Hepatitis C virus genomic RNA dimerization is mediated via a kissing complex intermediate

Abstract

With over 200 million people infected with hepatitis C virus (HCV) worldwide, there is a need for more effective and better-tolerated therapeutic strategies. The HCV genome is a positive-sense; single-stranded RNA encoding a large polyprotein cleaved at multiple sites to produce at least ten proteins, among them an error-prone RNA polymerase that confers a high mutation rate. Despite considerable overall sequence diversity, in the 3′-untranslated region of the HCV genomic RNA there is a 98-nucleotide (nt) sequence named X RNA, the first 55 nt of which (X55 RNA) are 100% conserved among all HCV strains. The X55 region has been suggested to be responsible for in vitro dimerization of the genomic RNA in the presence of the viral core protein, although the mechanism by which this occurs is unknown. In this study, we analyzed the X55 region and characterized the mechanism by which it mediates HCV genomic RNA dimerization. Similar to a mechanism proposed previously for the human immunodeficiency 1 virus (HIV-1) genome, we show that dimerization of the HCV genome involves formation of a kissing complex intermediate, which is converted to a more stable extended duplex conformation in the presence of the core protein. Mutations in the dimer linkage sequence loop sequence that prevent RNA dimerization in vitro significantly reduced but did not completely ablate the ability of HCV RNA to replicate or produce infectious virus in transfected cells.