAs (miRNA) and long non coding RNAs (lncRNA) in HCV infection. miRNAs can either directly bind to the HCV genome and regulate its life cycle or indirectly modulate the expression of host proteins required by the virus. miRNAs that are differentially regulated in virus infected tissues or body fluids of infected patients can also serve as biomarkers for diagnosis of various stages of the disease. Hence, it was planned to study the role of host proteins and miRNAs in the HCV life cycle and pathogenesis to have novel insights into the biology of HCV infection. Riboproteomic studies have identified several host proteins that directly interact with the 5‟ and/or 3‟UTRs of the HCV RNA. One of the RNA binding proteins that predominantly interact with the 3‟UTR of HCV RNA was found to be HuR. In the present study, we have extensively characterized the interaction between HuR and HCV 3‟UTR and studied its functional implications in HCV life cycle along with other host factors. Characterizing the HCV 3’UTR–HuR interaction and its role in HCV replication HuR is a ubiquitously expressed member of the Hu family which shuttles between the nucleus and cytoplasm in response to stress. Whole genome siRNA knockdown and other studies have suggested that HuR is essential for HCV replication. However, the molecular mechanism of its involvement in this process was not clear. We observed that siRNA mediated knockdown of HuR reduces the HCV RNA and protein levels. Immunofluorescence studies indicated that HuR relocalizes from the nucleus to the cytoplasm in HCV infected cells. Through confocal microscopy and GST pulldown assays, we have demonstrated that HuR co localizes with the viral polymerase, NS5B and directly interacts with the NS5B protein. Membrane flotation assays showed that HuR is present in the detergent resistant membrane fractions which are the active sites of HCV replication. In addition to the interaction of HuR with the viral protein NS5B, we also characterized its interaction with the viral RNA. Direct UV cross linking assays and UV cross linking immunoprecipitation assays were performed to demonstrate the interaction of HuR with the HCV 3‟UTR. The RRM3, hinge region and RRM1 of HuR were found to be important for binding. Further, we observed that HuR competes with PTB for binding to the 3‟UTR when cytoplasmic S10 extracts or recombinant proteins were used in UV cross linking assays. In contrast, the addition of HuR facilitated the binding of La protein to the HCV 3‟UTR in the above assays. Competition UV cross linking assays indicated that both HuR and PTB bind to the poly U/UC region of the 3‟UTR while La binds to the variable region. HuR and La showed higher affinities for binding to the 3‟UTR as compared to PTB in filter binding assays. Since HuR and PTB interact with the same region on the 3‟UTR and HuR showed ~4 fold higher affinity for binding, it could displace PTB from the 3‟UTR. Next, we investigated the roles of HuR, PTB and La in HCV translation and replication in cell culture using three different assay systems, HCV sub genomic replicon, HCV bicistronic SGR-JFH1/Luc replicon as well as the infectious HCV full length RNA (JFH1). Results clearly indicated that HuR and La are positive modulators of HCV replication. Interestingly, PTB facilitated HCV IRES mediated translation but appeared to have a negative effect on HCV replication. The positive effectors, HuR and La showed significant co localization with one another in the cytoplasm in immunofluorescence studies. GST pulldown and coimmunoprecipitation experiments indicated protein-protein interactions between HuR and La but not between HuR and PTB. Through quantitative IP-RT assays, we demonstrated that the overexpression of HuR in HCV RNA transfected cells increases the association of La with the HCV RNA while HuR knockdown reduces the association of La with the HCV RNA. Previous studies in our laboratory have shown that La helps in HCV genome circularization. The addition of HuR significantly increased La mediated interactions between the 5‟UTR and the 3‟UTR of HCV RNA as monitored by 5‟-3‟ co precipitation assays, suggesting a possible mechanism by which cooperative binding of HuR and La could positively regulate HCV replication. Taken together, our results suggest a possible interplay between HuR, PTB and La in the regulation of HCV replication. Studying the role of HuR- associated cellular RNAs in HCV infection HuR belongs to the category of mRNA turnover and translation regulatory proteins (TTR-RBPs), which are capable of triggering rapid and robust changes in cellular gene expression. HuR plays a role in several post transcriptional events such as mRNA splicing, export, stability and translation. In the present study, we have investigated the possible consequences of relocalization of