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

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.     

The Impact of Viral RNA on the Association Rates of Capsid Protein Assembly: Bacteriophage MS2 as a Case Study

A large number of single-stranded RNA viruses, which form a major class of all viruses, co-assemble their protein container and their genomic material. The multiple roles of the viral genome in this process are presently only partly understood. Recent experimental results indicate that RNA, in addition to its function as a repository for genetic information, could play important functional roles during the assembly of the viral protein containers. An investigation of the impact of genomic RNA on the association of the protein subunits may therefore provide further insights into the mechanism of virus assembly. We study here the impact of viral RNA on the association rates of the capsid proteins during virus assembly. As a case study, we consider the viral capsid of bacteriophage MS2, which is formed from 60 asymmetric (AB) and 30 symmetric (CC) protein dimers. Using Brownian dynamics simulations, we investigate the effect of the binding of an RNA stem-loop (the translational repressor) on the association rates of the capsid protein dimers. Our analysis shows that translational repressor binding results in self-association of AB dimers being inhibited, whilst association of AB with CC dimers is greatly enhanced. This provides an explanation for experimental results in which an alternating assembly pattern of AB and CC dimer addition to the growing assembly intermediate has been observed to be the dominant mode of assembly. The presence of the RNA hence dramatically decreases the number of dominant assembly pathways and thereby reduces the complexity of the self-assembly process of these viruses.