Specific Recognition of a Stem-Loop RNA Structure by the Alphavirus Capsid Protein

Alphaviruses are small enveloped viruses with positive-sense RNA genomes. During infection, the alphavirus capsid protein (Cp) selectively packages and assembles with the viral genomic RNA to form the nucleocapsid core, a process critical to the production of infectious virus. Prior studies of the alphavirus Semliki Forest virus (SFV) showed that packaging and assembly are promoted by Cp binding to multiple high affinity sites on the genomic RNA. Here, we developed an in vitro Cp binding assay based on fluorescently labeled RNA oligos. We used this assay to explore the RNA sequence and structure requirements for Cp binding to site #1, the top binding site identified on the genomic RNA during all stages of virus assembly. Our results identify a stem-loop structure that promotes specific binding of the SFV Cp to site #1 RNA. This structure is also recognized by the Cps of the related alphaviruses chikungunya virus and Ross River virus.