Le virus de l’hépatite C (HCV) est un problème majeur de santé publique. Le développement de nouveaux traitements pour lutter contre le HCV a été ralenti par l’absence de modèles d’études in vitro et in vivo convenables. Le but de mon travail de thèse a été, dans un premier temps, de caractériser les facteurs déterminant le tropisme hépatique du HCV. En exprimant des facteurs clés dans une lignée cellulaire humaine non-hépatocytaire, nous avons reconstitué in fine l’ensemble du cycle viral dans ces cellules. L’entrée du virus dans la cellule hôte fait intervenir différents récepteurs d’entrée dont CD81, occludin (OCLN), claudin-1 (CLDN1) et scavenger receptor class B type I (SR-BI). L’expression de ces quatre récepteurs sur cette lignée la rend hautement permissive à l’entrée du virus, mais ne permet pas de rétablir la réplication du virus. L’expression du micro-ARN 122, un micro-RNA important pour l’infection du HCV, dans les cellules exprimant les quatre récepteurs, restaure une forte réplication de l’ARN viral mais ne permet pas de détecter une production de particules infectieuses. L’expression de l’apolipoprotein E (apoE), jouant un rôle primordial dans l’assemblage et la sécrétion, rétablis cette dernière étape du cycle viral du HCV dans la lignée cellulaire humaine non-hépatocytaire. Dans un second temps, j’ai utilisé la stratégie, précédemment établie, pour étudier la spécificité d’espèce de l’infection du HCV dans plusieurs lignées hépatocytaires murines. Nous avons pu rendre ces cellules permissives à l’entrée du HCV et pu détecter une très faible réplication. L’ensemble de mes travaux apportent de nouvelles informations sur la compréhension des facteurs clés nécessaire au cycle viral du HCV dans des cellules murines et humaines. / Hepatitis C virus (HCV) is a global health burden. The development of new therapeutics to treat HCV infection has been hampered by the lack of convenient in vitro and in vivo model systems. The goal of my PhD work was, in a first time, to characterize the factors determining the hepatotropism of HCV. By expressing key factors within a non-hepatic cell line, we reconstituted in fine the full HCV life cycle in those cells. Virus entry into the host cell requires different entry factors which are CD81, occludin (OCLN), claudin-1 (CLDN1) and the scavenger receptor class B type I (SR-BI). The expression of these four factors in this cell line renders it highly permissive to viral entry, but does not allow restoring replication of the virus. The expression of miR-122, a micro-RNA important for HCV infection, into the cell lines expressing the four HCV entry factors restore a strong replication of the HCV RNA but does not allow detecting infectious viral particle production. Further expression of the apolipoprotein E (apoE), which plays a critical role in the assembly and release process, restore the last step of the HCV life cycle in a non-hepatic cell line. In a second part of my PhD, I have used the previously developed strategy to study the species specificity of HCV infection using different mouse hepatoma cell lines. We have been able to render these cell lines permissive to HCV entry and have been able to detect a slight replication. Altogether, my results bring new information on the understanding of key factors important for HCV life cycle in mouse and human cells.

