Balraj Krishnan Handa

Influenza virus endoribonuclease.

The influenza virus polymerase complex contains two associated enzymatic activities, an endoribonuclease and a RNA-dependent RNA polymerase activity. Both activities have so far been observed only with the complete polymerase complex consisting of three subunits, PB1, PB2, and PA. This chapter describes a robust and optimized procedure for the purification of active influenza virus polymerase in complex with genomic RNA and the single-stranded RNA-binding protein nucleoprotein from influenza virus particles. It also explains the synthesis of capped RNA molecules as substrates of the influenza virus endonuclease. The enzymatic properties of influenza virus-derived endoribonuclease activity have been characterized with a model RNA substrate of 20-nucleotide length, termed G20 RNA. The rate of RNA cleavage under steady state conditions appears to be limited by product dissociation. Therefore conditions have been optimized to study the chemical step of RNA cleavage under single turnover conditions. The enzyme requires divalent metal ions for activity and can use Mn(II), Co(II), and Fe(II) efficiently at pH 7, Mg(II) with intermediate efficiency, and Ni(II) and Zn(II) with lower efficiency. The reaction progress curves show slow binding of Zn(II) and Ni(II) to the protein, suggesting a conformational change of the active site as a prerequisite for endonuclease activity in the presence of these two metal ions. Low concentrations of the detergent DOC inhibit the activity and also disrupt the trimeric polymerase complex, whereas other detergents do not have a significant effect on the activity.