- Email: [email protected]
772 SINGLE NUCLEOTIDE POLYMORPHISMS IN HUMAN CYCLOPHILIN A AND THEIR INFLUENCE ON HCVRNA REPLICATION IN VITRO
POSTERS IFNa, surface expression of CD81 was increased following USP18 over-expression in Huh7.5 cells. Conclusions: These data indicate that USP18 expression does not directly alter HCV RNA replication, nor alter levels of cellular components critical for HCV replication, such as miRNA122. However, USP18 overexpression leads to increased HCV production in a manner that is independent of its catalytic activity, and in concert with increased surface expression of CD81. Thus, USP18, one of the ISGs increased in chronic HCV infection, favours HCV production and likely contributes to treatment failure by promoting HCV cell entry. 770 REGULATION OF HEPATITIS C VIRUS REPLICATION, VIRUS PRODUCTION AND INFECTIVITY BY BILE ACIDS P. Chhatwal, D. Bankwitz, J. Gentzsch, T. Pietschmann. Division of Experimental Virology, TWINCORE – Centre for Experimental and Clinical Infection Research, Hannover, Germany E-mail: [email protected] Background and Aims: Hepatitis C virus (HCV) particles are associated with low density and very low density lipoproteins. Bile acids (BAs) act as signaling molecules regulating triglyceride- and cholesterol homeostasis. Because HCV is dependent on lipoprotein metabolism, BAs might regulate the HCV life cycle. In-vitro experiments have established that RNA replication of some HCV strains is increased by BAs and that the response to IFN is modulated. The aim of this project is to analyze the influence of different BAs on HCV RNA replication, response to IFN, virus production and properties of released virus. Methods: We used human hepatoma cell lines including Huh7Lunet and HuH6, and measured replication and infectivity of HCV genotype 2a (GT2a) virus in the presence of BAs. We also monitored replication of subgenomic Con1 (GT1b) and JFH1 (GT2a) replicons and analyzed the influence of BAs on the antiviral effect of IFN. To dissect influences on different steps of the HCV life cycle we analyzed HCV cell entry employing HCV pseudoparticles, and assessed assembly and release of infectious progeny particles by repetitive cycles of freeze and thaw of virus producing cells. Results: In agreement with previous findings, high doses of BAs increased RNA replication of GT1b replicons up to 8-fold while BAs did not stimulate replication of subgenomic GT2a replicons. Cholic acid and chenodeoxycholic acid stimulated RNA replication of fulllength GT2a constructs 3-fold. Cell entry of HCV pseudoparticles was not modulated by BAs and the properties of particles were not changed. Finally, high doses of cholic acid enhanced susceptibility of GT2a viruses to IFN. Conclusions: Our data confirm a differential response of HCV strains to BAs and suggest that RNA replication of GT1b viruses may be stimulated. Additional experiments are ongoing to characterize strain- or genotype-specific modulation of HCV replication. Our findings suggest that within GT2a, BAs may differentially regulate replication of subgenomic versus full-length constructs. Since BAs stimulated replication of full-length RNAs also in cells which do not sustain HCV infection, but replication and assembly, we can exclude that this is attributable to secondary rounds of infection. Further studies dissecting the underlying mechanism are ongoing.
