Association of the polioviral RNA polymerase complex with phospholipid membranes.

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RESUMO

Polioviral RNA polymerase complex, which consists of enzyme, template, and nascent RNA, is membrane bound in vivo. The solubilized RNA polymerase complex associated spontaneously in vitro with phospholipid bilayer membranes (liposomes) of defined composition. The degree of association at 37 degrees C was greater for those membranes that were more fluid, suggesting that the binding involves the interaction of the RNA polymerase complex with the hydrocarbon chains in the interior of the lipid bilayer. The polymerase activity was not enhanced by addition of the lipid; in fact, the addition of some of the longer-chain lipids resulted in up to a 40% inhibition of the polymerase activity. Spin-label electron paramagnetic resonance experiments, which measured the membrane fluidity, and kinetic experiments on the rate of incorporation of tritiated UTP into RNA by the polymerase were performed as a function of temperature. The results indicated that the activity of the polymerase was not affected by the physical state of the phospholipid membrane and that its active site was not intimately associated with the membrane. Analysis of both the viral and host polypeptides associated with the smooth membrane-bound polymerase indicated that X was the primary viral polypeptide present. In addition, host polypeptides of molecular weight 86,000, 62,000, 54,000, and 46,000 were also present. If the membrane was disrupted with detergent, polypeptide X was released from the polymerase activity, suggesting that X may play a role in binding the polymerase to the membrane. In an analogous manner, polypeptide X associated spontaneously with phospholipid membranes to a greater extent than the capsid polypeptides. Analysis of both the host and viral polypeptides associated with the viral RNA polymerase purified by precipitation in 2 M LiCl indicated that host polypeptides of molecular weight 106,000, 38,000, 33,000, and 14,000 were the major constituents, whereas relatively small amounts of the viral polypeptides were present. It was confirmed that of the viral polypeptides found, polypeptide 4 was present in the largest amount.

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