Submitted by Ana Maria Fiscina Sampaio (fiscina@bahia.fiocruz.br) on 2012-07-18T19:50:32Z No. of bitstreams: 1 Felicidade Mota Pereira Avaliação da utilização do teste....pdf: 1069374 bytes, checksum: 6d5e3ef5ace61dfaf117c001157d5cec (MD5) / Made available in DSpace on 2012-07-18T19:50:32Z (GMT). No. of bitstreams: 1 Felicidade Mota Pereira Avaliação da utilização do teste....pdf: 1069374 bytes, checksum: 6d5e3ef5ace61dfaf117c001157d5cec (MD5) Previous issue date: 2011 / Fundação Oswaldo Cruz. Centro de Pesquisas Gonçalo Moniz. Salvador, Bahia, Brasil / A infecção pelo vírus da hepatite C (VHC) é comumente assintomática e apresenta uma elevada taxa de cronicidade, podendo evoluir para cirrose e carcinoma hepatocelular. O diagnóstico da hepatite C é realizado através da pesquisa de anticorpos pelo teste de ELISA (Enzyme Linked Immunosorbent Assay) e confirmado por testes suplementares, tais como o RIBA (Recombinant Immunoblot Assay) e westernblot e teste confirmatório, como a pesquisa do VHC-RNA. O objetivo deste trabalho foi avaliar a eficácia do RIBA no diagnóstico da infecção pelo VHC em doadores de sangue com anti-VHC reagente. Foram analisadas 102 amostras com resultado de anti-VHC reagente na HEMOBA, utilizando-se o teste anti-VHC Architect Abbott por quimioluminescência para detecção dos anticorpos anti-VHC, o RIBA III (CHIRON) como teste suplementar para as amostras anti-VHC reagentes e indeterminadas e a Reação em Cadeia da Polimerase (RT-PCR) convencional ou em tempo real (Amplicor Roche) para detecção do VHC-RNA. As amostras com VHC-RNA detectável foram genotipadas por hibridização reversa (LIPA; SIEMENS). Das 102 amostras analisadas no LACEN, 38,2% (39/102) foram reagentes, 57,8% (59/102) foram não reagentes e 3,9% (4/102) foram indeterminadas para o anti-VHC. Os resultados do RIBA foram 58,1% (25/43) positivos, 9,3% (4/43) negativos e 32,6% (14/43) indeterminados. Todas as amostras com resultado de RIBA indeterminado tiveram carga viral indetectável. As bandas predominantes nas amostras indeterminadas foram c33 e c22. Das amostras indeterminadas no RIBA, repetidas após seis meses com nova coleta, 20% (2/10) negativaram e 71,4% (10/14) permaneceram RIBA indeterminado. Destas, (8/10) continuaram indeterminadas com o mesmo padrão de bandas. O VHC-RNA foi realizado em todas as amostras do estudo (102) e foi detectável em apenas 22,5% (23/102). Todas as amostras com VHC-RNA detectável foram RIBA positivo e tinham índex maior que cinco na relação S/CO. Em apenas duas amostras que tiveram resultado de RIBA positivo, o VHC-RNA não foi detectado. As 23 amostras com VHC-RNA detectável foram genotipadas, sendo 78,3% (18/23) do genótipo 1, 17,4% (4/23) do genótipo 3 e 4,3% (1/23) do genótipo 2. A positividade do anti-VHC associou-se com o uso de droga intranasal (< 0,001), com drogas injetáveis (< 0,001)) e ocorrência de DST (< 0,05). Diante dos resultados encontrados, observa-se que o RIBA apresenta um elevado número de resultados indeterminados, sendo necessária a realização do VHC-RNA para a confirmação da infecção pelo VHC. Indivíduos com resultado de anti-VHC índex menor que cinco e resultado de RIBA indeterminado têm grande probabilidade de não apresentarem o VHC-RNA detectável e portanto, de não estarem infectados pelo VHC, mas devem ser acompanhados sorologicamente, de acordo com o critério médico. / The hepatitis C virus (HCV) infection is usually asymptomatic and has a high rate of chronicity, which may progress to cirrhosis and hepatocellular carcinoma. The hepatitis C diagnosis is realized by antibodies research using ELISA test (Enzyme Linked Immunosorbent Assay) and confirmed by additional serological tests, such as RIBA (Recombinant Immunoblot Assay) and westernblot, and confirmatory test, like HCV-RNA research. The objective of this study was to evaluate the RIBA efficacy in the diagnosis of HCV infection in blood donors with anti-HCV reagent. Were analyzed 102 samples with anti-HCV reagent in HEMOBA using the anti-HCV test Abbott Architect chemiluminescence for detection of anti-HCV antibodies, the RIBA III (Chiron) as a supplemental test for anti-HCV reactive and indeterminate samples and the Polymerase Chain Reaction test (RT-PCR) conventional or realtime (Roche Amplicor) for HCV-RNA detection. Samples with HCV-RNA detectable were genotyped by reverse hybridization (LIPA SIEMENS). Of the 102 samples analyzed in LACEN 38.2% (39/102) were positive, 57.8% (59/102) were negative and 3.9% (4 / 102) were indeterminate for anti-HCV. The RIBA results were 58.1% (25/43) positive, 9.3% (4 / 43) negative and 32.6% (14/43) indeterminate. All samples with indeterminate RIBA results had undetectable viral load. The predominant bands in the indeterminate RIBA samples were c33 and c22. Of the RIBA indeterminate samples, repeated after six months with a new collection, 20% (2 / 10) became negative and 71.4% (10/14) remained indeterminate. Of these, (8 / 10) remained undetermined with the same banding pattern. HCV-RNA was performed in all study samples (102) and was detectable in only 22.5% (23/102). All samples with detectable HCV-RNA were RIBA positive and had more than five in the ratio index S / CO. Only two samples had RIBA positive results with HCV-RNA not detected. The 23 samples with detectable HCV-RNA were genotyped, and 78.3% (18/23) were genotype 1, 17.4% (4 / 23) genotype 3 and 4.3% (1 / 23) genotype 2 . The anti-HCV positivity was associated with intranasal drug use (p<0.001), injectable drugs (p<0.001)) and STDs (p<0.05). Given the results, it is noted that the RIBA has a high number of indeterminate results, requiring the HCVRNA detection for HCV infection confirmation. Individuals with anti-HCV index below five, and indeterminate RIBA results are likely undetectable HCV-RNA and therefore are not infected with HCV, but must be serologically accompanied, according to medical criteria.