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771 HEPATITIS C VIRUS ACTIVATES, BUT DOES NOT INFECT, HUMAN HEPATIC STELLATE CELLS/MYOFIBROBLASTS (HSC/MFS) IN PRIMARY CULTURE 1 P. Chouteau1 , A. Florimond1 , E. Merour ´ , A. Mallat2 , S. Lotersztajn2 , J.-M. Pawlotsky1,3 . 1 Molecular Virology and Immunology – Team 18, 2 Hepatic Pathophysiology – Team 17, INSERM U955 and University of East-Paris, 3 National Reference Center for Viral Hepatitis B, C, and Delta, Creteil, France E-mail: [email protected] Chronic hepatitis C virus (HCV) infection is associated with the development of hepatic fibrosis. Fibrogenesis is believed to be in great part the result of inflammation induced by the local immune response to HCV infection. The direct role of HCV in the fibrogenetic process is unknown. Specifically, whether HCV is able to infect/activate hepatic stellate cells/myofibroblasts (HSC/MFs), the principal cellular actors of fibrogenesis, remains unexplored. Methods: We tested the ability of HSC/MFs to be infected and/or activated in vitro by HCV retroviral pseudoparticles expressing the folded HCV envelope glycoproteins (HCVpp) in both primary cultures of human HSC/MFs and the human hepatic stellate cell line LX-2. Huh7 cells were used as a positive control of infection by HCVpp. VSV pseudoparticules (VSVpp) and pseudoparticles lacking envelope proteins (NoENVpp) were used as controls for the specificity of infection. Results: HCVpp were not able to specifically infect primary human HSC/MFs and LX-2 cells in the different conditions tested. However, quantification by RT-qPCR of various transcripts involved in HSC/MFs activation showed that the levels of collagen 1a1, TGFb1 and MMP2 transcripts were significantly and specifically increased in the presence of HCVpp. In addition, gelatinolytic zymography showed increased extracellular activity of MMP2 in cells incubated with HCVpp, but not in those exposed to VSV-Gpp and NoENVpp. Conclusions: These results indicate that HSC/MFs are not susceptible to HCV infection. However, HCV can activate these cells in vitro, a finding suggesting that HCV could play a direct role in the fibrogenetic process, together with local inflammation. 772 SINGLE NUCLEOTIDE POLYMORPHISMS IN HUMAN CYCLOPHILIN A AND THEIR INFLUENCE ON HCV RNA REPLICATION IN VITRO T. von Hahn1 , B. Karavul1 , E. Steinmann2 , A. Potthoff1 , C.P. Strassburg1 , H. Wedemeyer1 , M.P. Manns1 , T. Pietschmann2 , S. Ciesek1,2 . 1 Gastroenterology, Hepatology and Endocrinology, Hannover Medical School, 2 Experimental Virology, Twincore, Centre for Experimental and Clinical Infection Research; a Joint Venture Between the Medical School Hannover (MHH) and the Helmholtz Centre for Infection Research (HZI), Hannover, Germany E-mail: [email protected] Background: Recently, human cyclophilin A was recognized as an essential cellular co-factor for Hepatitis C Virus (HCV). Moreover, the immunosuppressive drug cyclosporine A and its non immunosuppressive derivate alisorivir have been shown to interfere with HCV replication in vitro and in vivo by inhibiting cyclophilins. Numerous single nucleotide polymorphisms (SNPs) in the PPIA gene encoding cyclophilin A (CypA) have been described. These include six SNPs that alter the amino acid sequence of the CypA protein (coding non-synonymous SNPs). Here we investigated if these SNPs have an influence on permissiveness for HCV in vitro and on the clinical course of HCV infection in chronic HCV patients. Methods: Using shRNA-mediated knock down of endogenous CypA and subsequent lentiviral transduction of shRNA-resistant genes, we first generated Huh7.5 cell lines expressing CypA variants containing different coding non-synonymous SNP’s. Expression level of cyclophilins was monitored by western blot and SYBR
Journal of Hepatology 2011 vol. 54 | S209–S361
POSTERS Green assay. Permissiveness of these cells for HCV RNA replication and virus production was assessed by transfection of the cells with a luciferase reporter virus genome. The frequency of all SNPs was investigated in vivo by direct sequencing of DNA from 120 healthy controls. Results: Two out of six SNPs in the coding region of human CypA were associated with lower efficiency of HCV RNA replication and virus production in vitro while all other SNPs had no influence on HCV RNA replication. The two SNPs, that reduce HCV RNA replication, are located near the active site of cyclophilin A and also near the binding site of cyclosporine A and alisporivir. However, both of these SNPs were very rare in our Caucasian population (allele frequency <1%). Further work investigating a confirmatory cohort and cohorts of other ethnic origins is ongoing. Conclusion: These results highlight the important role of CypA for HCV and indicate that SNPs in human cyclophilin A may play an important role in vitro and also in vivo. 773 HEPATITIS C VIRUS AND LIPID DROPLETS: ROLE OF SEIPIN IN LIPID DROPLET MATURATION AND VIRAL LIFE CYCLE 1 S. Clement ´ , C. Fauvelle2 , S. Pascarella2 , S. Conzelmann2 , V. Kaddai2 , K. Minehira3 , F. Negro1,4 . 