HuR on cellular processes in the context of HCV infection. We observed that 72h post transfection of infectious HCV-JFH1 RNA, there is an increase in the mRNA levels of some of the validated targets of HuR including the vascular endothelial growth factor A (VEGFA), dual specificity phosphatise 1 (MKP1) and metastasis - associated lung adenocarcinoma transcript (MALAT1). IP-RT assays demonstrated that the association of HuR with VEGFA and MKP1 was higher in HCV-JFH1 RNA transfected cells as compared to the mock transfected cells indicating that increase in HuR association could probably help in stabilization of these mRNAs. Interestingly, we observed that the association of HuR with the lncRNA MALAT1 decreases in the presence of HCV RNA, while its RNA levels increased. Earlier it has been reported that MALAT1 interacts with HuR and was predicted to interact with La. We confirmed the interaction of both HuR and La proteins with MALAT1 RNA in vitro and in the cell culture system. Results from our time course experiments suggest that relocalization of HuR and La upon HCV infection might decrease their association with the nuclear retained MALAT1 RNA leading to significant reduction in MALAT1 RNA levels at the initial time points. However at later time points, MALAT1 was found to be unregulated through activation of the Wnt/beta-catenin pathway as demonstrated using a chemical inhibitor against β-catenin. Since MALAT1 is a known regulator of epithelial mesenchymal transition (EMT) and metastasis, we further studied the physiological consequence of the observed increase in MALAT1 levels upon HCV infection. Cell migration and cell invasion studies suggested that the knockdown of MALAT1 led to the inhibition of HCV- triggered wound healing and matrigel invasion and also rescued the down regulation of E-Cadherin protein levels, an EMT marker. Our study highlights the importance of the lncRNA, MALAT1 in HCV infection and suggests its possible involvement in HCV induced HCC. Investigating the role of miRNAs in HCV pathogenesis and replication miRNAs can also regulate HCV infection and pathogenesis in multiple ways. It is known that under disease conditions, there is aberrant expression of intracellular as well as circulating miRNAs. We have investigated the expression profile of 940 human miRNAs in HCV infected patient serum samples to identify the differentially regulated miRNAs. miR-320c, miR-483-5p and the previously reported miR-125b were found to be upregulated in the serum of cirrhotic and non-cirrhotic HCV infected patient serum samples. All three miRNAs were also unregulated in the cell culture supernatant of HCV infected cells as well as within the HCV infected cells. miR-483-5p was specifically enriched in the exosomes isolated from patient serum samples. Knockdown of miR-320c and miR-483-5p did not have significant effect on HCV replication while knockdown of miR-125b affected HCV replication through regulation of one of its target genes, HuR. We observed that with time, miR-125b levels in HCV-JFH1 RNA transfected cells increase while the HuR protein levels decrease. Using luciferase reporter constructs, we demonstrated that the decrease in HuR protein levels is indeed mediated by miR-125b. Mutations in the target site of miR-125b in the HuR 3‟UTR prevented the down regulation of luciferase activity. Next we tested the effect of silencing miR-125b on HCV replication. Knockdown of miR-125b prevented the reduction in HuR protein levels but with no significant effect on HCV replication. It appeared that the HuR protein already present in the cytoplasm could be sufficient to support HCV replication. Hence similar experiments were carried out in cells depleted of HuR using either siRNA against HuR or a chemical inhibitor of nucleocytoplasmic transport of HuR, Leptomycin B. We observed that when the intracellular levels of HuR are reduced using either of the two approaches, there is a decrease in HCV replication. This is in accordance with the results obtained in the first part of the thesis. However when miR-125b was silenced in HuR depleted cells, we noticed an upregulation in the HuR protein levels by western blot analysis and a consequent increase in HCV RNA levels as quantified by qRT-PCR. From our findings, we can conclude that miR-125b mediated regulation of HuR plays an important role in HCV replication. We hypothesize that this could be a cellular response to HCV infection to which the virus responds by inducing protein relocalization. Altogether, these studies outline the importance of host factors including cellular proteins and non-coding RNAs in the regulation of HCV life cycle and pathogenesis. Results reveal the mechanistic insights into how HCV infection triggers host defense pathways, which are evaded by the virus by counter strategies.

