Flu virus transcription caught in the act

Like a camera-shy teenager, influenza’s ribonucleoprotein (RNP) complex turns away whenever researchers point the camera at it. The RNP complex is extremely flexible, so scientists have historically had difficulty pinning down its full structure and mechanism. But this week, structural biologists report success in taking detailed candids of the whole RNP complex.

The team, led by Yi-Wei Chang at the University of Pennsylvania, combined two forms of cryo-electron microscopy (cryo-EM), single-particle analysis and cryo-electron tomography, to get atomic-level resolution snapshots of the complex as it transcribes (Science 2025, DOI: 10.1126/science.adq7597). The researchers’ efforts reveal the flu virus’s replication mechanism and its essential structural bits, knowledge that is important for designing paninfluenza drugs.

The RNP complex is the influenza virus’s essential package, the virus’s blueprints and a copy machine all rolled in one. The blueprints are the RNA genome, protected and carefully packed among stabilizing nucleoproteins arranged in a double helix, and the copy machine is a polymerase complex at the end.

The self-sufficient molecular machines are “all ready to replicate themselves once they infect,” Chang says. And their architecture is essential to that function.

Individual pieces of the RNP complex have previously been well-characterized, Chang says, yet the whole grouping is so flexible, images of the entire complex have gotten to only nanometer resolution. Chang and his colleagues used a new, combination approach to increase that resolution. They first made the shortest RNP complex, with the least amount of flexibility, and focused cryo-electron microscopy single-particle analysis on the smallest unit of each short RNP: four nucleoproteins, two on each helical strand, with a bit of the RNA genome. That focused concentration on the smallest symmetrical subunit got the resolution down to 5.1 Å and allowed the researchers to visualize, for the first time, the nucleoprotein helix protecting the RNA genome inside the minor groove, Chang says.

Next, the researchers turned to cryo-electron tomography, which allowed them to take detailed 3D snapshots of individual RNP macromolecules in action. They caught the polymerase complex sometimes resting at the head of RNP, while at other times it was on the side with a messenger RNA strand trailing off, showing that the polymerase was in the process of transcribing. In each case, even when transcribing, the nucleoprotein double helix stayed intact.

By analyzing thousands of RNP snapshots, Chang’s group discovered that the polymerase reads the RNA genome as the strands of the nucleoprotein double helix slide past each other. It’s like lines of walkers passing each other on a spiral staircase, one side going up and one side going down. The polymerase joins one of the lines—say, the group walking up—and interacts with the walkers going down just long enough to copy the RNA genome as they pass.

“This study clearly demonstrates the strand-sliding mechanism” and that RNA synthesis does not disrupt the double-helical structure, says Takeshi Noda, who leads the ultrastructural virology lab at Kyoto University and who was not involved in the study. Understanding this mechanism has “long been an unresolved issue.”

Chang says his group never could have resolved the entire complex and its mechanism without combining cryo-electron microscopy single-particle analysis for getting atomic close-ups and cryo-electron tomography for taking action shots. He is excited about the possibilities of combining the two technologies in the future.

Now Chang and his group are developing drug leads for targets that the new mechanism unveiled. They identified at least one key structure: tail loops connecting each nucleoprotein to its neighbor. When they took out the tail loops, the nucleoproteins stopped linking up altogether. And blocking the tail loops blocked virus replication. Chang’s group is now working on designing tail loop inhibitors that could be the basis of future paninfluenza antiviral drugs.