1 Division of Clinical Pathology, University Hospital, 2 Department of Pathology and Immunology, University of Geneva School of Medicine, Geneva, 3 Department of Physiology, University of Lausanne, Lausanne, 4 Division of Gastroenterology and Hepatology, University Hospital, Geneva, Switzerland E-mail: [email protected] Background and Aims: Hepatitis C virus (HCV) is a positivestrand RNA virus of the Flaviviridae family, whose life cycle is tightly associated with lipid metabolism. HCV assembly and maturation start at the surface of lipid droplets, and depend on very-low density lipoprotein assembly and secretion pathways. Furthermore, fatty acids can either stimulate or inhibit HCV replication, depending on their degree of saturation. Finally, steatosis is a frequent finding in hepatitis C, but its impact on HCV replication is unclear. We analyzed the relationship between HCV replication and either lipid droplet-associated proteins (perilipin and Adipose differentiation-related protein) or proteins involved in lipid droplet formation/maturation (seipin and ‘cell death inducing DNA fragmentation factor-a-like effector’ [CIDE] family members). Among these proteins, the seipin appeared to be one interesting candidate as its mRNA expression level was altered during the course of HCV full length infection. Methods: We established an in vitro system with Huh-7 cells transduced with lentiviral vector expressing seipin and monitored the effect of its overexpression on i. lipid droplet morphology and ii. JC1 full length replication and viral particle production. Results: The overexpression of seipin increasing dramatically the mean diameter of lipid droplets (by 40%). However, their number per cell was significantly decreased (by 60%). Together, these two effects resulted into a 34% reduction of the total outer surface area of lipid droplets per cell. Interestingly, the total triglyceride level per cell was not modified. Therefore, using this model, where changes in lipid droplet morphology occur without modification of their triglyceride content, we could determine which of these factors is crucial for HCV replication. Our data indicate that HCV replication (measured as intracellular HCV RNA and core protein level) and viral particle secretion (measured as secreted HCV RNA and HCV infectious titer of culture medium) decreased (by 40% and 60% respectively) in parallel with the outer surface area of lipid droplets. Conclusion: These findings suggest that the available outer surface of lipid droplets is a critical factor for HCV replication and release, rather than the cellular content of triglycerides.
774 SUBSTITUTION OF A CONSERVED AMINO ACID IN THE TRANSMEMBRANE DOMAIN OF E2 CONFERS RESISTANCE TO A NOVEL SMALL-MOLECULE INHIBITOR OF HCV ENTRY G.A. Coburn, D.N. Fisch, S. Moorji, J.D. Murga, J.-M. de Muys, D. Paul, A.Q. Han, Y. Rotshteyn, D. Qian, P.J. Maddon, W.C. Olson. Research and Development, Progenics Pharmaceuticals, Inc., Tarrytown, NY, USA E-mail: [email protected] Background: Viral entry represents a novel therapeutic target that has been validated clinically for other pathogenic viruses. Recently, we have discovered and optimized a novel series of small-molecule compounds that potently and selectively block HCV entry in vitro. Treatment of HCV cultures with a representative compound from this series resulted in a rapid decrease in the percentage of infected cells, and the effect was sustained for over three months in the presence of inhibitor. In one of the drug treated cultures, however, viral breakthrough was observed. In this study, we isolated viral variants with reduced susceptibility to a smallmolecule inhibitor of HCV entry and subsequently mapped the determinants of resistance. Methods: HCV cultures were maintained in the presence or absence of high concentrations of inhibitor for several weeks. At four day intervals, infected cell cultures were fixed, permeabilized and stained for NS3 expression. HCV positive cells were quantified in a flow cytometric-based assay. Envelope sequences were isolated, cloned and sequenced from control and drug-treated cultures. Individual mutations were then re-introduced into the H77 parental background and the fusogenic and drug susceptibility properties of the variant envelopes were determined. Results: Treatment of HCV infected cultures with a small-molecule HCV entry inhibitor resulted in the rapid clearance of HCV positive cells from the culture over the first three weeks of drug treatment. At day twenty-six, we observed viral