A 44-Nucleotide Region in the Chikungunya Virus 3' UTR Dictates Viral Fitness in Disparate Host Cells

Abstract

We previously reported that 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 embryonic 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.

Keywords: 3′ UTR; CHIKV; Chikungunya virus; RNA structure; SHAPE-MaP; viral fitness.

Figure 1

Organization of the Asian-lineage CHIKV genome with emphasis on 3′ untranslated region (UTR) elements. Diagram of the Asian-lineage CHIKV 3′ UTR highlighting the 44-nucleotide deletion, ∆44-UTR, identified in the virus isolated from the serum of persistently infected Rag1^−/− mice.

Figure 2

The ∆44-UTR mutation enhances CHIKV fitness in murine cells in vitro. In vitro viral fitness competitions of marked and unmarked viruses performed through five serial passages (p5) in WT MEFs: (A) similar starting inputs of E2-K200R* vs. E2-K200R or E2-K200R; ∆44-UTR viruses, (B) 10:1 starting ratio of E2-K200R* vs. E2-K200R; ∆44-UTR virus, (C) similar starting inputs of WT* vs WT or ∆44-UTR virus. For each sample, cDNA was synthesized from extracted RNA and used for PCR amplification of a region within the CHIKV nsP4 gene; PCR products were then digested with ApaI/PspOMI and run on an agarose gel. Virus identity is determined by the presence or absence of a synonymous genetic marking in the nsP4 gene (*) that confers susceptibility to ApaI/PspOMI restriction digest. The ratio of ApaI/PspOMI-resistant versus -susceptible PCR products was used to calculate % virus identity. Input indicates starting ratios of marked to unmarked virus in initial inoculum. Data are from 1–3 independent experiments performed in triplicate. Statistics are unpaired t-test; ****, p < 0.0001; ***, p < 0.001; *, p < 0.05; ns, not significant.

Figure 3

The ∆44-UTR mutation is deleterious in mosquito cells. In vitro viral fitness competitions of two viruses performed through five serial passages (p5) in C6/36 Aedes albopictus cells. Virus identity was determined by the presence or absence of a synonymous genetic marking in the nsP4 gene (*) that confers susceptibility to ApaI/PspOMI restriction digest. Input indicates starting ratios of marked to unmarked virus in initial inoculum. Data are from 2 independent experiments performed in triplicate. Statistics are unpaired t-test; ****, p < 0.0001; *, p < 0.05.

Figure 4

The fitness advantage conferred by 3′ UTR deletion in murine cells is independent of the type I interferon response and confers a fitness advantage in a mouse model of CHIKV disease. (A) In vitro viral fitness competitions of marked and unmarked CHIKV performed through five serial passages (p5) in Ifnar1^−/− MEFs. Data are from 1 experiment performed in triplicate. (B–D) For in vivo viral fitness competitions, mice were inoculated with 1000 PFU of each virus. Data are from 1–2 independent experiments performed with 4 mice per experiment. (B) Virus competition in WT C57BL/6 mice; virus proportions in spleen and contralateral ankle determined at 4 days post inoculation. (C,D) Virus competition in Ifnar1^−/− C57BL/6 mice; virus proportions in spleen and contralateral ankle determined at 3 days post inoculation. (A–D) Virus identity was determined by the presence or absence of a synonymous genetic marking in the nsP4 gene (*) that confers susceptibility to ApaI/PspOMI restriction digest. Input indicates starting ratios of marked to unmarked virus in initial inoculum. Statistics are unpaired t-test (A) or one-way ANOVA (B–D); ****, p < 0.0001; ***, p < 0.001; **, p < 0.01; ns, not significant.

Figure 5

The 44-nucleotide deletion in the CHIKV 3′ UTR is characterized by distinct RNA secondary structure. SHAPE-MaP informed models of (A) WT and (B) ∆44-UTR CHIKV 3′ UTRs. The 44-nucleotide region is highlighted in blue in (A). The nucleotide color corresponds to the SHAPE reactivity detected. SHAPE reactivities above 0.8 indicate likely unpaired bases (shown in red). SHAPE reactivities below 0.4 indicate likely paired and therefore unreactive bases (colored black). Intermediate reactivities (e.g., 0.4–0.8) are suggestive of unpaired bases (shown in yellow).