Virus Assembly Pathways: Straying Away but Not Too Far

Abstract

Non-enveloped RNA viruses pervade all domains of life. In a cell, they co-assemble from viral RNA and capsid proteins. Virus-like particles can form in vitro where virtually any non-cognate polyanionic cargo can be packaged. How only viral RNA gets selected for packaging in vivo, in presence of myriad other polyanionic species, has been a puzzle. Through a combination of charge detection mass spectrometry and cryo-electron microscopy, it is determined that co-assembling brome mosaic virus (BMV) coat proteins and nucleic acid oligomers results in capsid structures and stoichiometries that differ from the icosahedral virion. These previously unknown shell structures are strained and less stable than the native one. However, they contain large native structure fragments that can be recycled to form BMV virions, should a viral genome become available. The existence of such structures suggest the possibility of a previously unknown regulatory pathway for the packaging process inside cells.

Keywords: brome mosaic virus; charge detection mass spectrometry; cryo-electron microcopy; virus assembly.

Figure 1:

CDMS spectra of BMV CP assembled around polyA (A), and oligoB (B). The samples were prepared by mixing BMV CP to oligo at the molar ratio 15:1 at 4°C and low ionic strength. The data was collected 1 week after the assembly reaction was initiated. In the CDMS spectrum of CP assembled on polyA, there are 3 major peaks at 2.5 MDa, 3 MDa, and 3.4 MDa. BMV CP assembled with oligoB shows an additional peak at 1.9 MDa.

Figure 2:

Cryo-EM micrographs with representative assembly results for BMV-VLP/poly-A (A) and BMV-VLP/oligoB (B). In BMV-VLP/poly-A some individual particles show faceted morphology (white arrowhead), while a subset of particles with larger diameter appear to be more circular (black arrowhead). Some malformed complexes seem to share nucleic acid (black arrow). In BMV-VLP/oligoB, both small (white arrowhead) and large (black arrowhead) particles are observed. However, the interior seems to have less density compared to BMV-VLP/polyA. Some incomplete or aberrant particles were also observed (asterisk). Scale bar, 50 nm.

Figure 3:

Cryo-EM structure of the H8 assembly. (A) Isosurface representions of H8 rendered at different angles and the related views from symmetrical cage. The 6-fold symmetry axis is marked. Scale bar is 10 nm. (B) Selected reference-free 2D class averages show different orientations of the particle.

Figure 4:

Cryo-EM structure of the H15 assembly. (A) Isosurface representions of H15 rendered at different angles and the related views from symmetrical cage. The 5-fold symmetry axis is marked. (B) Selected reference-free 2D class averages show different orientations of the particle. Scale bar is 10 nm.