Tamarins (Saguinus species) infected by GB virus B (GBV-B) have recently been proposed as an acceptable surrogate model for hepatitis C virus (HCV) infection.The availability of infectious genomic molecular clones of both viruses will permit chimeric constructs to be tested for viability in animals. Studies in cells with parental and chimeric constructs would also be very useful for both basic research and drug discovery. For this purpose, a convenient host cell type supporting replication of in vitro-transcribed GBV-B RNA should be identified. We constructed a GBV-B subgenomic selectable replicon based on the sequence of a genomic molecular clone proved to sustain infection in tamarins. The corresponding in vitro-transcribed RNA was used to transfect the Huh7 human hepatoma cell line, and intracellular replication of transfected RNA was shown to occur, even though in a small percentage of transfected cells, giving rise to antibiotic-resistant clones. Sequence analysis of GBV-B RNA from some of those clones showed no adaptive mutations with respect to the input sequence, whereas the host cells sustained higher GBV-B RNA replication than the original Huh7 cells. The enhancement of replication depending on host cell was shown to be a feature common to the majority of clones selected. The replication of GBV-B subgenomic RNA was susceptible to inhibition by known inhibitors of HCV to a level similar to that of HCV subgenomic RNA.A problem that remains unsolved in the search for new therapeutic agents against hepatitis C virus (HCV) infection (12,20,25) is the availability of a small-animal model suitable for pharmacological studies, since the known host range of this virus includes only humans and chimpanzees. An interesting murine model based on the repopulation of mouse liver with human HCV hepatocytes has recently been proposed as sustaining HCV infection and yielding significantly high serum titers (17). The current version of this model, however, presents several drawbacks, including the difficulty of identifying an unlimited source of human hepatocytes and the low success rate, due in part to the peculiar genetic background of the mice used. Albeit very promising, this model needs improvement to be turned into a method accessible to most of the laboratories concerned.In the absence of a direct and convenient small-animal model, over the last few years a model for HCV infection alternative to the chimp has been proposed by various research groups. That model is based on the use of a surrogate virushost system, tamarins (Saguinus species) infected by GB virus B (GBV-B). A growing body of data about the enzymatic activity of GBV-B proteins has corroborated the hypothesis that useful information for research on anti-HCV drugs can be derived from experiments with GBV-B in tamarins. The identification of in vivo infectious cDNA has provided an indispensable tool to engineer the virus genome by insertion of HCV regions of interest. The availability of a small nonhuman primate GBV-B host and the perspective of using c...

Hepatitis C virus (HCV) and GB virus B (GBV-BThe simian flavivirus GB virus B (GBV-B) is in principle a useful surrogate model for human hepatitis C virus (HCV) (3): the genome organization and enzymatic activities are similar in the two viruses (6,7,16,(19)(20)(21)(22)(23), and GBV-B offers the advantage of infecting primates more suitable for research than the chimpanzee, which is the only host for HCV (3,5). A valuable achievement for drug discovery purposes would be the construction of viable chimeric HCV/GBV-B viruses with the host range of GBV-B. An intermediate step, important to further validate the use of GBV-B as a model for HCV and as a scaffold for chimeric constructs, is the development of comparable cell-based systems for GBV-B and for HCV.We have recently described a subgenomic dicistronic GBV-B replicon system (9, 18) working in Huh7-derived human hepatoma cell lines and similar to that available for HCV (2,4,14), which allows analysis of the replication and the synthesis/processing of the viral nonstructural proteins. So far no successful attempt at identifying cells different from Huh7 cells as the host for HCV or GBV-B has been described (1, 14). Nonetheless, the availability of more than one host cell type would be important to study the molecular features underlying cell permissiveness for these viruses and as an additional tool in the drug discovery process.In this paper we describe the identification of cells permissive to GBV-B replicons derived from the human hepatoma Hep3B cell line