SHAPE MATTERS: EFFECT OF POINT MUTATIONS ON RNA SECONDARY STRUCTURE

A suitable model to dive into the properties of genotype-phenotype landscapes is the relationship between RNA sequences and their corresponding minimum free energy secondary structures. Relevant issues related to molecular evolvability and robustness to mutations have been studied in this framework. Here, we analyze the one-mutant neighborhood of the predicted secondary structure of 46 different RNAs, including tRNAs, viroids, larger molecules such as Hepatitis-δ virus, and several random sequences. The probability distribution of the effect of point mutations in linear structural motifs of the secondary structure is well fit by Pareto or Lognormal probability distributions functions, independent of the origin of the RNA molecule. This extends previous results to the case of natural sequences of diverse origins. We introduce a new indicator of robustness, the average weighted length of linear motifs (AwL) and demonstrate that it correlates with the average effect of point mutations in RNA secondary structures. Structures with a high AwL value display the highest structural robustness and cluster in particular regions of sequence space.

Efficient delivery and regulable expression of hepatitis C virus full-length and minigenome constructs in hepatocyte-derived cell lines using baculovirus vectors

Baculovirus vectors have been used as efficient delivery vehicles for constitutive gene expression in a variety of mammalian cells. We have further developed the system to allow for regulable expression by placing the gene of interest under the control of an inducible promoter, and complementing it with a second baculovirus vector providing the control elements necessary for promoter activity. We have used this system to express (a) the lacZ gene, (b) a ‘minigenome’ derived from hepatitis C virus (HCV) and carrying lacZ or (c) the full-length HCV viral genome, in human hepatocyte cell lines in an inducible fashion. Control systems that rely on either the absence of tetracycline or presence of ponasterone to induce gene expression were tested. Expression of lacZ was controlled by ponasterone, but β-galactosidase activity was limited to 10–20% of cells. In contrast, the tetracycline-controlled expression system gave a low basal activity and was highly inducible in almost 100% of cells. Inducible expression was also obtained in almost 100% of cells infected with baculoviruses in which an HCV minigenome was placed downstream of the tetracycline-inducible promoter and upstream of either a hammerhead or hepatitis δ virus ribozyme. Northern blot analysis was consistent with accurate cleavage of the minigenome transcript by the hepatitis δ virus ribozyme. Finally, regulable transcript production and viral polypeptide processing could be demonstrated in HepG2 cells infected with baculoviruses bearing the full-length HCV genome. This system thus provides a novel tool for the analysis of HCV replication and host–cell interactions.