Structure and function analysis of the essential 3′X domain of hepatitis C virus

The 3′X domain of hepatitis C virus has been reported to control viral replication and translation by modulating the exposure of a nucleotide segment involved in a distal base-pairing interaction with an upstream 5BSL3.2 domain. To study the mechanism of this molecular switch, we have analyzed the structure of 3′X mutants that favor one of the two previously proposed conformations comprising either two or three stem–loops. Only the two-stem conformation was found to be stable and to allow the establishment of the distal contact with 5BSL3.2, and also the formation of 3′X domain homodimers by means of a universally conserved palindromic sequence. Nucleotide changes disturbing the two-stem conformation resulted in poorer replication and translation levels, explaining the high degree of conservation detected for this sequence. The switch function attributed to the 3′X domain does not occur as a result of a transition between two- and three-stem conformations, but likely through the sequestration of the 5BSL3.2-binding sequence by formation of 3′X homodimers.