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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.
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This content will become publicly available on August 19, 2026
Measuring the selective packaging of RNA molecules by viral coat proteins in cells
Some RNA viruses package their genomes with extraordinary selectivity, assembling protein capsids around their own viral RNA while excluding nearly all host RNA. How the assembling proteins distinguish viral RNA from host RNA is not fully understood, but RNA structure is thought to play a key role. To test this idea, we perform in-cellulo packaging experiments using bacteriophage MS2 coat proteins and a variety of RNA molecules inEscherichia coli. In each experiment, plasmid-derived RNA molecules with a specified sequence compete against the cellular transcriptome for packaging by plasmid-derived coat proteins. Following this competition, we quantify the total amount and relative composition of the packaged RNA using electron microscopy, interferometric scattering microscopy, and high-throughput sequencing. By systematically varying the input RNA sequence and measuring changes in packaging outcomes, we are able to directly test competing models of selective packaging. Our results rule out a longstanding model in which selective packaging requires the well-known translational repressor (TR) stem-loop, and instead support more recent models in which selectivity emerges from the collective interactions of multiple coat proteins and multiple stem-loops distributed across the RNA molecule. These findings establish a framework for studying and understanding selective packaging in a range of natural viruses and virus-like particles, and lay the groundwork for engineering synthetic systems that package specific RNA cargoes.
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- Award ID(s):
- 2443955
- PAR ID:
- 10657491
- Publisher / Repository:
- National Academy of Sciences
- Date Published:
- Journal Name:
- Proceedings of the National Academy of Sciences
- Volume:
- 122
- Issue:
- 33
- ISSN:
- 0027-8424
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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