In vitro protein polymerization and nucleoprotein reconstitution of tobacco rattle virus

Abstract

Investigation of the effects of pH, ionic strength, and temperature on coat protein polymerization of tobacco rattle virus (TRV) have been examined and correlated with conditions optimal for nucleoprotein rod reconstitution. In acidified aqueous solution, TRV protein existed predominantly in the subunit form (<10 S). A discrete 35 S aggregate formed when the pH was raised (pH 6.5–8.5) in low ionic strength buffers at 4 °C. The formation of larger aggregates (>50 S) occurred at higher temperature (21 °C) at low ionic strength. Reconstitution of acetic-acid-extracted protein and phenol-extracted RNA occurred optimally in 0.25 M glycine buffer (pH 8.0) at 4–10 °C. Reconstituted nucleoprotein and native virus sedimented at the same rate after sucrose density gradient centrifugation. The participation of 35 S protein aggregates in reconstitution is suggested.