Researchers identify proteins hijacked by respiratory syncytial virus

This electron micrograph depicts the Respiratory Syncytial Virus (RSV) pathogen.Image courtesy of the Centers for Disease Control and Prevention

Although the human respiratory syncytial virus is the most common cause of bronchiolitis and pneumonia in infants, no vaccine is available. Research groups worldwide are seeking possible drug targets for the disease, which also is particularly dangerous for the elderly and those with compromised immune systems.

One international team of researchers recently reported in the journal Molecular and Cellular Proteomics that it had found 24 proteins that may serve as drug targets, given their direct interactions with one of the most crucial viral proteins. Monika Bajorek at the Imperial College London and Doron Gerber at the Bar Ilan University in Israel led the study.

Four fundamental RSV proteins — fusion, matrix, phosphor and nucleo — are responsible for the virus’s replication. The matrix, or M, protein specifically plays key roles in the viral life cycle through inhibition of host transcription and facilitation of viral transcription, assembly and budding. However, until now, only two of the host proteins engaged in viral replication had been known to interact directly with the M protein. The researchers reporting their findings in MCP uncovered another 24.

Using a high-throughput microfluidics screen, the researchers expressed 500 human proteins previously reported to be manipulated for RSV replication. After immobilizing the human proteins on a chip, they labeled them with a fluorescent tag. They expressed the M protein separately and labeled it with a different tag before flowing it over the chip to capture any human interactors. Finally, they detected all interactions with fluorescence from the human and M proteins.

The researchers verified some interactions with a different microfluidics screen and then verified 71 percent of them with a co-immunoprecipitation technique. In the latter technique, they precipitated the M protein out of the cells, which had been lysed, before capturing it onto affinity beads, and then they used the beads to co-precipitate human interactors from the samples before detecting the interactions with Western blotting.

After demonstrating the host-virus protein interactions in a cell-free approach, the researchers went on to prove that four human proteins — Caveolin 1, Caveolin 2, Cofilin 1 and a zinc finger protein — directly interacted with the M protein in a cellular environment. They did that by showing that all four co-localized with the M protein at an intracellular site where the M protein was expected to play its specific role in RSV replication. The researchers say that the four human proteins and other direct interactors could be potential targets of anti-RSV therapies, given that knocking down the genes encoding the four proteins significantly reduced viral infectivity.

The team already is planning future avenues of investigation. “The next step will be focusing on some of the other factors from the list with the goal of detailed mechanistic analysis. Although we showed they interact with M, we still need to see which factors are critical for virus replication and what the mechanism is,” Bajorek said. She said her team is now set to crystallize some of the M-host protein complexes to analyze the interactions in detail in hope of designing drugs that inhibit or foil the interactions.

Gerber added: “We would like to solve one of the puzzling issues with RSV. What is the matrix protein doing in the nucleus?” To answer the question, the team will screen 5,000 host nuclear proteins to investigate their interactions with the RSV M protein.

Vivian Tang Vivian Tang is a graduate student at the School of Pathology and Laboratory Medicine at the University of Western Australia.