Removing the Polyanionic Cargo Requirement for Assembly of Alphavirus Core-Like Particles to Make an Empty Alphavirus Core

Abstract

The assembly of alphavirus nucleocapsid cores requires electrostatic interactions between the positively charged N-terminus of the capsid protein (CP) and the encapsidated polyanionic cargo. This system differs from many other viruses that can self-assemble particles in the absence of cargo, or form “empty” particles. We hypothesized that the introduction of a mutant, anionic CP could replace the need for charged cargo during assembly. In this work, we produced a CP mutant, Minus 38 (M38), where all N-terminal charged residues are negatively-charged. When wild-type (WT) and M38 CPs were mixed, they assembled into core-like particles (CLPs). These “empty” particles were of similar size and morphology to WT CLPs assembled with DNA cargo, but did not contain nucleic acid. When DNA cargo was added to the assembly mixture, the amount of M38 CP that was assembled into CLPs decreased, but was not fully excluded from the CLPs, suggesting that M38 competes with DNA to interact with WT CPs. The composition of CLPs can be tuned by altering the order of addition of M38 CP, WT CP, and DNA cargo. The ability to produce alphavirus CLPs that contain a range of amounts of encapsidated cargo, including none, introduces a new platform for packaging cargo for delivery or imaging purposes.

Self-assembly of dengue virus empty capsid-like particles in solution

“… 46 Remarkably, mutations in Ross River Virus (RRV) CP disordered N-terminal domain that replaced 4 lysine residues (K104-107) by aspartic residues (4D mutant) led CP to assemble in empty capsids, 31 confirming that CP N-terminal charge neutralization is the trigger for NC assembly. Additionally, when an anionic RRV CP mutant, in which all the N-terminal positively charged residues were replaced by negatively charged ones, was mixed with the WT protein, they assembled into CLPs, 32 further supporting the CP N-terminal charge neutralization mechanism for alphaviruses NC assembly.…”

“… 29 Indeed, either for flaviviruses or for alphaviruses, which are also small enveloped +ssRNA viruses, attempts for reproducing NC assembly in vitro required capsid protein charge neutralization. 28 , 30 , 31 , 32 Thus, it is expected that genomic RNA elements, namely negative charges, and long length, drive the orientation of capsid proteins to allow the protein-protein interaction necessary to build the capsid. Our TEM results showed an average diameter size of ∼30 nm for the CLPs formed by the R85C ox mutant.…”

The Structural Biology of Eastern Equine Encephalitis Virus, an Emerging Viral Threat

“…Presently, it is not known what causes this deviation in nucleocapsid core symmetry, the extent to which these differences in structural uniformity are important for assembly/disassembly, and if this non-uniformity is a widely conserved feature in alphaviruses. Recent data on genome-less capsid cores add another layer of complexity to the role of the viral genome in driving nucleocapsid assembly [ 102 , 103 ]. Overall, the understanding of uncoating [ 104 ], symmetry, organization, assembly of alphavirus nucleocapsid cores is limited despite recent advances.…”

Specific Recognition of a Stem-Loop RNA Structure by the Alphavirus Capsid Protein

“…Cp binds RNA through its primarily unstructured poly-basic N-terminal domain, while its C-terminal structured domain binds the cytoplasmic domain of the E2 envelope protein [ 10 ]. Cp’s oligomerization and assembly into NC is dependent on charge neutralization contributed by the RNA [ 10 , 11 ] and a motif in the Cp linker region that is required for cytoplasmic NC assembly [ 12 , 13 ]. The infectious viral NC consists of 240 copies of the Cp protein organized around a single copy of the gRNA.…”