HCV genome is a positive sense single-stranded RNA containing a single open reading frame (ORF) flanked by untranslated regions (UTRs), 5’UTR and 3’UTR.Initiation of HCV RNA translation is mediated by internal ribosome entry site (IRES) present in 5’ UTR and this process is independent of cap-structure and requires only a small subset of canonical initiation factors. Hence, HCV IRES-mediated translation initiation mechanism is quite different from canonical cellular mRNA translation initiation. The IRES is organized into highly structured domains, namely domain II, III and IV. High affinity interactions between structured RNA elements present in the IRES and 40S ribosomal proteins mediate 40S recruitment to HCV IRES. However, details of the RNA elements and region of ribosomal proteins involved in these interactions are poorly understood. In recent days, RNA-based molecules like siRNAs, antisense RNAs and RNA decoys have become promising candidates for antiviral molecules. So designing short RNA molecules that target unique HCV translation initiation mechanism might help in developing novel anti-HCV molecules. HCV 3’UTR and antisense-5’ UTRs serve as sites for replication initiation to synthesize negative and positive strand and this process is catalyzed by NS5B protein (RNA-dependent RNA polymerase). Hence, host proteins binding to both 3’UTR and antisense-5’UTR might play important role in HCV replication. This puts the study of HCV RNA–host protein interactions and its role in viral translation and replication in perspective. Studying the HCV IRES-ribosomal protein S5 interactions and its role in HCV IRES function Previous studies from our laboratory have demonstrated that binding of La protein to GCAC close to initiator AUG enhances ribosomal protein S5 (RPS5) binding with HCV IRES and stimulates HCV translation. However in-detail study on HCV IRES–RPS5 interactions and its implication on HCV translation initiation were lacking. In present study computational modelling suggested that domain II and IV interact majorly with the beta hairpin structure and C-terminal helix of RPS5. Filter-binding and UV cross-linking studies with peptides derived from predicated RNA-binding region of RPS5 and mutational studies with RPS5 demonstrated that beta hairpin structure present in RPS5 is critical for IRES–RPS5 interaction. In parallel, we have studied RNA elements involved in the IRES–RPS5 interactions using deletions and substitution mutations, which we had generated on the basis of the computational model. Direct and competition UV cross-linking experiments performed with these IRES mutants and 40S subunits as a source of RPS5 suggested that structure and sequence of both domain II and IV play crucial role in IRES–RPS5 interactions. We further investigated the effect of these mutations on IRES activity by in vitro translation assay and found that all the mutants that were compromised in binding to RPS5 showed reduced IRES activity. Moreover, ribosome assembly experiments on HCV IRES demonstrated that mutations affecting IRES–RPS5 interactions result in reduction of 80S peak and slight increase of 48S peak. Since the 40S subunit had been previously reported to bind with HCV 3’UTR, we explored the possible interaction of RPS5 with HCV 3’UTR. From direct and competition UV cross-linking assays, we found that RPS5 does not bind to 3’UTR and the interaction is unique to IRES (5’UTR). Interestingly, partial silencing of RPS5 preferentially inhibited HCV translation with marginal effect on cap-dependent translation. Recently, reduction in 40S subunit abundance was reported to preferentially inhibit HCV translation. So, we investigated the abundance of free 40S subunit upon silencing RPS5 and results showed reduction in free 40S subunit level. So, we hypothesize that silencing of RPS5 reduces free 40S abundance to inhibit HCV translation. Taken together, results identified specific RNA elements present in HCV IRES that are critical for IRES–RPS5 interactions and demonstrated the role of these interactions in HCV translation initiation. Targeting ribosome assembly on HCV IRES using short RNAs Stem-loops (SL) IIIe and IIIf of HCV IRES are known to play an important role in stable IRES–40S complex formation. However interaction of these stem-loops with 40S subunit in isolation, independent of other regions of HCV IRES, was not studied. In this study, using electrophoretic mobility shift assay (EMSA) and sucrose gradient centrifugation experiments, we demonstrate that short RNA containing both SLIIIe and SLIIIf together (SLRef RNA) binds to 40S subunit, while short RNAs containing either of the stem-loops (SLRe RNA and SLRf RNA) lose their ability to interact with 40S subunit. Further, SLRef RNA inhibited ribosome assembly on the IRES, whereas SLRe and SLRf RNA failed to inhibit the same. Since SLRef RNA is derived from IRES, we investigated the interaction SLRef RNA with IRES–trans-acting factors (ITAFs). UV cross-linking of radio-labelled HCV IRES with