A 44-nucleotide region in the chikungunya virus 3′ UTR dictates viral fitness in disparate host cells
Description
Abstract: We previously reported that the deletion of a 44-nucleotide element in the 3′ untranslated region (UTR) of the Chikungunya virus (CHIKV) genome enhances the virulence of CHIKV infection in mice. Here, we find that while this 44-nucleotide deletion enhances CHIKV fitness in murine em-bryonic fibroblasts in a manner independent of the type I interferon response, the same mutation decreases viral fitness in C6/36 mosquito cells. Further, the fitness advantage conferred by the UTR deletion in mammalian cells is maintained in vivo in a mouse model of CHIKV dissemination. Finally, SHAPE-MaP analysis of the CHIKV 3′ UTR revealed this 44-nucleotide element forms a distinctive two-stem-loop structure that is ablated in the mutant 3′ UTR without altering additional 3′ UTR RNA secondary structures. Viral Fitness Competitions and Analyses: Cells or mice were inoculated with a mixture of genetically marked (introduction of ApaI/PspOMI restriction site in the nsP4 gene) and unmarked viruses. In vitro, virus was blind-passaged every 24 h for a total of 5 passages; in vivo, samples from infected mice were collected at 3-4 dpi. To determine the percent virus identity (ratio of marked and unmarked virus), RNA was isolated from samples, and random hexamers were used to generate cDNA. After generating cDNA, the nsP4 region of the viral genome was PCR-amplified using GoTaq polymerase and the following primers: 5′-ATATCTAGACATGGTGGA-3′ and 5′-TATCAAAGGAGGCTATGTC-3′. PCR products were digested with ApaI and PspOMI at room temperature for 30 min followed by 2–3 h at 37 °C. The redundancy of the double digestion with the neoschizomers ApaI and PspOMI ensured complete digestion of the genetically marked PCR products within each sample. Digested PCR products were analyzed on a 1% TAE gel, stained with ethidium bromide, imaged, and band intensities per lane were quantified. Marked virus generates a PCR product susceptible to restriction enzyme digestion (cleaving the PCR product in half), while unmarked virus is resistant to cleavage.