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.

Le virus de l'hépatite C (HCV) est un problème majeur de santé publique. Le développement de nouveaux traitements pour lutter contre le HCV a été ralenti par l'absence de modèles d'études in vitro et in vivo convenables. Le but de mon travail de thèse a été, dans un premier temps, de caractériser les facteurs déterminant le tropisme hépatique du HCV. En exprimant des facteurs clés dans une lignée cellulaire humaine non-hépatocytaire, nous avons reconstitué in fine l'ensemble du cycle viral dans ces cellules. L'entrée du virus dans la cellule hôte fait intervenir différents récepteurs d'entrée dont CD81, occludin (OCLN), claudin-1 (CLDN1) et scavenger receptor class B type I (SR-BI). L'expression de ces quatre récepteurs sur cette lignée la rend hautement permissive à l'entrée du virus, mais ne permet pas de rétablir la réplication du virus. L'expression du micro-ARN 122, un micro-RNA important pour l'infection du HCV, dans les cellules exprimant les quatre récepteurs, restaure une forte réplication de l'ARN viral mais ne permet pas de détecter une production de particules infectieuses. L'expression de l'apolipoprotein E (apoE), jouant un rôle primordial dans l'assemblage et la sécrétion, rétablis cette dernière étape du cycle viral du HCV dans la lignée cellulaire humaine non-hépatocytaire. Dans un second temps, j'ai utilisé la stratégie, précédemment établie, pour étudier la spécificité d'espèce de l'infection du HCV dans plusieurs lignées hépatocytaires murines. Nous avons pu rendre ces cellules permissives à l'entrée du HCV et pu détecter une très faible réplication. L'ensemble de mes travaux apportent de nouvelles informations sur la compréhension des facteurs clés nécessaire au cycle viral du HCV dans des cellules murines et humaines.

No description available.