breakthrough in one HCV culture which was denoted by a marked increase in HCV positive cells. Over the following three weeks, HCV infection and replication continued to increase to levels that were comparable to the vehicle-treated control. Sequencing of variant envelopes revealed the presence of two unique amino acid substitutions in the drugtreated culture. Introduction of one amino acid substitution into the H77 background resulted in a pronounced decrease in susceptibility to a novel small-molecule inhibitor of HCV entry. Conclusions: We have demonstrated that substitution of a conserved amino acid in the transmembrane domain of the E2 glycoprotein can confer resistance to small-molecule inhibitors of HCV entry. This amino acid residue may define a drug-binding site or protein-protein interaction required for mediating HCV entry. 775 HCV REPLICATION REGULATES GENES IMPLICATED IN MRNA TRANSLATION AT THE POST-TRANSCRIPTIONAL LEVEL H. Colman1 , C. Scoul1 , C. Hernandez2 , S. Pierredon3 , A. Bihoudee ´ 4, R. Houlgatte4 , S. Vagner3 , A. Rosenberg2 , C. Feray1 . 1 INSERM U.948, Nantes, 2 INSERM U.567, Paris, 3 INSERM U.563, Toulouse, 4 INSERM U.915, Nantes, France E-mail: [email protected] Introduction: The consequences of HCV infection on cellular gene transcription (mRNA) have been studied in clinical samples, in replicon models and in Huh7 cell infected by JFH1 strain. The influence of HCV replication on the protein translation of cellular mRNAs has never been systematically analyzed. Methods: Huh7.5.1 cells infected with JFH1 (MOI: 1), treated or not with protease inhibitor (telaprevir), and uninfected cells were grown for 72 hours. After stopping the mRNA translation (cycloheximide) cell lysate was fractionated on a 15–50% sucrose gradient to separate the translated mRNA (polysomal mRNA: bound to ribosomes) of untranslated mRNA (free). Polysomal
Journal of Hepatology 2011 vol. 54 | S209–S361
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771 HEPATITIS C VIRUS ACTIVATES, BUT DOES NOT INFECT, HUMAN HEPATIC STELLATE CELLS/MYOFIBROBLASTS (HSC/MFS) IN PRIMARY CULTURE 1 P. Chouteau1 , A. Florimond1 , E. Merour ´ , A. Mallat2 , S. Lotersztajn2 , J.-M. Pawlotsky1,3 . 1 Molecular Virology and Immunology – Team 18, 2 Hepatic Pathophysiology – Team 17, INSERM U955 and University of East-Paris, 3 National Reference Center for Viral Hepatitis B, C, and Delta, Creteil, France E-mail: [email protected] Chronic hepatitis C virus (HCV) infection is associated with the development of hepatic fibrosis. Fibrogenesis is believed to be in great part the result of inflammation induced by the local immune response to HCV infection. The direct role of HCV in the fibrogenetic process is unknown. Specifically, whether HCV is able to infect/activate hepatic stellate cells/myofibroblasts (HSC/MFs), the principal cellular actors of fibrogenesis, remains unexplored. Methods: We tested the ability of HSC/MFs to be infected and/or activated in vitro by HCV retroviral pseudoparticles expressing the folded HCV envelope glycoproteins (HCVpp) in both primary cultures of human HSC/MFs and the human hepatic stellate cell line LX-2. Huh7 cells were used as a positive control of infection by HCVpp. VSV pseudoparticules (VSVpp) and pseudoparticles lacking envelope proteins (NoENVpp) were used as controls for the specificity of infection. Results: HCVpp were not able to specifically infect primary human HSC/MFs and LX-2 cells in the different conditions tested. However, quantification by RT-qPCR of various transcripts involved in HSC/MFs activation showed that the levels of collagen 1a1, TGFb1 and MMP2 transcripts were significantly and specifically increased in the presence of HCVpp. In addition, gelatinolytic zymography showed increased extracellular activity of MMP2 in cells incubated with HCVpp, but not in those exposed to VSV-Gpp and NoENVpp. Conclusions: These results indicate that HSC/MFs are not susceptible to HCV infection. However, HCV can activate these cells in vitro, a finding suggesting that HCV could play a direct role in the fibrogenetic process, together with local inflammation. 772 SINGLE NUCLEOTIDE POLYMORPHISMS IN HUMAN CYCLOPHILIN A AND THEIR INFLUENCE ON HCV RNA REPLICATION IN VITRO T. von Hahn1 , B. Karavul1 , E. Steinmann2 , A. Potthoff1 , C.P. Strassburg1 , H. Wedemeyer1 , M.P. Manns1 , T. Pietschmann2 , S. Ciesek1,2 . 