Bacteriophage MS2 genomic RNA encodes an assembly instruction manual for its capsid

Abstract

Using RNA-coat protein crosslinking we have shown that the principal RNA recognition surface on the interior of infectious MS2 virions overlaps with the known peptides that bind the high affinity translational operator, TR, within the phage genome. The data also reveal the sequences of genomic fragments in contact with the coat protein shell. These show remarkable overlap with previous predictions based on the hypothesis that virion assembly is mediated by multiple sequences-specific contacts at RNA sites termed Packaging Signals (PSs). These PSs are variations on the TR stem-loop sequence and secondary structure. They act co-operatively to regulate the dominant assembly pathway and ensure cognate RNA encapsidation. In MS2, they also trigger conformational change in the dimeric capsomere creating the A/B quasi-conformer, 60 of which are needed to complete the T=3 capsid. This is the most compelling demonstration to date that this ssRNA virus, and by implications potentially very many of them, assemble via a PS-mediated assembly mechanism.

Keywords: RNA bacteriophage; RNA packaging signals; virion assembly.

Figure 1.

PS-mediated assembly of STNV. (A) Multiple, short, degenerate CP-recognition motifs within the STNV genome (highlighted in red) in the primary sequence (top), when presented appropriately by RNA folding into stem-loops, are bound co-operatively at high affinity (low nM) by cognate CPs. This is revealed by smFCS assays with oligos dye-labeled at one end (middle). An individual PS, shown to the left of the smFCS trace in blue, stimulates sequence-specific assembly when titrated with increasing CP concentrations. Titration points are shown below the trace also in blue. At a threshold concentration, ˜5 nM, the Rh shifts from around 3 nm to around 7 nm, consistent with formation of an RNA-CP capsomere, which we have shown contains 3 CP subunits. Thereafter, a T = 1 VLP is created, due to CP-CP interactions with this initiation complex that is only complete by 3 µM CP. In contrast, a viral fragment encompassing the first 127 nt at the 5′ end of the genome and predicted to form 5 PSs, reveals the co-operativity between these CP binding sites. The initial Rh is unchanged until the CP concentration reaches ˜120 nM, where it declines by about 20% mimicking effects we have seen on the full length genome (titration points shown in black above the trace). As CP concentration increases there is a rapid and complete transition to a T = 1 capsid, reflecting the co-operative interactions between CPs being mediated by the PSs. There are at least 2 stages of assembly, a rapid initial collapse of the genome preparing it for encapsidation into the limited space of its T=1 capsid (cartoon, bottom), and a slower assembly completion stage. (B) The molecular basis of PS action is revealed by the crystal structure of VLPs assembled around an RNA encompassing a high affinity PS, B3. In the presence of PSs, a region of the CP toward the N-terminus that is normally disordered forms an additional turn of α-helix. This region is rich in basic amino acids suggesting that PS binding overcomes an electrostatic barrier preventing CP-CP interaction from forming the trimeric capsomere.

Figure 2.

PS-mediated assembly of the T = 3 MS2 virion. (A) The capsomere in this case is a CP dimer, which in the absence of RNA is symmetrical in solution, consistent with it resembling the C/C quasi-dimer of the capsid. Binding to TR, and other related stem-loops, triggers a conformational change to the A/B-like dimer. Such RNA switching is required up to 60 times to make the T = 3 shell. (B) There is only a single copy of TR within the MS2 genome but many variant stem-loops can be formed that contain some of the important recognition features for CP. Note, when these interactions (arrowed in figure) are measured in vitro their affinities are lowered significantly (shown as relative reduced affinities compared to TR). However, the concentration of a single stem-loop in the volume of the capsid is ˜0.3 mM. This compares with the K_d for TR binding to a CP dimer which is 1–10 nM, depending on the assays used. In vivo therefore many of the variant stem-loops can be expected to act as PSs. The locations of these variant PSs have been predicted and are shown below on the genetic map of the phage. Those that are also independently identified by the recent cross-linking within infectious phage are highlighted in red.

Figure 3.

The implications of PS-mediated assembly for the earliest stages of infection. Shown is the asymmetric tomographic structure of MS2, at low resolution, bound to its initial cellular receptor, the E.coli F-pilus. This structure shows extensive density for the viral RNA, confirming the idea that PS-mediated assembly leads to almost identical RNA conformations within each viral particle. The contact to the pilus is mediated by the maturation protein, MP, which also binds to specific sites close to either end of the viral genome. Only the MP-RNA complex enters the bacterial cell, leaving the CP shell largely intact. MP replaces a C/C capsomere in an otherwise icosahedral protein shell.