Hepatitis C virus (HCV) infects 170 million people worldwide. Current therapy is relatively poorly effective, comprising treatment with interferon and ribavirin, and is genotype specific. New antiviral drugs are being developed but, as with other RNA viruses, resistance is expected. We urgently require novel therapeutic targets, in particular those that are highly conserved in a range of divergent virus genotypes.
We are interested in fundamental aspects of the replication cycle of HCV. Since all genotypes replicate in the same way e.g. using similar RNA stemloop structures to recruit viral and cellular proteins required for replication, these may be suitable targets to develop antiviral therapies in the future. In particular we are interested in understanding the ‘switch’ between translation and replication of the genome. These processes must occur on the same template – at least initially – and are mutually exclusive; the ribosome progressing 5’ to 3’ on the genome strand and the viral polymerase synthesising a negative strand starting at the 3’ end of the genome.
In many other RNA viruses hydrogen-bonded stemloop structures are involved in the initiation and control of the translation and replication processes. We have therefore used bioinformatic studies to predict the conserved stemloop structures in the HCV genome (Tuplin et al., 2002) and used biophysical (Tuplin et al., 2004) and reverse genetic (Diviney et al., 2008) methods to confirm their presence and test their function. “Reverse genetics” refers to the modification of the genotype and subsequent analysis of the phenotype.
Our studies have defined a number of conserved stemloop structures in the 5’ and 3’ ends of the HCV genome, some of which we and others have demonstrated are critical for virus replication. In recent studies we have demonstrated that the structure of one such stemloop differs significantly in the two widely-used laboratory systems for analysis of HCV replication (Tuplin et al., 2012). The two conformations adopted by the extended pseudoknot, designated SL9266, may represent the two positions of a riboswitch that controls the translation and subsequent replication of the virus genome.
These studies use a combination of bioinformatic, biophysical and reverse genetic approaches to dissect the structure and function of the virus genome.
Tuplin, A., Struthers, M., Simmonds, P. & Evans, D. J. (2012). A twist in the tail: SHAPE mapping of long-range interactions and structural rearrangements of RNA elements involved in HCV replication. Nucleic Acids Research 40, 6908-6921.
Tuplin, A., Evans, D. J., Buckley, A., Jones, I. M., Gould, E. A. & Gritsun, T. S. (2011). Replication enhancer elements within the open reading frame of tick-borne encephalitis virus and their evolution within the Flavivirus genus. Nucleic Acids Research 39, 7034-7048.
Diviney, S., Tuplin, A., Struthers, M., Armstrong, V., Elliott, R. M., Simmonds, P. & Evans, D. J. (2008). Hepatitis C virus cis-acting replication element forms a long-range RNA-RNA interaction with upstream RNA sequences in NS5B. Journal of Virology 82, 9008-9022.
Tuplin, A., Wood, J., Evans, D., Patel, A. & Simmonds, P. (2002). Thermodynamic and phylogenetic prediction of RNA secondary structures in the coding region of hepatitis C virus. Rna-a Publication of the Rna Society 8, 824-841.