1 Gastroenterology, Hepatology and Endocrinology, Hannover Medical School, 2 Experimental Virology, Twincore, Centre for Experimental and Clinical Infection Research; a Joint Venture Between the Medical School Hannover (MHH) and the Helmholtz Centre for Infection Research (HZI), Hannover, Germany E-mail: [email protected] Background: Recently, human cyclophilin A was recognized as an essential cellular co-factor for Hepatitis C Virus (HCV). Moreover, the immunosuppressive drug cyclosporine A and its non immunosuppressive derivate alisorivir have been shown to interfere with HCV replication in vitro and in vivo by inhibiting cyclophilins. Numerous single nucleotide polymorphisms (SNPs) in the PPIA gene encoding cyclophilin A (CypA) have been described. These include six SNPs that alter the amino acid sequence of the CypA protein (coding non-synonymous SNPs). Here we investigated if these SNPs have an influence on permissiveness for HCV in vitro and on the clinical course of HCV infection in chronic HCV patients. Methods: Using shRNA-mediated knock down of endogenous CypA and subsequent lentiviral transduction of shRNA-resistant genes, we first generated Huh7.5 cell lines expressing CypA variants containing different coding non-synonymous SNP’s. Expression level of cyclophilins was monitored by western blot and SYBR
Journal of Hepatology 2011 vol. 54 | S209–S361
POSTERS Green assay. Permissiveness of these cells for HCV RNA replication and virus production was assessed by transfection of the cells with a luciferase reporter virus genome. The frequency of all SNPs was investigated in vivo by direct sequencing of DNA from 120 healthy controls. Results: Two out of six SNPs in the coding region of human CypA were associated with lower efficiency of HCV RNA replication and virus production in vitro while all other SNPs had no influence on HCV RNA replication. The two SNPs, that reduce HCV RNA replication, are located near the active site of cyclophilin A and also near the binding site of cyclosporine A and alisporivir. However, both of these SNPs were very rare in our Caucasian population (allele frequency <1%). Further work investigating a confirmatory cohort and cohorts of other ethnic origins is ongoing. Conclusion: These results highlight the important role of CypA for HCV and indicate that SNPs in human cyclophilin A may play an important role in vitro and also in vivo. 773 HEPATITIS C VIRUS AND LIPID DROPLETS: ROLE OF SEIPIN IN LIPID DROPLET MATURATION AND VIRAL LIFE CYCLE 1 S. Clement ´ , C. Fauvelle2 , S. Pascarella2 , S. Conzelmann2 , V. Kaddai2 , K. Minehira3 , F. Negro1,4 . 1 Division of Clinical Pathology, University Hospital, 2 Department of Pathology and Immunology, University of Geneva School of Medicine, Geneva, 3 Department of Physiology, University of Lausanne, Lausanne, 4 Division of Gastroenterology and Hepatology, University Hospital, Geneva, Switzerland E-mail: [email protected] Background and Aims: Hepatitis C virus (HCV) is a positivestrand RNA virus of the Flaviviridae family, whose life cycle is tightly associated with lipid metabolism. HCV assembly and maturation start at the surface of lipid droplets, and depend on very-low density lipoprotein assembly and secretion pathways. Furthermore, fatty acids can either stimulate or inhibit HCV replication, depending on their degree of saturation. Finally, steatosis is a frequent finding in hepatitis C, but its impact on HCV replication is unclear. We analyzed the relationship between HCV replication and either lipid droplet-associated proteins (perilipin and Adipose differentiation-related protein) or proteins involved in lipid droplet formation/maturation (seipin and ‘cell death inducing DNA fragmentation factor-a-like effector’ [CIDE] family members). Among these proteins, the seipin appeared to be one interesting candidate as its mRNA expression level was altered during the course of HCV full length infection. Methods: We established an in vitro system with Huh-7 cells transduced with lentiviral vector expressing seipin and monitored the effect of its overexpression on i. lipid droplet morphology and ii. JC1 full length replication and viral particle production. Results: The overexpression of seipin increasing dramatically the mean diameter of lipid droplets (by 40%). However, their number per cell was significantly decreased (by 60%). Together, these two effects resulted into a 34% reduction of the total outer surface area of lipid droplets per cell. Interestingly, the total triglyceride level per cell was not modified. Therefore, using this model, where changes in lipid droplet morphology occur without modification of their triglyceride content, we could determine which of these factors is crucial for HCV replication. Our data indicate that HCV replication (measured as intracellular HCV RNA and core protein level) and viral particle secretion (measured as secreted HCV RNA and HCV infectious titer of culture medium) decreased (by 40% and 60% respectively) in parallel with the outer surface area of lipid droplets. Conclusion: These findings suggest that the available outer surface of lipid droplets is a critical factor for HCV replication and release, rather than the cellular content of triglycerides.
