Figure 3. Two-stage assembly of cognate viral RNAs. Time-resolved changes in the apparent R_h of MS2 (black) and STNV (red) genomic RNAs are shown before and after addition (black arrow) of stoichiometric amounts of MS2 (A) or STNV (B) coat proteins, i.e., sufficient protein to allow each RNA to form a complete capsid. Blue stars denote the end of the respective compaction stages for interactions between cognate molecules. For MS2, this is followed by a slower increase in R_h correlated with formation of the capsids with high yield and fidelity. Capsid formation may already be complete for STNV following stage 1. Non-cognate interactions do not lead to collapse but do lead to inefficient formation of mostly aberrant aggregates. Electron micrographs of negatively stained assembly reactions at defined points in the pathway are shown. ( (A) i, assembly intermediates observed ~1 min after addition of MS2 CP to MS2 RNA; ii, T = 3 MS2 capsids present at the end of stage 2, assembled with MS2 RNA; iii, aberrant assembly products and aggregates formed by co-assembly of MS2 CP and STNV RNAs (scale bars 50 nm). (B) i, T = 1 STNV capsids at the end of the assembly reaction and ii, aberrant aggregates formed by co-assembly of STNV CP and MS2 genomic RNA. (C) Effects of multivalent cations on the apparent R_h of the MS2 3′ RNA sub-genomic fragment (Fig. 1) shown as size (R_h) distributions. RNA in a monovalent buffer is shown in black, followed by separate reactions in which divalent (Mg²⁺, 10 mM, blue) or trivalent (spermidine, 1mM, red) ions were added. (D) Condensation by counterions or coat protein subunits are not equivalent processes. The MS2 3′ RNA undergoes condensation upon addition of 1 mM spermidine. Addition of 200 nM MS2 CP₂ to this sample (red) or one in the monovalent buffer (black) has no effect or results in compaction and then recovery, respectively. The latter sample produces capsids, whereas no assembly occurs under these conditions in the presence of spermidine unless the coat protein concentration is raised significantly (not shown).