Journal:
Journal of Physics: Condensed Matter
Abstract:
In vitro assembly of alphavirus nucleocapsid cores, called core-like particles
(CLPs), require a polyanionic cargo. There are no sequence or structure requirements
to encapsidate single-stranded nucleic acid cargo. In this work, we wanted to determine
how the length of the cargo impacts the stability and structure of the assembled CLPs.
We hypothesized that cargo neutralizes the basic region of the alphavirus capsid protein
and if the cargo is long enough, it will also act to scaffold the CP monomers together.
Experimentally we found that CLPs encapsidating short 27mer oligonucleotides were
less stable than CLPs encapsidating 48mer or 90mer oligonucleotides under different
chemical and thermal conditions. Furthermore, cryo EM studies showed there were
structural differences between CLPs assembled with 27mer and 48mer cargo. To mimic
the role of the cargo in CLP assembly we made a mutant (4D) where we substituted a
cluster of 4 Lys residues in the CP with 4 Asp residues. We found that these few amino
acid substitutions were enough to initiate CLP assembly in the absence of cargo. The
cargo-free 4D CLPs show higher resistance to ionic strength and increased temperature
compared to wild-type cargo containing CLPs suggesting their CLP assembly
mechanism might also be different.
Date:
2017
Journal link:
keywords:
Virology
Biophysics
Physics
Computational Modeling
Structural Biology