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.
Highlights
Alphaviruses are enveloped, positive-sense RNA viruses that are members of the Togaviridae family [1]
When DNA cargo was added to the assembly mixture, the amount of Minus 38 (M38) capsid protein (CP) that was assembled into core-like particles (CLPs) decreased, but was not fully excluded from the CLPs, suggesting that M38 competes with DNA to interact with WT CPs
Despite the limited extent of assembly, the 4D CP was still able to interact with anionic cargo to form CLPs, demonstrating its ability to assemble both with itself and with nucleic acid cargo
Summary
Alphaviruses are enveloped, positive-sense RNA viruses that are members of the Togaviridae family [1]. The virion has three concentric layers: (1) a nucleocapsid core consisting of the RNA genome surrounded by 240 copies of the capsid protein (CP) to form the inner-most shell, (2) a host-derived lipid membrane surrounding that core, and (3) 80 trimeric glycoprotein spikes, comprised of the E2 and E1 proteins, that are embedded in the lipid bilayer and present on the surface of the complete virion [2,3]. The CP has two domains with unique functions: a disordered, positively-charged N-terminal domain that interacts with the negatively-charged genome to form cores during early assembly [9,10,11,12], and an ordered C-terminal domain that interacts with neighboring C-terminal domains and with the E2 protein during the later budding stage [13,14,15,16]. One hypothetical model of core assembly is that the viral RNA acts to neutralize the cationic N-terminus of the CP and provide a scaffold to hold CP monomers together until a full core is assembled [19]
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