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

Abstract

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...

Alphavirus Nucleocapsid Packaging and Assembly

“…In a recent study, the sequence in SD1 was both randomized as well as mutated into EEEV and CHIK SD1 sequences. Compensatory mutations resulted in SD1 and SD2 from the passaging of these viruses, which was interpreted together with structural data from Zhang et al, 2011 [13], to suggest that SD1 and SD2 synergistically form a central core interaction critical for NC assembly [43]. SD3 was described as a negative regulator of assembly.…”

“…Recently, studies using VEEV have re-defined the N-terminal domain into four separate sub-domains called SD1-4 ( Table 1) [40]. SD1 represents the N-terminal peptide of VEEV (amino acids 1-37) and SD2 (38)(39)(40)(41)(42)(43)(44)(45)(46)(47)(48)(49)(50)(51), the location of a peptide which is similar to helix I in SINV. SD3 represents the sequence with the greatest positive charge and is followed by SD4 (111-126), which is highly conserved among alphaviruses.…”

Venezuelan Equine Encephalitis Virus Capsid—The Clever Caper

“…Capsid’s N-terminal region can be further divided into four subdomains, referred to as SD1–4, that are critical for nucleocapsid formation [ 55 ]. SD1 (aa.…”

“…While it has very few positively charged amino acids, no predicted secondary structure, and is diverse among alphavirus species, VEEV SD1 is critical for nucleocapsid assembly and viral assembly. Its deletion or substitution of a similar sequence from other alphaviruses has a deleterious effect on infectious virus release [ 55 ]. SD2 (aa.…”

“…SD2 (aa. 38–51) contains an α-helix, and in VEEV, works in synergy with SD1 to drive nucleocapsid formation through forming a central core and determining viral genomic RNA interactions [ 47 , 52 , 55 ]. Its deletion leads to nonspecific encapsidation of other RNAs [ 52 ].…”