Chikungunya Virus Overcomes Polyamine Depletion by Mutation of nsP1 and the Opal Stop Codon To Confer Enhanced Replication and Fitness

Abstract

Polyamines, which are small positively charge molecules present in all cells, play important roles in the replication of DNA and RNA viruses. Chikungunya virus (CHIKV) relies on polyamines for translation of the viral genome upon viral entry, and pharmacological depletion of polyamines limits viral replication. However, the potential development of antiviral resistance necessitates a better understanding of how polyamines function and can be targeted via compounds that alter polyamine levels. We have isolated CHIKV that is resistant to polyamine depletion and contains two mutations in the nonstructural protein 1 (nsP1)-coding region in combination with a mutation to the opal stop codon preceding nsP4. These mutations, in addition to promoting viral replication in polyamine-depleted cells, confer enhanced viral replication in vitro and in vivo. The nsP1 mutations enhance membrane binding and methyltransferase activities, while the stop codon mutation allows increased downstream translation. These mutations, when combined, enhance viral fitness, but individual mutants are attenuated in mosquitoes. Together, our results suggest that CHIKV can evolve resistance to polyamine depletion and that pharmaceuticals targeting the polyamine biosynthetic pathway may be best used in combination with other established antivirals to mitigate the development of resistance.IMPORTANCE Chikungunya virus is a mosquito-borne virus that has infected millions worldwide. Its expansion into the Americas and rapid adaptation to new mosquito hosts present a serious threat to human health, which we can combat with the development of antiviral therapies as well as understanding how these viruses will mutate when exposed to antiviral therapies. Targeting polyamines, small positively charged molecules in the cell, may be a potential strategy against RNA viruses, including chikungunya virus. Here, we have described a virus that is resistant to polyamine depletion and has increased fitness in cells and in full organisms. Mutations in viral genome capping machinery, membrane binding activity, and a stop codon arise, and their altered activities enhance replication in the absence of polyamines. These results highlight strategies by which chikungunya virus can overcome polyamine depletion and emphasize continued research on developing improved antiviral therapies.KEYWORDS antiviral, antiviral resistance, chikungunya virus, polyamines, viral replication C hikungunya virus (CHIKV) is an arthropod-borne virus (arbovirus) from the Alphavirus family that has caused several severe outbreaks worldwide. Clinical manifestations can be severe and long lasting, including fever and chronic joint pain, and rapid

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

“…Both R171Q and opal524R had been previously reported in CHIKV isolates such as MADOPY1, StBI and StVE (GenBank accession numbers KP003808.1, KP003811.1, and KP003810.1 respectively) which all had been passaged in cell culture prior to sequencing [33]. 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].…”

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

“…A recent study by Ashbrook et al found that inhibitors of sodium-potassium ATPases antagonize CHIKV replication and that drug-resistant variants often contain mutations in the opal termination codon ( 44 ). Drug-resistant variants containing the Opal524R mutation were also obtained when CHIKV was instead passaged in the presence of difluoromethylornithine (DFMO), a drug that inhibits polyamine biosynthesis and thereby restricts viral replication ( 45 , 46 ). Interestingly, the Opal524R mutant did not replicate as well as the parental strain in either Aedes albopictus mosquitos or zebrafish, but when combined with additional drug-resistant mutations, the Opal524R mutation enhances replication in vivo ( 46 ).…”

“…Drug-resistant variants containing the Opal524R mutation were also obtained when CHIKV was instead passaged in the presence of difluoromethylornithine (DFMO), a drug that inhibits polyamine biosynthesis and thereby restricts viral replication ( 45 , 46 ). Interestingly, the Opal524R mutant did not replicate as well as the parental strain in either Aedes albopictus mosquitos or zebrafish, but when combined with additional drug-resistant mutations, the Opal524R mutation enhances replication in vivo ( 46 ). Although these drug-resistant variants have not, to our knowledge, been tested for virulence in a mouse model, our results suggest that these viruses would cause less tissue damage than the parental virus.…”

6′-β-Fluoro-Homoaristeromycin and 6′-Fluoro-Homoneplanocin A Are Potent Inhibitors of Chikungunya Virus Replication through Their Direct Effect on Viral Nonstructural Protein 1

“…An additional mutation of the opal stop codon to an arginine codon (*524R) at the end of the nsP3-coding sequence further increased resistance, but this appeared to be a nonspecific effect (cell culture adaptation). In an independent study with the polyamine inhibitor difluoromethylornithine (DFMO), Mounce et al found that the G230R mutation in CHIKV nsP1 contributes to resistance to polyamine depletion (37). This might reflect the interconnectedness of pathways linked to methionine metabolism and polyamine biosynthesis (17,38).…”