“I’m a virus guy, for sure,” says Dr. Tamanash Bhattacharya, a postdoctoral fellow in the Malik Lab of the Fred Hutch Basic Sciences division, with a broad smile. The Malik Lab specializes in studying the mechanisms and consequences of genetic conflicts, and there is perhaps no more famous biological battleground than that of the host-virus evolutionary arms race. And while some viruses primarily ‘battle’ a single host species, others boast intricate lifecycles involving distinct life stages in two or more distinct hosts. The prototypical example of such viruses are alphaviruses, whose ranks include Sindbis Virus, Chikungunya Virus, and Eastern Equine Encephalitis Virus, among others. While alphaviruses can infect and cause disease in humans, natural alphavirus reservoirs continuously cycle between vertebrate hosts (like small mammals or birds) and invertebrate hosts—most commonly, mosquitoes.
Dual-host viruses raise a host (no pun intended) of fascinating evolutionary questions: because these viruses must cycle through both vertebrate and invertebrate hosts to replicate, their evolution represents a constant battle on two distinct biological fronts. “From the perspective of a virus,” notes Bhattacharya, “mammal cells and mosquito cells are hugely different from each other—you can imagine that an adaptation that helps a virus infect or survive in vertebrate cells might actually hinder its fitness in a mosquito cell, for instance. There are clearly some complex interactions influencing viral fitness in both hosts, but in general, we still lack a detailed mechanistic understanding of the ways in which dual-host viruses optimize for fitness in these very different biological settings.”
Reporting their results in Science Advances, Bhattacharya and colleagues set out to address this question, focusing on a specific alphavirus called Sindbis Virus. Specifically, they zoomed in on a specific gene in the Sindbis genome—non-structural protein 3 (nsP3)—and went even further to focus on just a single codon in this gene. To understand the importance of this codon, we need to take a little dive into viral biology:
“One of the things that makes viruses so cool is how efficient they are,” notes Bhattacharya. Indeed, like biological Swiss-army knives, viruses must pack a lot of functionality into genomes which are orders of magnitude smaller than their hosts’. One of the tricks that viruses use to stretch a meager supply of genes is to translate multiple adjacent proteins into long polyproteins, which are then further processed into their constituent subunits. This arrangement affords viruses more functional diversity from the same genome (polyproteins can carry out distinct functions than their individual constituent subunits), and it also provides an efficient means of regulating the stoichiometry (relative amounts) of proteins that they produce. Four Sindbis proteins (nsP1-4) are regulated in this manner: these four proteins appear in order in the viral genome under the control of one promoter before nsP1, and there is a STOP codon at the end of nsP3 (see below).
“Wait a second,” my astute readers might interject at this point. “Surely you just made a mistake—you just said there are four subunits, but there’s a STOP codon after nsP3? How does the nsP4 protein ever get made??” You read that right—there is a STOP codon between nsP3 and nsP4, but it’s no ordinary STOP codon. This particular codon is called an opal codon (UGA), and it differentiates itself from the other two types of STOP codon (called amber, UAG and ochre, UAA), by being… well, pretty bad at its job! While the amber and ochre STOP codons recruit termination factors that efficiently cause a translating ribosome to detach from its mRNA molecule, the opal codon’s sequence can be outcompeted by sense tRNAs in the cell, causing the ribosome to ‘ignore’ the STOP codon in a phenomenon termed ribosomal readthrough. Thus, the presence of an opal codon at the end of the nsP3 sequence means that Sindbis produces two flavors of this polyprotein: a shorter, nsP1-nsP2-nsP3 (abbreviated P123) variant if the opal codon is obeyed, and a longer nsP1-nsP2-nsP3-nsP4 (P1234) variant if the opal codon is ignored.
“On one hand, nsP4 encodes the RNA polymerase that the virus uses to replicate its genome, and nsP4 levels are known to be rate-limiting for viral replication. But on the other hand, you have this exotic opal codon that acts to suppress the amount of nsP4 that the virus translates (the opal codon is read through less than 20% of the time). It also turns out that this opal codon is almost universally conserved among alphaviruses, suggesting that it confers some selective fitness advantage. Previous work has revealed some interesting clues suggesting that the opal codon impacts viral fitness differently in different host contexts (i.e. in vertebrate versus invertebrate settings), but the exact fitness advantage and its source remained unclear,” says Bhattacharya.
