Capsid-E2 Interactions Rescue Core Assembly in Viruses That Cannot Form Cytoplasmic Nucleocapsid Cores.

Alphavirus capsid proteins (CPs) have two domains: the N-terminal domain (NTD), which interacts with the viral RNA, and the C-terminal domain (CTD), which forms CP-CP interactions and interacts with the cytoplasmic domain of the E2 spike protein (cdE2). In this study, we examine how mutations in the CP NTD affect CP CTD interactions with cdE2. We changed the length and/or charge of the NTD of Ross River virus CP and found that changing the charge of the NTD has a greater impact on core and virion assembly than changing the length of the NTD. The NTD CP insertion mutants are unable to form cytoplasmic cores during infection, but they do form cores or core-like structures in virions. Our results are consistent with cdE2 having a role in core maturation during virion assembly and rescuing core formation when cytoplasmic cores are not assembled. We go on to find that the isolated cores from some mutant virions are now assembly competent in that they can be disassembled and reassembled back into cores. These results show how the two domains of CP may have distinct yet coordinated roles. IMPORTANCE Structural viral proteins have multiple roles during entry and assembly. The capsid protein (CP) of alphaviruses has one domain that interacts with the viral genome and another domain that interacts with the E2 spike protein. In this work, we determined that the length and/or charge of the CP affects cytoplasmic core formation. However, defects in cytoplasmic core formation can be overcome by E2-CP interactions, thus assembling a core or core-like complex in the virion. In the absence of both cytoplasmic cores and CP-E2 interactions, CP is not even packaged in the released virions, but some infectious particles are still released, presumably as RNA packaged in a glycoprotein-containing membrane shell. This suggests that the virus has multiple mechanisms in place to ensure the viral genome is surrounded by a capsid core during its life cycle.

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

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.

Revisiting an old friend: new findings in alphavirus structure and assembly.

Alphaviruses are transmitted by an arthropod vector to a vertebrate host. The disease pathologies, cellular environments, immune responses, and host factors are very different in these organisms. Yet, the virus is able to infect, replicate, and assemble into new particles in these two animals using one set of genetic instructions. The balance between conserved mechanisms and unique strategies during virus assembly is critical for fitness of the virus. In this review, we discuss new findings in receptor binding, polyprotein topology, nucleocapsid core formation, and particle budding that have emerged in the last five years and share opinions on how these new findings might answer some questions regarding alphavirus structure and assembly.