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.

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24The oncometabolite L-2-hydroxyglutarate (L-2HG) is considered an abnormal 25 product of central carbon metabolism that is capable of disrupting chromatin 26 architecture, mitochondrial metabolism, and cellular differentiation. Under most 27 circumstances, mammalian tissues readily dispose of this compound, as aberrant L-28 2HG accumulation induces neurometabolic disorders and promotes renal cell 29 carcinomas. Intriguingly, Drosophila melanogaster larvae were recently found to 30 accumulate high L-2HG levels under normal growth conditions, raising the possibility 31 that L-2HG plays a unique role in insect metabolism. Here we explore this hypothesis by 32 analyzing L-2HG levels in 18 insect species. While L-2HG was present at low-to-33 moderate levels in most of these species (<100 pmol/mg; comparable to mouse liver), 34Dipteran larvae exhibited a tendency to accumulate high L-2HG concentrations (>100 35 pmol/mg), with the mosquito Aedes aegypti, the blow fly Phormia regina, and three 36representative Drosophila species harboring concentrations that exceed 1 nmol/mg -37 levels comparable to those measured in mutant mice that are unable to degrade L-2HG. 38Overall, our findings suggest that one of the largest groups of animals on earth 39 commonly generate high concentrations of an oncometabolite during juvenile growth, 40 hint at a role for L-2HG in the evolution of Dipteran development, and raise the 41 possibility that L-2HG metabolism could be targeted to restrict the growth of key disease 42 vectors and agricultural pests. 43 44