Intermediates of Bacteriophage MS2 Assembly In Vivo

Abstract

The in vivo process of virion assembly was studied in rifampin-treated, MS2-infected Escherichia coli during late times of infection—after 18 min postinfection. Differential sucrose gradient sedimentation of infected-cell lysates taken at various times after radioactive labeling indicated a definite temporal order of appearance of phage-specific protein in assembly-related structures. Labeled MS2 protein appears first as a low-molecular-weight peak at the tops of gradients, then as a peak at 40 S and as a large number of almost unseparable structures between 40 and 80 S , and finally as 80 S mature phage particles. During the chase of a short labeling period, radioactive phage protein was found to disappear from gradients in the same temporal order as it appeared; the soluble peak disappears first, followed by the 40 to 70 S region. The chased label appears quantitatively in the 80 S phage peak. Labeled phage RNA was found to appear first in the 40 S peak, then in the structures between 40 and 70 S , and finally in 80 S phage particles. The order of disappearance of labeled phage RNA during a chase is the same as its appearance. Resedimentation of the 40 to 70 S region indicated the presence of distinct structures at 60 and 70 S and many indistinct ones between 40 and 60 S . The smaller intermediates exhibit separable maturation protein-rich and coat protein-rich segments, indicating nonrandom binding of the two proteins during the initial steps of assembly. Larger, discrete intermediates appear at 60 and 70 S . Treatment of the various structures with pancreatic RNase results in destruction of those from 40 through 60 S; treatment of the 70 S structure results in the conversion of some of it to a 45 S peak, presumably the complete capsid. A small fraction of the 80 S phage peak is also sensitive to RNase, resulting in a similar 45 S peak. Pulse-chase experiments indicate that structures from 40 through 60 S as well as the RNase-sensitive 70 S structure are assembly intermediates, but that the RNase-insensitive 70 S and the RNase-sensitive 80 S structures are not.