and the spontaneous occurrence of an interesting adapted mutant replicon lacking the stem-loop structure at the immediate 5Ј terminus. MATERIALS AND METHODSCell lines and culture conditions. Human hepatoma cell lines HepG2, Hep3B, Huh7 (our laboratory stocks), and derived cell lines (9) were grown in highglucose Dulbecco's modified Eagle's medium (Life Technologies) supplemented with 2 mM L-glutamine, 100 U of penicillin per ml, 100 g of streptomycin per ml, and 10% fetal bovine serum. Cells were subcultivated twice a week with a 1:5 split ratio. Cells transfected with neomycin-resistant constructs were selected and used, whenever appropriate, in the presence of 0.250 mg of G418 per ml. Nonetheless they were tested for resistance up to 1 mg of G418 per ml.Plasmids. The neo-RepB plasmid (9) encodes a subgenomic dicistronic GBV-B replicon bearing the neomycin phosphotransferase (neo) gene, which confers resistance to G418. The 4-29⌬-neo-RepB mutant construct was obtained by replacing the BamHI-AscI fragment of neo-RepB with an equivalent fragment produced by primer-based mutagenesis with a sense primer spanning the deletion of nucleotides 4 to 29 identified in the resident replicon molecules of the cell line B6/Hep3B, one of the Hep3B-derived cell lines identified in this study. The GBV-B wild-type replicon bearing the ␤-lactamase reporter gene was described previously (9, 18), and the corresponding nucleotide-4-to-29 deletion mutant was constructed with a similar strategy. HCV replicon plasmids were kindly pr...

. 76: [7736][7737][7738][7739][7740][7741][7742][7743][7744][7745][7746] 2002). In this report, we have dissected this phenomenon, examining the effects of the insertion of core sequences of different lengths on GBV-B IRES-dependent translation and RNA replication by using experimental approaches aimed at analyzing these two aspects independently. The results achieved indicate that an enhancement of translation efficiency does occur and that it correlates with the length of the inserted core sequences. Interestingly, the insertion of these sequences also has a direct similar effect on the efficiency of replication of the GBV-B replicon. These results suggest that in GBV-B replicon RNA and potentially in the complete viral genome, the core coding sequences not only are part of the IRES but also take part in the replication process, independently of the presence of the corresponding whole protein.Selectable dicistronic replicons that replicate in cultured cells independently from the infection capability of the corresponding virus have been described for flaviviruses and pestiviruses (5,10,23,26,32,40). The description of a replicon system for hepatitis C virus (HCV) (26) represented a real milestone for the study of the severe worldwide disease provoked by this virus (31), since a true infection system was lacking. The use of HCV subgenomic replicons is supplying a wealth of information about important features of HCV replication and virus-cell interactions (1,6,11,14,15,24,28,34,41) as well as about the mechanisms of antiviral drugs (12,13,18,33). Full-length genomic HCV replicons, including the genetic information for capsid and envelope proteins, have also been described (7,13,20,33) and might represent an efficient way to select for cells suitable as hosts for viral maturation and infection.A subgenomic replicon of GB virus B (GBV-B) that replicates in cell lines selected from human hepatoma Huh7 cells was previously described (10). The main interest in developing cell-based systems for GBV-B lies in the possibility of using this virus as an indirect but valid alternative to using HCV in animal models (4). In fact, HCV infects only chimpanzees, considered the nearest relative to humans, a fact which sets obvious limitations on the development of in vivo studies. On the contrary, the similar flavivirus GBV-B infects small monkeys, such as tamarins (Saguinus spp.) (3, 9, 39) and owl monkeys (Aotus spp.) (8), which present several advantages for research over apes (4).The construction of chimeric molecules in which part of the GBV-B genome is substituted by its HCV counterpart would be a valuable tool for studying HCV in nonhuman primates, taking advantage of the capability of GBV-B to replicate in these animals. It is conceivable, however, that these chimeric species would not replicate as efficiently as the wild-type virus, since the necessary interactions among viral genomes and virus and/or host factors would involve elements derived from different parental viruses. Preliminary cell-based experiments are thu...