Chikungunya virus evolution following a large 3′UTR deletion results in host-specific molecular changes in protein-coding regions

Abstract

The 3′untranslated region (UTR) in alphavirus genomes functions in virus replication and plays a role in determining virus host range. However, the molecular evolution of virus UTRs is understudied compared to the evolution of protein-coding regions. Chikungunya virus (CHIKV) has the longest 3′UTR among the alphaviruses (500–700 nt), and 3′UTR length and sequence structure vary substantially among different CHIKV lineages. Previous studies showed that genomic deletions and insertions are key drivers of CHIKV 3′UTR evolution. Inspired by hypothesized deletion events in the evolutionary history of CHIKV, we used experimental evolution to examine CHIKV adaptation in response to a large 3′UTR deletion. We engineered a CHIKV mutant with a 258 nt deletion in the 3′UTR (ΔDR1/2). This deletion reduced viral replication on mosquito cells, but did not reduce replication on mammalian cells. To examine how selective pressures from vertebrate and invertebrate hosts shape CHIKV evolution after a deletion in the 3′UTR, we passaged ΔDR1/2 virus populations strictly on primate cells, strictly on mosquito cells, or with alternating primate/mosquito cell passages. We found that virus populations passaged on a single host cell line increased in fitness relative to the ancestral deletion mutant on their selected host, and viruses that were alternately passaged improved on both hosts. Surprisingly, whole genome sequencing revealed few changes in the 3′UTR of passaged populations. Rather, virus populations evolved improved fitness through mutations in protein coding regions that were associated with specific hosts.

Design and Use of Chikungunya Virus Replication Templates Utilizing Mammalian and Mosquito RNA Polymerase I-Mediated Transcription

“…The missing part of the CHIKV 3= UTR contains repeated sequence motifs which have only a minimal effect on CHIKV replication in mammalian cells (60). In contrast, however, a deletion of this region caused a prominent reduction of CHIKV replication in both Aedes aegypti and Aedes albopictus cells (61). To the best of our knowledge, the molecular basis of this defect has not been reported.…”

Cross-utilisation of template RNAs by alphavirus replicases

“…Most of this variation is due to different copy numbers of repeated sequence motifs. For CHIKV the presence of repeated motifs is essential for efficient replication in mosquito cells but has little, if any, impact on replication in vertebrate cells [21][22][23]. The 3' UTR interacts with host protein HuR increasing the stability of viral RNAs and promoting infection both in vertebrate and mosquito cells [24].…”

Differential alphavirus defective RNA diversity between intracellular and encapsidated compartments is driven by subgenomic recombination events

“…These CHIKV 3’UTR recombination events mostly consisted of duplications, especially in intracellular populations. Duplications in the CHIKV 3’UTR are well-documented and are thought to influence CHIKV host adaptability, particularly in the insect vector (36, 38, 73). Filomatori and Bardossy and colleagues additionally found that these recombination events arise from a mixture of homologous- and nonhomologous-template switching (39), although the relative quantities of each varied depending on host type.…”