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BACKGROUND: We previously reported high prevalence of hepatitis C virus genotype 5a (HCV 5) (14%) in Central France. AIM: To identify the risk factors associated with HCV5 infection and to characterize local HCV5 lineages. METHOD: A case-control study and phylogenetic analysis were conducted. RESULTS: In all, 131 HCV5 and 343 HCV non 5 infected patients were enrolled. No HCV5 patient was born in sub-Saharan Africa and only two were injection drug user. HCV5 contamination was associated with living in a rural area called Vic le Comte (VLC) in non-transfused patients (OR = 17.7), with transfusion in patients living outside VLC (OR = 3.8) and with receiving injections in patients from VLC (OR = 3.1). More than 80% of the patients from outside VLC were contaminated by transfusion and those from VLC mainly by an iatrogenic factor - injections performed before 1972 by the local physician. Phylogenetic analysis of HCV5 isolates evidenced no distinct genetic cluster, but close relationships between the isolates of spouse pairs and between blood donors and recipients. CONCLUSIONS: Our results suggest that HCV5 spread in our district by iatrogenic route before 1972 and then via transfusion to the whole district. Collaborative studies are underway to study viral sequences from different parts of Africa and Europe to estimate the origin of our HCV 5a strains.

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We compared sustained virological response (SVR) in chronic hepatitis C patients with severe fibrosis treated with pegylated interferon (Peg-IFN) alpha-2b 1.5 microg/kg/week or 0.75 microg/kg/week in combination with ribavirin 800 mg/day for 48 weeks. This was a multicentre randomized controlled study. SVR was observed in 44.5% (45/101) of patients treated with the standard dose of Peg-IFN and 37.2% (38/102) of patients treated with the low dose (NS). In patients with genotypes 1, 4 and 5, SVR was observed in 25.0% of patients who received the standard dose and 16.9% of patients who received the low dose of Peg-IFN (P = NS). In patients with genotypes 1, 4 and 5 and low viraemia, SVR was obtained in 27.3% of patients treated with the standard dose and 25.8% of patients treated with the low dose (P = NS). In the high-viraemia subgroup, SVR was obtained in 24.0% and 9.1% of patients, respectively. In patients with genotypes 2 and 3, SVR was similar in both groups (73.2%vs 73.0%). Thus, (1) patients with genotypes 2 and 3 and severe fibrosis can be treated with low dose of Peg-IFN and ribavirin, (2) this study suggests that patients with genotypes 1, 4 and 5 and high viraemia could receive a standard dose of Peg-IFN associated with ribavirin for 48 weeks, (3) side effects limit the efficacy of the treatment with standard dose of Peg-IFN in patients with genotypes 1, 4 and 5 and low viraemia, (4) more studies are needed for patients with genotype 2 or 3 to define the optimal duration (24 or 48 weeks) in patients with severe fibrosis.

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The binding of a specific tRNA (acylated or not) to the 40S subunits in the presence of the proper codon was shown to produce two striking effects on the subunits. First, the subunits were no longer able to dimerize at low ionic strength. Second, they became fully resistant to 1.25 M LiCl treatment: bound tRNA prevented subunit inactivation as measured by polyphenylalanine synthesis; it also prevented large sedimentation changes of subunits and ribosomal protein release induced by LiCl. The number of protected proteins far exceeded that of the proteins crosslinked with tRNA after irradiation at 254 nm (A.M. REBOUD, S. DUBOST and J.P. REBOUD (1983) FEBS Lett. 158, 285-288). These results strongly suggest that tRNA binding induces modifications of rRNA-protein interactions in large domains of the subunits. A weak interaction of tRNA with the 40S subunit was demonstrated in the absence of the codon.

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When rat liver 60S ribosomal subunits were heated in phosphate buffer in the presence of MgCl2, 5S RNA was released in the form of a nucleoprotein complex (RNPH), which was isolated either by electrophoresis in polyacrylamide gel or centrifugation through a sucrose gradient. In addition to L5 several proteins of functional significance were identified in the complex: the acidic phosphoproteins P1-P2 and, as weaker spots, L3-L4, L6-L7 and L22. Most of these proteins were also found, but only as traces, in the RNPEDTA used as a control. RNPH was able to associate with 40S subunits. Our results support the interpretation that RNPH is located at the subunits' interface, at or near the peptidyl-transferase center.

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High concentrations of sucrose have a strong protective effect on heat-induced modifications of rat liver ribosomal subunits. They prevent to a large extent subunit inactivation, measured by poly (U)-dependent [14C] Phe tRNA binding (40S subunits) and puromycin reaction (60S subunits), subunit unfolding into light forms, and the release of both free and protein-complexed 5S RNA. They also increase the temperature at which subunits start to melt. Our data indicate that sucrose affects subunit conformation.

