Abstract
The conditions for the in vitro reconstitution of papaya mosaic virus (PMV) from its isolated constituents are described and are related to the formation of coat protein subassembly products. PMV assembles best in 0.01 M, pH 8.0, Tris buffer at 25° at a protein to RNA ratio of 20:1 (w/w). At lower pH levels, faulty, segmented particles are formed which are sensitive to ribonuclease. The assembly process at pH 8.0 is composed of two energetically distinct phases corresponding to helix initiation and elongation. Both reactions may be stopped by low levels of NaCl, whereas only elongation requires elevated temperatures. If the growth of elongating particles is stopped by lowering the temperature, long and thin “extended particles” are detected; if NaCl is used, the arrested particles terminate with a “brush” at one end only. Under virus assembly conditions, the coat protein exists in an equilibrium among several polymeric species, most notably 14 S and 25 S polymers. The latter is not required for reconstitution. The equilibrium mixture is very sensitive to changes in pH, ionic strength, temperature, and protein concentration.
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