The development of specific antiviral drugs directly targeting the hepatitis C virus (HCV) is clinically important, as the current standard interferon/ribavirin combination treatment is only partially effective, expensive and often associated with severe side effects. Inhibitors of the NS3 protease (PI) therefore represent a promising alternative or additional therapy. To date, the development and in vitro evaluation of PIs is restricted to the genotype 1/2 based replicon and the genotype 2a full length viral cell culture system. However, proteases of the different HCV genotypes vary substantially in their amino acid sequence and secondary structure and require separate evaluation of their efficacy before they go into clinical trials. To address this issue, a panel of intra- and intergenotypic recombinants based on the recombinant infectious clone Jc1 (pFK JFH1/J6/C-846) was developed in this work. The viability of these recombinants was assessed in the Huh7.5 cell culture system, where replicating viruses were detected by HCV-NS5A immunostaining. Intergenotypic recombinants containing genotype 1a, 1b, 3a, 4a and 6a derived proteases were replication defective, whereas the recombinant with genotype 5a derived protease replicated efficiently after acquiring cell culture adaptive mutations. The replacement of not only the NS3 protease gene region, but also its cofactor NS4A, allowed the generation of replication competent intra- and intergenotypic recombinants for all 6 major genotypes. Replacing the NS3 protease of the recombinants with that of patientderived proteases also generated replicating recombinants, greatly expanding the panel of intergenotypic recombinants available for phenotyping and PI evaluation. However, intra- and intergenotypic recombinants showed substantial differences in their replication kinetics, which may be influenced by naturally occurring polymorphism between genotypes and the differential requirement of adaptive/attenuating cell culture mutations. Genotype 1a recombinants replicated very poorly, which may be due to incompatibilities between the type 1a NS3/4A protease and the type 2a backbone. 50% inhibitory concentrations (IC50) of different PIs were measured using Foci Forming Units/ml (FFU/ml) reductions and replication inhibition assays. The different recombinants showed consistent, genotype-associated differences in their susceptibility to the PI BILN 2061, with genotypes 2a, 3a and 5a derived recombinants showing approximately 100-fold lower susceptibility than genotype 1b, 4a and 6a derived recombinants. These observations are consistent with major differences in response rates found in recent treatment trials of genotype 1, 2 and 3 infected patients. Differences in susceptibility were also observed for VX-950, with genotype 1b, 2a and 6a derived recombinants being twice as susceptible than genotype 3a, 4a and 5a derived recombinants. Passaging the intra- and intergenotypic recombinants under increasing concentrations of PI allowed the identification of PI resistance mutations. Resistance mutations to BILN 2061 mapped to the previously identified positions 156 and 168 within the NS3 protease, with a great diversity of amino acid substitutions observed within each genotype. Reintroduction of the identified resistance mutations into the original recombinant viruses conferred increased resistance towards BILN 2061 and some mutations also affected replication kinetics of the recombinants. The developed system will be of major value for the phenotypic characterisation of naturally occurring and treatment induced resistance mutations within all 6 major HCV genotypes towards different PIs. This will allow treatment response predictions for newly developed PIs before they enter clinical trials and the development of individually tailored antiviral treatment regimes.

Hepatitis C virus recurrence (which is defined by detection of HCV RNA in serum) in post-transplanted liver is universal but the progression of infection remains unpredictable, varying from case to case. It has been estimated that 75%-80% of the HCV recurrence patients will suffer chronic hepatitis C infection and up to a third of them will progress into the development of fibrosis and cirrhosis within 5 years post-transplantation. Therefore, finding ways to predict early on the progression of fibrosis can contribute to better prognoses. Recent literatures have mentioned that the hepatitis C virus relies on the host microRNA-122 (miR-122) for assistance in replication of the viral genome in hepatocytes. Experimental depletion of miRNA-122 in the cell line Huh 7 has shown up to an 80% decrease in HCV whereas an increase of miRNA-122 has shown an increase of HCV. Since miRNAs are known to have numerous indirect roles by the binding of the target messenger RNAs (mRNAs) and repressing the expression of their proteins, we hypothesized that the elucidation of associations between miRNA-122 and the histological severity in HCV recurrence post-liver transplantation might serve as a biomarker in predicting the outcome of HCV recurrence severity in patients. We also evaluated the expression levels of BCAP31 (a predicted target of miRNA-122), and CD4 (T cell surface molecules involved in immune response) among the HCV recurrence severity groups. RNA samples were isolated from FFPE liver samples from patients with HCV recurrence post-transplantation, and Reverse Transcription and TaqMan Real-Time PCR were carried out for qualitative analysis. We did not see any association between the levels of miRNA-122 expression and severity of HCV recurrence, but we did find a positive correlation between the miRNA-122 expression and the HCV viral load in Group 3 (Severe) at time of HCV recurrence, which supports previous studies of the role of miRNA-122 in HCV replication. We did not find any associations between the expression of BCAP31 and the severity of HCV recurrence but we did discovery an inverse relationship between miRNA-122 and BCAP31 in Group 3 (Severe) at time of HCV recurrence, confirming our assumption of miRNA:mRNA interaction. Also, we did find CD4 expression being statistically significant between Group 1 (Benign) versus Group 3 (Severe), which may support the hypothesis that strong, adequate CD4+ T-cell response is associated with better outcome post-liver transplantation.

