RNA Replication

HCV and its related viruses replicate entirely in the cytoplasm in close association with intracellular membranes. The virally encoded RNA-dependent RNA polymerase (NS5B) and additional replicase proteins are responsible for amplifying the genomic RNA. One such replicase protein is NS5A, a zinc-binding phosphoprotein of essential but unclear function. We have recently solved the X-ray crystal structure of NS5A domain I and observed a groove potentially involved in RNA binding; further functional and biochemical characterization of this and other replicase proteins is ongoing. A detailed molecular understanding of the proteins involved in viral RNA replication will assist in the development of therapeutic drugs targeting these essential enzymes.

Replication is also dependent on signals within the viral RNA molecule itself. These cis-acting RNA elements direct translation of the viral polyprotein and orchestrate initiation of positive and negative strand RNA synthesis. Although nontranslated sequences at each end of the genome are known to be required, we have also identified important RNA structures within the polyprotein-coding region. A stem-loop structure within the core gene as well as a cruciform element in the NS5B coding sequence have both been shown to play roles in replication. Interestingly, the NS5B element forms a long-range kissing-loop interaction with sequences in the 3’ nontranslated region. We have recently shown that this tertiary association and the low complexity sequence that intervenes the interacting partners have distinct and separable roles in RNA replication. These studies highlight the fact that the HCV genomic RNA molecule is not only a substrate for replication, but also an integral and dynamic player in the RNA accumulation process. Increased understanding of the functions of these RNA elements, as well as of their interactions with viral or cellular proteins may lead to additional targets for replication inhibitors.