Length of encapsidated cargo impacts stability and structure ofin vitroassembled alphavirus core-like particles

Abstract

In vitro assembly of alphavirus nucleocapsid cores, called core-like particles (CLPs), requires 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 four Lys residues in the CP with four 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.

Identification of Chikungunya virus nucleocapsid core assembly modulators

“…CLPs can be encapsidated by viral glycoproteins, and the newly formed virus-like particles are able to enter and disassemble within a new cell (50,51). A 27mer DNA oligo interacts in a 1:1 molar ratio to neutralize the basic N-terminus of the capsid protein (52,53) to initiate CLP assembly. CLPs are 40 nm in diameter and structurally similar to cores in virions (52)(53)(54)(55).…”

“…A 27mer DNA oligo interacts in a 1:1 molar ratio to neutralize the basic N-terminus of the capsid protein (52,53) to initiate CLP assembly. CLPs are 40 nm in diameter and structurally similar to cores in virions (52)(53)(54)(55). The efficiency of CLP assembly can be regulated by ionic strength.…”

“…In this study, we sought to identify small molecule modulators of CHIKV CLP assembly (48,53,57). We monitored the fluorescence quenching of fluorophore-labelled oligonucleotides to monitor nucleic acid packaging.…”

“…Cells were grown for an additional 4 hours. Cells were pelleted and resuspended in 20 mM sodium phosphate buffer containing 2μg/ml leupeptin, 2 μg/ml aprotinin, and 1mM PMSF to a final cell density of 0.06-0.08 g/ml (53). Samples were lysed using a cell cracker or a sonicator, sodium chloride was added to a concentration of 500 mM, and then lysates centrifuged at in a Beckman JA17 rotor at 23000 x g or 45 minutes at 4°C.…”

“…In addition, the properly assembled core interacts with the cytoplasmic domain of the viral glycoprotein E2 (66) suggesting the assembly of the core is critical for particle budding. In vitro assembled CLPs are structurally and functionally similar to nucleocapsid cores from infected cells (50, 51, 54) making them ideal systems for studying core assembly (48,49,52,53,59,(67)(68)(69).…”