Hepatitis C virus (HCV) is the major cause of chronic hepatitis worldwide and a leading cause for liver transplant. Unfortunately, graft HCV infection is a universal phenomenon despite of pre-transplant prophylactic strategies. Acute HCV infection and innate immune responses elicit an inflammatory scenario that triggers the recruitment of adaptive immune response cells. Of those chronically infected, 30% experience accelerated fibrosis with concomitant cirrhosis development within 5 years post-LT and require re-transplant. With many patients responding unfavorably to antivirals and ineffective vaccines, much attention is now placed on T cell immunity in controlling HCV infection. This study represents a retrospective analysis that examined the association of T cells with respect to liver fibrosis severity progression in a prospective cohort of biopsy samples taken from 27 patients at the time of HCV recurrence disease diagnosis post-LT. For those patients, the fibrosis progression was scored 36 months post-LT by Metavir scoring system. Liver biopsies were classified based on fibrosis severity as Mild (G1; n = 12), Moderate (G2; n = 6), and Severe (G3; n = 9). Additionally, an independent set of liver biopsy samples, taken according to fibrosis severity progression, was classified (G1; n =3, G3; n = 4) and used as a validation set for CD4 gene expression. Real time PCR was performed to study the expression of immune-related genes using the Taqman® probe system. From the results analysis, the CD4 T cell marker encoding gene was down-regulated (2.9-fold) in G3 with respect to G1; although, only borderline significant (p = 0.052). This suggests an inverse relationship of CD4+T cell related-genes expression with respect to worse fibrosis progression in HCV recurrence diagnosed recipients. The validation samples showed a similar trend (1.8 fold decrease in G3 with respect to G1), although not significant. This may be due to impaired T cell function resulting from T cell exhaustion, poor dendritic cell priming and activation, or the use of immunosuppressant drugs. To conclude, CD4 could be a potential biomarker to help in identifying HCV recurrent patients with a high risk of fibrosis development soon after LT.

MicroRNAs (miRNAs) are non-coding small RNAs that regulate eukaryotic gene activity at the post-transcriptional level by a process termed miRNA gene suppression. MicroRNA-122 (miR-122) is predominantly expressed in human liver cells and recent studies indicated that miR-122 promotes Hepatitis C Virus (HCV) replication and translation through physical interaction with two tandem binding sites located in the 5 untranslated region (5UTR) of the HCV genome (Jopling, et al., 2006; Jopling, et al., 2008). It has been reported that host genes that are also implicated in the miRNA gene suppression pathway are key regulators of HCV replication (Randall, et al., 2007). Two proteins, Dicer, a key RNaseIII enzyme, and its binding partner TRBP are essential proteins for miRNA activity. They are part of a protein complex called the RNA induced silencing complex (RISC) which also includes Argonaute proteins, and function in miRNA biogenesis loading the miRNA into RISC. As such, they are intriguing targets to study host-viral interplay during HCV replication.<p> In our study, we designed siRNAs to knock down Dicer and TRBP and then observed the effects of gene knockdown on full length J6/JFH-1-RLuc HCV (genotype 2a chimeric genome) replication and translation. The results showed that knocking down Dicer and TRBP reduced wild type (wt) J6/JFH-1-RLuc replication but had almost no effects on HCV translation in human liver cells. However, since knocking down Dicer and TRBP did not significantly alter miR-122 levels in the cell, it appears that the role of Dicer and TRBP was not solely the biogenesis of miR-122. This was confirmed by an experiment in which we observed that knocking down Dicer and TRBP also attenuated replication of a mutant virus in which replication is dependent on a exogenously supplied miRNA instead of endogenous miR-122.<p> Taken together, the results supported the hypotheses that Dicer and TRBP facilitate HCV infection mainly through HCV replication but not translation. The effects of Dicer and TRBP on HCV replication are not solely due to miR-122 biogenesis, and may be due to RISC loading functions in steps of miRNA gene suppression.<p> This study has set some essential groundwork for investigating potential roles of host factors in the RNAi machinery modulating HCV replication/translation and exploring novel antiviral targets.

Many bioinformatics problems are inference problems: Given partial or incomplete information about something, use that information to infer the missing or unknown data. This work addresses two inference problems in bioinformatics. The rst problem is inferring viral quasispecies sequences and their frequencies from 454 pyrosequencing reads. The second problem is inferring the structure of signal transduction networks from observations of interactions between cellular components. At first glance, these problems appear to be unrelated to each other. However, this work successfully penetrates both problems using the machinery of ow networks and transitive reduction, tools from classical computer science that prove useful in a wide array of application domains.