Chikungunya, Sindbis, and Equine Encephalitis viruses can infect humans and cause symptoms ranging from mild flu-like fever and aches to severe, deadly brain-swelling. They belong to a genus of RNA viruses collectively known as alphaviruses.
Alphaviruses are transmitted between individuals via mosquitoes, meaning they must live and thrive in two divergent hosts. They face the challenge of maintaining replicative fitness across temperatures from 37°C in humans to variable, but lower, ambient temperatures in mosquitoes, while also evading different immune defenses: interferon-driven antiviral responses in humans and RNA interference (RNAi) in mosquitoes.
Surprisingly, alphaviruses contain a conserved premature stop codon within the gene that encodes viral nonstructural proteins, just before the sequence encoding an essential viral RNA Polymerase. While ribosomal readthrough during translation usually yields sufficient RNA polymerase for replication, researchers in Dr. Harmit Malik’s lab in the Basic Sciences Division became curious about why the stop codon is so prevalent, especially since some stop codon substitutions are well tolerated at lower temperatures in mosquito cells. Postdoc Dr. Tamanash Bhattacharya explained, “this unusual arrangement has been conserved throughout alphavirus evolution. We set out to ask why.” The results of the study were recently published in Science Advances.
The researchers performed a series of experiments using two Sindbis virus variants, one with the wild type premature stop codon – UGA – in the nonstructural protein gene, and the other with a substitution to a cysteine-encoding sense codon – UGC – at the same site. The team investigated infections in both mosquito and human cells.
In mosquito cells, the wild type UGA variant had a higher rate of infection and outcompeted the UGC variant, indicating a higher replicative fitness. However, this was only true in mosquito cells with functional Dcr2, a key component of the mosquito RNAi antiviral defense.
The protein Dcr2 recognizes double-stranded viral RNA replication intermediates and cuts them into fragments. These fragments are loaded onto the protein Ago2 and used as bait to detect more viral RNA. Once bound to Ago2, additional host machinery degrades the viral RNA through the process of RNA interference, or RNAi, thereby inhibiting viral replication.
If either Dcr2 or Ago2 were absent, infection by the two Sindbis virus variants was indistinguishable, suggesting that the RNAi pathway selects against viruses with the UGC sense codon. The single nucleotide change (UGA to UGC) in the entire 12kb viral genome triggered a substantially greater immune response in RNAi-competent cells.
These results were confirmed with infection experiments in live mosquitoes. Infection with the UGA stop codon variant produced higher viral RNA levels than infection with the UGC variant in mosquitoes with functional Dcr2, while both variants produced similarly high viral RNA levels in mosquitoes without Dcr2.
The results indicate that the UGA stop codon gives the virus a replicative advantage in mosquitoes by helping it evade RNAi immune defense. Because the codon is located within the gene encoding viral nonstructural proteins, the researchers thought the mechanism may involve replication spherules, structures composed of viral nonstructural proteins where RNA replication occurs. The spherules normally shield viral RNA intermediates from host cell detection.
Using fluorescence imaging, the team compared spherule formation in cells infected with either the wild type UGA stop codon viral variant or the UGC sense codon viral variant. Replication spherules in cells infected with the UGC variant had compromised integrity, allowing fluorescent proteins from the surrounding cytoplasm to enter. In contrast, the spherules in the UGA variant-infected cells formed an impermeable barrier with the cytoplasm.
Overall, these results suggest that in mosquitoes, the premature UGA stop codon protects spherule integrity and shields replicating viral RNA from RNAi detection. Replacing it with the UGC sense codon disrupts spherule formation, exposing viral RNA to immune detection and reducing replicative fitness.
Does a similar mechanism exist in humans?
Unlike mosquitoes, human cells detect viral infections via pathogen recognition receptors that, when engaged, trigger an antiviral interferon signaling cascade. To understand if the UGA premature stop codon protects spherule formation and helps the virus evade immune detection in the human host, the researchers measured the expression of interferon and interferon-stimulated genes in human cell lines infected with either the wild-type UGA stop codon virus or the UGC sense codon variant. Cells infected with the UGC variant had a stronger interferon response. Knockout of the pathogen recognition receptors RIG-I and MDA5 eliminated the interferon response, so that infections with both viral variants produced similarly low interferon levels. The results indicate that the UGA stop codon in the nonstructural protein gene allows alphaviruses to evade immune detection in human hosts, just as it protects them from RNAi-mediated defense in mosquitoes.
Dr. Bhattacharya commented, “The UGA codon allows alphaviruses to shield themselves from being detected by host immunity – a clever strategy devised almost entirely by a single codon.” This all highlights the power of immune responses to control viral replication – and how viruses can fight back against immunity using similar simple mechanisms across divergent hosts.
Dr. Bhattacharya explained that moving forward, the lab plans to “determine whether related viruses use similar or alternate evasion strategies, since evading host detection must be a universal requirement for all RNA viruses that replicate inside host cells.”
Fred Hutch/University of Washington/Seattle Children’s Cancer Consortium Member Dr. Harmit Malik contributed to this research.
The spotlighted research was funded by a Helen Hay Whitney Fellowship, the National Institutes of Health, and the Howard Hughes Medical Institute.
Bhattacharya T, Freeman TS, Alleman EM, Wang F, Chechik L, Emerman M, Myles KM, Malik HS. 2026. Conserved in-frame stop codon acts as multipotent defense mechanism in alphaviruses. Science Advances. doi: 10.1126/sciadv.aeee6015
Science Spotlight writer Ashley Person is a PhD candidate in the Cohn lab in the Vaccine and Infectious Disease Division at Fred Hutch. She studies how HIV-infected cells persist over time in people living with HIV on long term treatment.