Disruption of the Opal Stop Codon Attenuates Chikungunya Virus-Induced Arthritis and Pathology

Abstract

Chikungunya virus (CHIKV) is a mosquito-borne alphavirus responsible for several significant outbreaks of debilitating acute and chronic arthritis and arthralgia over the past decade. These include a recent outbreak in the Caribbean islands and the Americas that caused more than 1 million cases of viral arthralgia. Despite the major impact of CHIKV on global health, viral determinants that promote CHIKV-induced disease are incompletely understood. Most CHIKV strains contain a conserved opal stop codon at the end of the viral nsP3 gene. However, CHIKV strains that encode an arginine codon in place of the opal stop codon have been described, and deep-sequencing analysis of a CHIKV isolate from the Caribbean identified both arginine and opal variants within this strain. Therefore, we hypothesized that the introduction of the arginine mutation in place of the opal termination codon may influence CHIKV virulence. We tested this by introducing the arginine mutation into a well-characterized infectious clone of a CHIKV strain from Sri Lanka and designated this virus Opal524R. This mutation did not impair viral replication kinetics in vitro or in vivo. Despite this, the Opal524R virus induced significantly less swelling, inflammation, and damage within the feet and ankles of infected mice. Further, we observed delayed induction of proinflammatory cytokines and chemokines, as well as reduced CD4+ T cell and NK cell recruitment compared to those in the parental strain. Therefore, the opal termination codon plays an important role in CHIKV pathogenesis, independently of effects on viral replication.

Genome-Scale Phylogeny and Evolutionary Analysis of Ross River Virus Reveals Periodic Sweeps of Lineage Dominance in Western Australia, 1977–2014

“…Replacement of the opal stop codon by an arginine and cysteine residue, as was seen in isolates K3011 and P42214, respectively, has been described for other alphaviruses, including CHIKV and SINV (34,36), but this is the first time these mutations have been identified in the RRV genome. In addition, the loss of otherwise well-conserved proline-rich domains within the hypervariable domain (HVD) and the nucleotide deletions of various lengths that were observed in multiple isolates may have significant implications in terms of RRV replicative fitness, virulence, vector competence, and human pathogenesis that will need to be investigated.…”

“…Such opal stop codon-to-arginine transitions have been observed in several strains of other alphaviruses, including O'nyong-nyong virus (ONNV) (33) and CHIKV, and this is the first documented observation of such a transition in RRV. Introduction of an arginine codon into a CHIKV isolate significantly reduced joint morbidities in infected mice compared to that in mice infected with the wild type (34). Virus replication did not appear to be affected.…”

Functional and structural characterization of the chikungunya virus translational recoding signals

“…3A). Reports in the literature have also indicated that substitution of an arginine codon for the UGA codon of the CHIKIV TCR signals promoted reduced viral pathogenesis (13). When the TCR opal termination codon was mutated to an AGA arginine, 83.6 and 77.0% read-through efficiencies were observed for the Af/As and Carib consensus sequences, respectively (Fig.…”

“…A closely associated downstream stimulatory element is thought to prevent efficient association of the eRF1-eRF3 complex with the ribosomes stalled at this codon, increasing the likelihood of a TCR event (12). The specific stop codon identity for this signal is critical for optimal alphavirus functionality as substitutions to amber or ochre stop codons or an arginine read-through have been associated with lowered transmission in mosquito vectors and significantly attenuated pathogenesis (13)(14)(15). The Ϫ1 PRF signal is located in the subgenomic RNA that encodes a polyprotein that is subsequently processed into structural proteins.…”

Suramin Inhibits Chikungunya Virus Replication by Interacting with Virions and Blocking the Early Steps of Infection

“…The mutation opal524R in the nsP3-coding region was reported also by Mounce et al in the context of resistance selection against the compound DFMO [34], alongside with other nsP mutations. In the case of CHIKV and other alphaviruses (SFV, ONNV), evolutionary pressures have maintained both variants (stop and arginine codon) as it offers a fitness advantage when switching between vertebrate and invertebrate hosts [35][36][37]. The combination of the R171Q and opal524R mutations was also found independently during the selection of CHIKV variants that were resistant to an unrelated compound [38], and they merely appear to reflect adaptation to repeated passaging in mammalian cells (under stress).…”