Download Mechanisms of assembly and genome packaging in an RNA virus

Mechanisms of assembly and genome packaging in an RNA virus revealed
by high-resolution cryo-EM
Emma Hesketh, Rebecca Thompson and Neil Ranson
Introduction
A crucial step in virus assembly is the specific encapsidation of their genomes. This is a
particular challenge for single-stranded RNA viruses, as they must preferentially select their
genomes from a high background of cellular mRNA. CPMV, a plant infecting member of the
order Picornavirales, has a bipartite, positive-sense, single-stranded RNA genome. Its
icosahedral particles have a maximum diameter of ~30 nm and are comprised of 60 copies
each of a Large (L) and Small (S) coat protein. The mechanisms by which RNA is selected
and packaged are poorly understood. The only portion of the CPMV capsid proteins currently
implicated in RNA packaging is a segment of 24 amino acids at the C-terminus of the S
subunit. CPMV is a ‘biotechnology workhorse’ and is being developed for many
biotechnology applications, including targeted nano-containers for drug delivery,
nanomaterials, imaging agents and as a platform for novel vaccine development. However,
despite much research on CPMV, its true potential in biotechnology may not be realised until
we achieve a full understanding of the mechanisms that underlie capsid assembly and
genome encapsidation.
Results
Although the C-terminal extension to the S subunit is implicated in capsid assembly and
RNA packaging, understanding these roles has been difficult because the normal maturation
of wild type (WT) CPMV involves its cleavage and dissociation. As a result no structural
information for these residues is currently available. To address this lack, we examined the
structure of the CPMV empty virus like particle (eVLP). Crucially, eVLPs completely lack
RNA encapsidation and undergo C-terminal cleavage more slowly than WT virions, raising
the possibility that we could determine the structure of an eVLP that retains the C-terminal
segment using cryo-EM and single particle image processing. We therefore collected a cryoEM dataset for CPMV eVLP on an FEI Titan Krios microscope, using a direct electron
detector. Iterative rounds of 2D and 3D classification were then used to select a homogeneous
subset of particles for 3D structure refinement. The resulting density map was refined to 3.04
Å resolution (Fig. 1).
Figure 1: EM density map of CPMV
empty virus-like particle (eVLP)
determined by cryo-EM to 3.04Å
resolution (EMDB-3014). The L
subunit is shown in green, the S
subunit in blue and the C-terminal
13 amino acids of S subunit in
magenta. The density for an
individual β strand is shown in a
mesh representation with the EMderived atomic model within,
showing clear resolution of the side
chains.
Existing structural information for the CPMV capsid show the C-terminus of S subunit after
cleavage (ending at residue 189) in an extended conformation running across the exterior
Figure 2: (a) EM density map of unsharpened WT CPMV coloured as described previously. The C-terminus is
yellow (residues 184-189). (b) EM density map of the unsharpened eVLP map. The density corresponding to
residues 184-189 is coloured yellow. This portion of the C-terminal moves in the eVLP map compared to the
WT CPMV map (see yellow segment in (a). Coloured magenta is the newly resolved 13 residues (190-202 in
the S subunit). (c) Zoom-in of the C-terminal extension from the sharpened EM density map with the new
atomic model is shown.
surface of the capsid toward a cleft between the S subunits that form the turret at an
icosahedral 5-fold vertex (Fig. 2a). In the eVLP map, we see additional density in this cleft
that does not match the previously deposited structure. The density that would correspond to
residues 184-189 in the C-terminus is very weak suggesting this segment is poorly ordered in
the particle in solution (yellow segment Fig. 2b). However, it is clear that the polypeptide
chain takes a steeper path along the edge of the cleft than it does once C-terminal cleavage
(between residues 189 & 190) has occurred (compare yellow segments in Fig. 2a and 2b).
The C-terminal segment then becomes ordered once more, and we see density corresponding
to Leu190 to Arg202, residues absent from previous structures. A loop runs from the top of
the S subunit back into the cleft between subunits, before forming two turns of α-helix
running out of the cleft towards the bulk solvent (magenta segment Fig. 2b). The bottom of
this segment appears to be very well ordered, with clear density for side chains that make
intimate contacts to the neighbouring S subunit around the penton (Fig. 2c). The density then
becomes disordered once more, with Arg202 as the last ordered residue, suggesting that the
11 C-terminal residues are disordered in solution. The ordered C-terminal segment described
for the first time here forms an intimate network of interactions with the neighbouring S
subunit
around
the
pentameric
ring
that
forms
Figure 3: A Model for CPMV Assembly. The
the 5-fold vertex of the
L (green) and S (blue) subunit, associate to
particle.
Mutational
form a coat protein penton. The Canalysis
of
amino
acids in
terminal extension (magenta circle)
appears to act a dab of ‘molecular glue’,
this region has shown that
stabilising the formation of the penton
hydrophobic interactions
due to extensive interactions with the
play a central role in this
neighbouring S subunit.
binding, allowing us to
propose a model (Fig. 3).
Publications
Hesketh E.L., Meshcheriakova Y., Dent K.C., Saxena P., Thompson R.F., Cockburn J.J.,
Lomonossoff G.P. & Ranson N.A. (2015) Mechanisms of assembly and genome packaging in
an RNA virus revealed by high-resolution cryo-EM. Nat. Commun. 6: 10113.
Funding
Funded by the BBSRC (BB/L020955/1) and The Wellcome Trust (096685/Z/11/Z).
Collaborators
University of Leeds: J. Cockburn.
External: G. Lomonossoff, Y. Meshcheriakova and P. Saxena (John Innes Centre, Norwich),
K. Dent (Present affiliation: Diamond Light Source).