With these possibilities in mind, Bhattacharya and team used a high-throughput approach to systematically mutate the nsP3 opal codon and measured the relative fitness of the resulting viruses in two biological settings: Vero cells (derived from an African green monkey) and C3/36 mosquito cells. They also took this one step further, however, by considering biological factors that are different between vertebrate and invertebrate hosts. “One underappreciated factor that differs between mammal and mosquito cells is the temperature—mammals are endotherms and maintain an approximately constant 37C body temperature, while mosquitos are ectotherms and thus operate near ambient temperature. Interestingly, it’s been shown that ribosomal readthrough of opal codons increases at lower temperatures, so we wondered how temperature would influence the relative fitness of the opal codon versus alternatives.” To get at this, the team included another condition in their screen: all of the viral variants cultured in Vero cells, but at the mosquito-like temperature of 28C instead of 37C.
As one might predict given the evolutionary conservation of the opal codon, Bhattacharya and colleagues found that viruses with opal outperformed all other alternatives in Vero cells at 37C—however, they were interested to find that the degree to which opal rose above the rest of the codons was significantly reduced in mosquito cells, suggesting that the main selective pressure to keep opal comes from the vertebrate host. But did this difference arise due to some inherent biological difference between monkey and mosquito cells? “Surprisingly, we also noticed that the gulf between opal and its alternatives similarly narrowed in Vero cells cultured at 28C,” noted Bhattacharya, “suggesting that the main variable influencing the selective advantage of opal has less to do with biological differences between monkeys and mosquitoes and more to do with the temperature at which the virus is passaged.” Measuring the relative amounts of nsP4 protein produced in viruses with each of the three STOP codons (opal, amber, and ochre), the team found a strong correlation: more nsP4 protein equated with more fit viruses, and vice versa.
But if more nsP4 protein correlated with higher viral fitness, why did replacing the opal codon with sense codons (which should result in the highest nsP4 production, since there is no longer any kind of STOP codon preceding it), reduce fitness? An assemblage of mechanistic experiments led Bhattacharya and team to an explanation: too much P1234 can actually be bad, because it effectively ‘gums up’ the further processing of these polyproteins during the viral infection cycle. As Bhattacharya explains, “Alphaviruses process the P123 and P1234 polyproteins in a regulated and stepwise manner, and the timing of this processing is crucial for viral reproduction. We found that too much P1234—which happens when opal is replaced with sense codons—sequesters important processing factors and leads to overall impaired polyprotein processing and slower replication kinetics.”
Overall, the team’s findings support a model whereby selective pressure from vertebrate hosts maintains the alphavirus nsP3 opal codon because this codon keeps nsP4 levels in a Goldilocks zone: enough to replicate the viral genome but not too much as to slow down further viral genome processing steps. Another way to think of the opal codon, as the authors put it in this study, is as a ‘crude but effective thermometer for viral replication”: at the higher temperatures found in vertebrate hosts, the opal codon caps the amount of nsP4 produced without shutting it off entirely. “Beyond more ‘traditional’ factors that are known to influence viral fitness—like host immune systems—these results also point to temperature as an important but sometimes overlooked host-specific factor to consider in some contexts,” notes Dr. Harmit Malik.
Malik and Bhattacharya are quick to note that since opal codons are found in other viral families, they might represent a broader adaptive mechanism that allows viruses to thrive in disparate host environments. In all, their results shed light on a fascinating and elegant mechanism of genome regulation employed by alphaviruses that informs our fundamental understanding of how these viruses function as well as our capacity to combat them in situations where they threaten human health.
The spotlighted work was funded by the National Institutes of Health, a Helen Hay Whitney Postdoctoral Fellowship, and the Howard Hughes Medical Institute.
Fred Hutch/University of Washington/Seattle Children’s Cancer Consortium Member Dr. Harmit Malik contributed to this research.
Bhattacharya, T., Alleman, E. M., Freeman, T. S., Noyola, A. C., Emerman, M., & Malik, H. S. (2025). A conserved opal termination codon optimizes a temperature-dependent trade-off between protein production and processing in alphaviruses. Science Advances, 11(16), eads7933.
Science Spotlight writer David Sokolov is a graduate student in the Sullivan Lab at the Fred Hutch. He studies how cancer cells modify their metabolism to facilitate rapid proliferation and accommodate tumor-driving mitochondrial defects. He's originally from the east coast and has bachelors' and masters' degrees from West Virginia University. Outside of the lab, you'll find him enjoying the outdoors, playing music, or raising composting worms in his front yard.
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