cytoplasmic extract (S10) in presence of unlabelled short RNAs suggested possible interactions of La and RPS5 proteins with SLRef RNA. Studies with recombinant La protein and RPS5 further confirmed their interaction with SLRef RNA. Ex vivo experiments with HCV bicistronic RNA suggested that SLRef RNA specifically inhibits HCV translation. In addition to that SLRef RNA inhibited the HCV RNA synthesis in JFH1 HCV cell culture system. Moreover, specific delivery of pSUPER construct expressing SLRef RNA (pSUPERSLRef) to mice liver along with HCV bicistronic construct using Sendai virosomes demonstrated specific inhibition of HCV IRES activity by SLRef RNA in mice hepotocytes. In summary, short RNA derived from HCV IRES was shown to bind with La protein and RPS5 to inhibit ribosome assembly on HCV IRES. Further, targeted delivery of SLRef RNA into mice liver using Sendai virosome resulted in inhibition of HCV RNA translation in mice hepatocytes. Characterizing the interaction of host proteins with antisense-5’UTR and 3’UTR and its significance in HCV replication Antisense-5’UTR and 3’UTR of HCV RNA are the sites of replication initiation. Hence, host proteins binding to both of these RNA sequences are potential candidates for regulation of HCV replication. In this study, we have investigated host proteins binding with antisense-5’UTR and 3’UTRof HCV RNA by performing UV cross-linking experiments with cytoplasmic extract of Huh7 cells, and found that a protein of ~42kDa protein interacts with both antisense-5’UTR and 3’UTR. Based on earlier report, we predicted that the ~42kDa protein could be hnRNPC1/C2. Results of UV cross-linking followed by immuno pull-down (UV-IP assay) and UV cross-linking experiments with recombinant hnRNPC1 protein confirmed that hnRNPC1 indeed binds to antisense-5’UTR and 3’UTR. Further, filter-binding experiments demonstrated that hnRNPC1 protein binds to 3’UTR with higher affinity compared to antisense-5’UTR. Subsequently, we investigated the regions within 3’UTR and antisense-5’UTR that interact with hnRNPC1protein. Results demonstrated that poly-(U/UC) region of 3’UTR and region containing stem-loops SL-IIIa’, SL-IIIb’, SL-IIIcdef’ and SL-IV’ in antisense-5’UTR were mostly involved in the interaction. Interestingly, studies with confocal microscopy suggested that hnRNPC1/C2 re-localizes from nucleus to cytoplasm upon JFH1 infection, which might in turn influence HCV replication. To investigate the role of hnRNPC1/C2 in HCV replication, partial silencing of hnRNPC1/C2 was performed in HCV cell culture system (JFH1) and results demonstrated that hnRNPC1/C2 is critical for HCV RNA synthesis. However experiments with HCV bicistronic RNA suggested that hnRNPC1/C2 does not play significant role in HCV translation. Taken together, results suggested that hnRNPC1/C2 re-localizes from nucleus to cytoplasm upon JFH1 infection and binds to HCV 3’UTR and antisense- 5’UTR to regulate HCV replication. In summary, this thesis provides novel insights into the interaction of host proteins with HCV RNA and its significance in HCV translation and replication. Inhibition of the ribosome assembly and consequent reduction in HCV translation with mutations interfering with IRES–RPS5 interaction, reported in the present study, unfolds the novel role of this interaction in HCV translation. Further, results obtained in the present study with a small RNA SLRef, derived from HCV IRES, provide proof of concept for using short RNAs to specifically inhibit HCV translation. In addition, studies of interaction of hnRNPC1/C2 with HCV RNA and its re-localization upon HCV infection sheds light on the significance of host–virus interaction in viral RNA replication.

One of the important tasks of virology and immunology is to explore the species- and cell-barriers preventing virus horizontal transmission and reveal the ways how viruses overcome these barriers and "adapt" to different species. This work is based on a well- established retroviral model - avian Rous sarcoma virus (RSV) and studies virus replication blocks in mammalian cells at both pre- and post-integration level. Interaction of the viral envelope glycoprotein (Env) with a specific cellular receptor mediates virus entry into cells. Although mammalian orthologues of specific chicken receptors do not support RSV entry, it was observed that some RSV strains are able to enter mammalian cells. Several RSV-transformed rodent cells lines were described and analysis of provirus H20- RSV in one these cells lines (hamster H-20 tumor cell line) showed multiple mutations including two crucial amino acid substitutions in different regions of Env. Substitutions D32G and L378S confer virus transmission to hamster, human and also chicken cells lacking the appropriate receptor. Altered conformation of H20-RSV Env is similar to a receptor-primed (activated) state of Env. This observation indicates that virus can circumvent the need of original cell receptor because of spontaneous Env activation caused by single...