774 SUBSTITUTION OF A CONSERVED AMINO ACID IN THE TRANSMEMBRANE DOMAIN OF E2 CONFERS RESISTANCE TO A NOVEL SMALL-MOLECULE INHIBITOR OF HCV ENTRY G.A. Coburn, D.N. Fisch, S. Moorji, J.D. Murga, J.-M. de Muys, D. Paul, A.Q. Han, Y. Rotshteyn, D. Qian, P.J. Maddon, W.C. Olson. Research and Development, Progenics Pharmaceuticals, Inc., Tarrytown, NY, USA E-mail: [email protected] Background: Viral entry represents a novel therapeutic target that has been validated clinically for other pathogenic viruses. Recently, we have discovered and optimized a novel series of small-molecule compounds that potently and selectively block HCV entry in vitro. Treatment of HCV cultures with a representative compound from this series resulted in a rapid decrease in the percentage of infected cells, and the effect was sustained for over three months in the presence of inhibitor. In one of the drug treated cultures, however, viral breakthrough was observed. In this study, we isolated viral variants with reduced susceptibility to a smallmolecule inhibitor of HCV entry and subsequently mapped the determinants of resistance. Methods: HCV cultures were maintained in the presence or absence of high concentrations of inhibitor for several weeks. At four day intervals, infected cell cultures were fixed, permeabilized and stained for NS3 expression. HCV positive cells were quantified in a flow cytometric-based assay. Envelope sequences were isolated, cloned and sequenced from control and drug-treated cultures. Individual mutations were then re-introduced into the H77 parental background and the fusogenic and drug susceptibility properties of the variant envelopes were determined. Results: Treatment of HCV infected cultures with a small-molecule HCV entry inhibitor resulted in the rapid clearance of HCV positive cells from the culture over the first three weeks of drug treatment. At day twenty-six, we observed viral breakthrough in one HCV culture which was denoted by a marked increase in HCV positive cells. Over the following three weeks, HCV infection and replication continued to increase to levels that were comparable to the vehicle-treated control. Sequencing of variant envelopes revealed the presence of two unique amino acid substitutions in the drugtreated culture. Introduction of one amino acid substitution into the H77 background resulted in a pronounced decrease in susceptibility to a novel small-molecule inhibitor of HCV entry. Conclusions: We have demonstrated that substitution of a conserved amino acid in the transmembrane domain of the E2 glycoprotein can confer resistance to small-molecule inhibitors of HCV entry. This amino acid residue may define a drug-binding site or protein-protein interaction required for mediating HCV entry. 775 HCV REPLICATION REGULATES GENES IMPLICATED IN MRNA TRANSLATION AT THE POST-TRANSCRIPTIONAL LEVEL H. Colman1 , C. Scoul1 , C. Hernandez2 , S. Pierredon3 , A. Bihoudee ´ 4, R. Houlgatte4 , S. Vagner3 , A. Rosenberg2 , C. Feray1 . 1 INSERM U.948, Nantes, 2 INSERM U.567, Paris, 3 INSERM U.563, Toulouse, 4 INSERM U.915, Nantes, France E-mail: [email protected] Introduction: The consequences of HCV infection on cellular gene transcription (mRNA) have been studied in clinical samples, in replicon models and in Huh7 cell infected by JFH1 strain. The influence of HCV replication on the protein translation of cellular mRNAs has never been systematically analyzed. Methods: Huh7.5.1 cells infected with JFH1 (MOI: 1), treated or not with protease inhibitor (telaprevir), and uninfected cells were grown for 72 hours. After stopping the mRNA translation (cycloheximide) cell lysate was fractionated on a 15–50% sucrose gradient to separate the translated mRNA (polysomal mRNA: bound to ribosomes) of untranslated mRNA (free). Polysomal
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