HEPATITIS C VIRUS: Effect Of NS5A On The NS5B in vitro Replication Reaction

The Hepatitis C virus replicates its viral RNA template by a process that involves a protein complex containing the RNA dependent RNA polymerase (RdRp) NS5B, the helicase NS3, the membrane inserted protein NS4, and the phospho‐protein NS5A along with a number of host proteins. In vitro studies of the replication reaction have shown that the rate of nucleotide incorporation of NS5B is slow when compared to other DNA dependent RNA polymerases, suggesting that other factors such as host or non structural proteins, membrane structures, cis‐elements in the template or a combination of these factors are required to achieve higher rates. To understand how NS5A stimulates NS5B, we have developed an in vitro assay to study the effect of NS5A in the replication reaction. The NS5A recombinant protein was expressed and highly purified from bacterial cells. Using gel filtration, two isoforms of different molecular weight were isolated. The multimeric form stimulates the in vitro NS5B activity in a concentration and in an order of addition dependent manner. The monomeric isomer also will be tested. A pre‐incubation step with NS5B is required for the enhancement effect of NS5A on the replication reaction. Using the most terminal 98 nucleotides of the viral 3’UTR genome, NS5A stimulates the replication reaction three fold. Further studies using templates containing structural elements of the 3’UTR of the positive strand and negative strand are being tested. Stimulation is also seen with a synthetic RNA template. With this latter template, individual steps in replication can be studied; and preliminary data suggests that the activation by NS5A seems to occur at the elongation phase. In addition, the pause profile changes when NS5A is present in the reaction. We will explore in detail these features of the stimulation mechanism as well as analyze the affinity of the template to the complex NS5A‐NS5B.