Probing the Early Temporal and Spatial Interaction of the Sindbis Virus Capsid and E2 Proteins with Reverse Genetics

Abstract

A 7-Å cryoelectron microscopy-based reconstruction of Sindbis virus (SINV) was recently generated. Fitting the crystal structure of the SINV capsid protein (Cp) into the density map revealed that the F2-G2 loop of the Cp was shifted away from cytoplasmic domain of E2 (cdE2) in the 7-Å reconstruction relative to its position in the Cp crystal structure. Furthermore, the reconstruction demonstrated that residue E395 in region I of the cytoplasmic domain of the E2 envelope protein (cdE2-RI) and K252 of Cp, part of the Cp F2-G2 loop, formed a putative salt bridge in the virion. We generated amino acid substitutions at residues K250 and K252 of the SINV Cp and explored the resulting phenotypes. In the context of cells infected with wild-type or mutant virus, reversing the charge of these two residues resulted in the appearance of Cp aggregates around cytopathic vacuole type I (CPV-I) structures, the absence of nucleocapsid (NC) formation, and a lack of virus particle release in the infected mammalian cell. However, expressing the same Cp mutants in the cell without the envelope proteins or expressing and purifying the mutants from an Escherichia coli expression system and assembling in vitro yielded NC assembly in all cases. In addition, second-site mutations within cdE2 restored NC assembly but not release of infectious particles. Our data suggest an early temporal and spatial interaction between cdE2-RI and the Cp F2-G2 loop that, when ablated, leads to the absence of NC assembly. This interaction also appears to be important for budding of virus particles.

The Amino-Terminal Domain of Alphavirus Capsid Protein Is Dispensable for Viral Particle Assembly but Regulates RNA Encapsidation through Cooperative Functions of Its Subdomains

“…The commonly accepted hypothesis regarding alphavirus particle assembly is that NCs are formed in the cytoplasm of infected cells, and this process is mediated by interaction of the capsid protein with the RNA packaging signal (PS), which is present in genomic, but not subgenomic, RNA. The NCs are then transported to the plasma membrane either by themselves or on the surfaces of membranous vesicles, the so-called CPVII, and then bud from the cells while acquiring their lipid envelope with glycoprotein spikes (27,28). NC itself has an icosahedral geometry and apparently determines the final alphavirus T4 symmetry.…”

The SD1 Subdomain of Venezuelan Equine Encephalitis Virus Capsid Protein Plays a Critical Role in Nucleocapsid and Particle Assembly

“…The studies performed on different alphaviruses also indicated the existence of two mechanisms in virion assembly. The first mechanism suggests preformation of the nucleocapsids in the cytoplasm, followed by their migration to the plasma membrane either by passive diffusion or at the surface of previously described cytopathic vacuoles (CPVII) (28)(29)(30)(31). Such preformed NCs and CPVII vesicles are readily detectable by EM in the cytoplasm of virus-infected cells.…”

The Alphavirus E2 Membrane-Proximal Domain Impacts Capsid Interaction and Glycoprotein Lattice Formation

“…isolated from infected cells versus those isolated from virus particles (28). Consideration of these and other data suggested that the interaction of the envelope proteins with the capsid proteins could promote nucleocapsid maturation during budding of virus particles (5,11,29,30). The importance of envelope interactions in nucleocapsid maturation is in keeping with a temperature-sensitive capsid mutant, in which unstable cytoplasmic nucleocapsids are stabilized upon budding into virus particles (19).…”

“…Both the capsid protein and the envelope proteins form organized lattices, but the relative importance of the nucleocapsid lattice versus the envelope protein lattice in promoting particle assembly and budding is unclear (reviewed in reference 4). Nucleocapsids assemble in the cytoplasm of infected cells in the absence of envelope protein expression (10,11), and purified capsid protein can also assemble into nucleocapsid-like structures when incubated with RNA in vitro (12)(13)(14)(15). However, interaction of intracellular nucleocapsids with the envelope proteins is clearly required for virus particle production, as discussed above.…”

“…Capsid protein mutants that are deficient in capsid-capsid interactions do not form cytoplasmic nucleocapsids but are still able to form nucleocapsids at the plasma membrane and bud into virus particles, indicating that the glycoprotein lattice itself can promote nucleocapsid formation (5,(16)(17)(18). The evidence also suggests that cytoplasmic nucleocapsids undergo rearrangements during budding (5,11,19), suggesting that the mature particle structure may be influenced by interactions between the two lattices. While residues important in formation of the capsid protein lattice have been identified (reviewed in reference 20), less is known about the role of specific residues in mediating interactions within the envelope protein lattice.…”