Abstract
Alphaviruses are arthropod-borne viruses that represent a significant threat to public health at a global level. While the formation of alphaviral nucleocapsid cores, consisting of cargo nucleic acid and the viral capsid protein, is an essential molecular process of infection, the precise interactions between the two partners are ill-defined. A CLIP-seq approach was used to screen for candidate sites of interaction between the viral Capsid protein and genomic RNA of Sindbis virus (SINV), a model alphavirus. The data presented in this report indicates that the SINV capsid protein binds to specific viral RNA sequences in the cytoplasm of infected cells, but its interaction with genomic RNA in mature extracellular viral particles is largely non-specific in terms of nucleotide sequence. Mutational analyses of the cytoplasmic viral RNA-capsid interaction sites revealed a functional role for capsid binding early in infection. Interaction site mutants exhibited decreased viral growth kinetics; however, this defect was not a function of decreased particle production. Rather mutation of the cytoplasmic capsid-RNA interaction sites negatively affected the functional capacity of the incoming viral genomic RNAs leading to decreased infectivity. Furthermore, cytoplasmic capsid interaction site mutants are attenuated in a murine model of neurotropic alphavirus infection. Collectively, the findings of this study indicate that the identified cytoplasmic interactions of the viral capsid protein and genomic RNA, while not essential for particle formation, are necessary for genomic RNA function early during infection. This previously unappreciated role of capsid protein during the alphaviral replication cycle also constitutes a novel virulence determinant.
Highlights
Alphaviruses are positive-sense RNA viruses that exhibit a broad host range; and as evidenced by the emergence of chikungunya virus (CHIKV), represent a significant burden on the public health systems of developed and underdeveloped communities [1,2,3,4,5,6,7,8,9]
Mature infectious alphavirus particles are approximately 70 nm in diameter, and consist of two concentric protein shells divided by a host derived lipid envelope [14, 15]
The assembly of mature alphavirus particles is a highly selective process as, to date, many characterizations of alphaviral particles have agreed that the viral genomic RNA is the predominant RNA molecule within the nucleocapsid core [16,17,18,19,20]
Summary
Alphaviruses are positive-sense RNA viruses that exhibit a broad host range; and as evidenced by the emergence of chikungunya virus (CHIKV), represent a significant burden on the public health systems of developed and underdeveloped communities [1,2,3,4,5,6,7,8,9]. Several studies have identified a region of the genomic RNA associated with the selective packaging of the viral genome during the assembly of infectious particles This element, termed the Packaging Signal, consists of a highly-structured region found within the open reading frame of the nonstructural polyprotein [16,17,18, 21,22,23]. While these studies provided an excellent definition of the cis-acting elements involved in the selection of the cargo RNA, they did not identify nor describe the interaction between the capsid protein and the viral RNA cargo [18]. It should be noted that the direct interaction between the viral capsid protein and viral RNA has not been exhaustively characterized in the cytoplasm of infected cells, or in mature viral particles leaving our understanding of the molecular interactions between these essential components of the virus incomplete
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