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Irradiation at 254 nm of the rat liver 40 S ribosomal subunit-poly(U)-Phe[32P]tRNA complex induced a covalent linkage of tRNA with a limited number of ribosomal proteins. After RNA hydrolysis, S10 was found to be the protein most highly labeled by radioactive nucleotides. Some radioactivity was also associated with protein S6-6a, S3a, S2 and S13-15.

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[3H]Tetracycline was covalently incorporated into rat liver ribosomes and isolated 40-S and 60-S subunits on irradiation at 254 nm. The antibiotic was almost exclusively incorporated into ribosomal proteins. At least some of these proteins are assumed to be involved in ribosomal function, since photoincorporated tetracycline was found to inhibit the activity of 40-S and 60-S subunits in the poly(U)-directed protein-synthesizing system as well as that of the 40-S subunit in the poly(U)-mediated [14C]Phe-tRNA binding. The results from simultaneous one-dimensional and two-dimensional gel electrophoreses showed a small distribution of label among ribosomal proteins in 60-S subunits and in 80-S ribosomes, L10 being the most radioactive protein. As non-acylated tRNA partly competed with this labeling, it is likely that tetracycline interaction with these proteins occurred at a functional site. L10 has already been found to interact with puromycin [Reboud, A. M., Dubost, S., Buisson, M. & Reboud, J. P. (1981) Biochemistry, 20, 5281-5288]. In the case of feed 40-S subunits the label distribution was wider among ribosomal proteins. No particular role has yet been found for the most labeled protein, S12, but protein S3a, which was also highly labeled, has already been reported to be involved in subunit function.

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[3H]Puromycin was covalently incorporated into rat liver ribosomes and isolated 40S and 60S subunits on irradiation at 254 nm. A study of the concentration dependence of this photolytic incorporation suggested that it arose from specific sites on isolated subunits but also from unspecific ones in the case of ribosomes, these sites being probably located on contaminant nonribosomal proteins. Puromycin was incorporated simultaneously into ribosomal proteins and rRNAs. The results from simultaneous one-dimensional and two-dimensional gel electrophoreses showed a small distribution of label among ribosomal proteins in 60S subunits and in 80S ribosomes, L10 being the most radioactive protein. Some antibiotics, which act on the peptidyltransferase center (amicetin and gougerotin), and also tetracycline competed with this labeling. Therefore, it was concluded that puromycin interaction with protein L10 occurred most likely at a functional site. In the case of free 40S subunits, labeling distribution among proteins was much wider. The possibility that proteins S3 and perhaps S23-24, which were significantly labeled in crude ribosomes too, also belong to a specific site interacting with puromycin is discussed.

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We have shown recently that, in the absence of mRNA, 1 molecule of nonacylated tRNA binds to the large ribosomal subunit of rat liver with a high affinity constant (Buisson, M., Reboud, A.M., Dubost, S., and Reboud, J. P. (1979) Biochem. Biophys. Res. Commun. 90,634-640). In this paper, free and tRNA-bound 60 S subunits were treated with increasing concentrations of LiCl to obtain information on tRNA binding site. The rationale for using deacylated tRNA was that it is assumed to bind to the peptidyl donor site. We observed that tRNA has a strong protective effect on subunit modifications produced by LiCl: tRNA prevents subunit inactivation as measured by puromycin reaction and polyphenylalanine synthesis and it shifts the Li+/Mg2+ ratio value needed to reach 50% inactivation, from 60 to 250; it also prevents ribosomal protein and 5 S RNA release and large sedimentation changes of subunits, induced by LiCl. To explain the mechanism of 60 S subunit stabilization by tRNA, two hypotheses are considered: stabilization can be consequent on direct interaction of tRNA with specific proteins, or on maintenance on subunits of essential cations which are otherwise displaced by Li+, or both.

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RNA-protein interactions in the 80-S rat liver ribosomes were studied by measuring cross-linking of proteins to rRNAs induced by ultraviolet radiation, as already reported for free 40-S and 60-S subunits. Our results are compatible with the model in which most of the ribosomal proteins are accessible to rRNAs in the native conformational state of the ribosomes. Subunit association in 80-S ribosomes does not seem to induce modifications in protein-RNA interactions as measured by this irradiation technique. However, two proteins, S9 and S13, appeared to be significantly less cross-linked with RNA in ribosomes than in free subunits. Ribosomes which had been frozen and thawed several times were highly sensitive to ultraviolet radiation. Such treatment in the cold chiefly modified their 60-S subunit moiety.

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