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

Venezuelan equine encephalitis virus (VEEV) is a pathogenic alphavirus, which circulates in the Central, South, and North Americas, including the United States, and represents a significant public health threat. In recent years, strong progress has been made in understanding the structure of VEEV virions, but the mechanism of their formation has yet to be investigated. In this study, we analyzed the functions of different capsid-specific domains and its amino-terminal subdomains in viral particle formation. Our data demonstrate that VEEV particles can be efficiently formed directly at the plasma membrane without cytoplasmic nucleocapsid preassembly. The entire amino-terminal domain of VEEV capsid protein was found to be dispensable for particle formation. VEEV variants encoding only the capsid's protease domain efficiently produce genome-free VEEV virus-like particles (VLPs), which are very similar in structure to the wild-type virions. The amino-terminal domain of the VEEV capsid protein contains at least four structurally and functionally distinct subdomains, which mediate RNA packaging and the specificity of packaging in particular. The most positively charged subdomain is a negative regulator of the nucleocapsid assembly. The three other subdomains are not required for genome-free VLP formation but are important regulators of RNA packaging. Our data suggest that the positively charged surface of the VEEV capsid-specific protease domain and the very amino-terminal subdomain are also involved in interaction with viral RNA and play important roles in RNA encapsidation. Finally, we show that VEEV variants with mutated capsid acquire compensatory mutations in either capsid or nsP2 genes.

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confidence: 99%

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

Lulla

Kim

Frolova

et al. 2013

“…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.…”

Section: Discussionmentioning

confidence: 91%

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

Reynaud

Lulla

Kim

et al. 2016

Venezuelan equine encephalitis virus (VEEV) is an important human and animal pathogen, for which no safe and efficient vaccines or therapeutic means have been developed. Viral particle assembly and budding processes represent potential targets for therapeutic intervention. However, our understanding of the mechanistic process of VEEV assembly, RNA encapsidation, and the roles of different capsid-specific domains in these events remain to be described. The results of this new study demonstrate that the very amino-terminal VEEV capsid-specific subdomain SD1 is a critical player in the particle assembly process. It functions in a virus-specific mode, and its deletion, mutation, or replacement by the same subdomain derived from other alphaviruses has strong negative effects on infectious virus release. VEEV variants with mutated SD1 accumulate adaptive mutations in both SD1 and SD2, which result in a more efficiently replicating phenotype. Moreover, efficient nucleocapsid and particle assembly proceeds only when the two subdomains, SD1 and SD2, are derived from the same alphavirus. These two subdomains together appear to form the central core of VEEV nucleocapsids, and their interaction is one of the driving forces of virion assembly and budding. The similar domain structures of alphavirus capsid proteins suggest that this new knowledge can be applied to other alphaviruses. IMPORTANCEAlphaviruses are a group of human and animal pathogens which cause periodic outbreaks of highly debilitating diseases. Despite significant progress made in understanding the overall structure of alphavirus and VEEV virions, and glycoprotein spikes in particular, the mechanistic process of nucleocapsid assembly, RNA encapsidation, and the roles of different capsid-specific domains in these processes remain to be described. Our new data demonstrate that the very amino-terminal subdomain of Venezuelan equine encephalitis virus capsid protein, SD1, plays a critical role in the nucleocapsid assembly. It functions synergistically with the following SD2 (helix I) and appears to form a core in the center of nucleocapsid. The core formation is one of the driving forces of alphavirus particle assembly. V enezuelan equine encephalitis virus (VEEV) is a representative member of the New World (NW) alphaviruses, which circulate mostly in Central and South America (1). In nature, VEEV is transmitted by mosquito vectors between vertebrate hosts, in which it causes a highly debilitating disease, often resulting in meningoencephalitis and a frequently lethal outcome (2). Among humans, the mortality rates are below 1%, mostly among the elderly and the very young, but the disease often leads to neurological sequelae (3, 4). Due to its user-friendly characteristics, VEEV has the potential for development as a biological warfare agent. It can be propagated to very high infectious titers in many commonly used cell lines, is very stable in lyophilized form, and is exceptionally efficiently transmitted by aerosol. Therefore, some of the VEEV serotypes a...

“…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).…”

Section: Figmentioning

confidence: 83%

“…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.…”

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confidence: 99%

“…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.…”

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confidence: 99%

See 1 more Smart Citation

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

Byrd

Kielian

2019

Alphaviruses are small enveloped RNA viruses that bud from the host cell plasma membrane. Alphavirus particles have a highly organized structure, with a nucleocapsid core containing the RNA genome surrounded by the capsid protein, and a viral envelope containing 80 spikes, each a trimer of heterodimers of the E1 and E2 glycoproteins. The capsid protein and envelope proteins are both arranged in organized lattices that are linked via the interaction of the E2 cytoplasmic tail/endodomain with the capsid protein. We previously characterized the role of two highly conserved histidine residues, H348 and H352, located in an external, juxtamembrane region of the E2 protein termed the D-loop. Alanine substitutions of H348 and H352 inhibit virus growth by impairing late steps in the assembly/budding of virus particles at the plasma membrane. To investigate this budding defect, we selected for revertants of the E2-H348/352A double mutant. We identified eleven second-site revertants with improved virus growth and mutations in the capsid, E2 and E1 proteins. Multiple isolates contained the mutation E2-T402K in the E2 endodomain or E1-T317I in the E1 ectodomain. Both of these mutations were shown to partially restore H348/352A growth and virus assembly/budding, while neither rescued the decreased thermostability of H348/352A. Within the alphavirus particle, these mutations are positioned to affect the E2-capsid interaction or the E1-mediated intertrimer interactions at the 5-fold axis of symmetry. Together, our results support a model in which the E2 D-loop promotes the formation of the glycoprotein lattice and its interactions with the internal capsid protein lattice. IMPORTANCE Alphaviruses include important human pathogens such as Chikungunya and the encephalitic alphaviruses. There are currently no licensed alphavirus vaccines or effective antiviral therapies, and more molecular information on virus particle structure and function is needed. Here, we highlight the important role of the E2 juxtamembrane D-loop in mediating virus budding and particle production. Our results demonstrated that this E2 region affects both the formation of the external glycoprotein lattice and its interactions with the internal capsid protein shell.