image: topology of M protein
Credit: ©Science China Press
Beta-coronaviruses, including SARS-CoV-2, have caused global health problems. How the virus infects host cells and triggers immunological responses has been investigated extensively. However, how the virus replicates and assembles in the cell is largely unknown. Researchers at the Institute of Biophysics, Chinese Academy of Sciences have uncovered a conserved mechanism that drives viral assembly, which not only provides a novel target for developing broad-spectrum antiviral strategies but also sheds light on membrane protein biogenesis. These findings were published in Science Bulletin.
Beta-coronaviruses have three structural components on their surface, that is the spike protein, membrane protein, and envelope protein. Among them, the spike protein is the most studied one, it can bind to the receptor on the surface of host cells, thus initiating infection. But spike is easily mutated, which hinders the effectiveness of vaccines. Targeting the more conserved intracellular pathway would potentially lead to a broadband anti-viral strategy.
“We aim to understand the viral assembly process and searched lots of papers. It was generally assumed that coronaviruses assemble at the ERGIC and the most abundant M protein plays a role in viral assembly. Many reports are controversial and problematic when investigating different beta-coronaviruses by different methods.” said Dr. Junjie Hu, who is the leader of this project.
An unconventional topology of M
It is believed that M contains three transmembrane domains. The team started by analyzing the membrane topology of M and found both its N- and C-termini face the cytosol; the first "transmembrane domain" is not embedded in the membrane. This unconventional topology prevents glycosylation of M and plays a critical role in recruiting S and E proteins. Importantly, this is evolutionarily conserved among other beta-coronaviruses.
“At that time, we were shocked and puzzled by these findings, because several recently determined M structures all exhibit a three-transmembrane topology,” said Rui Lyu, one of the first authors of the paper. They then further analyzed the amino acid sequence of M and found the topology of M is highly sensitive to N terminal amino acid composition, and the addition of N-terminal tags, even as small as HA or Flag, would promote mis-integration. Additionally, under high expression conditions during purification, the three-transmembrane conformation is more stable than the two-transmembrane form, which may lead to the enrichment of wrongly integrated proteins.
A variety of trafficking motifs
When the three proteins are expressed individually, only the M protein predominantly localizes to the trans-Golgi network (TGN), suggesting that viral assembly may occur at the Golgi apparatus. Further analysis reveals that the M protein contains two ER export signals capable of direct binding to COPII components, as well as a Golgi retention signal that mediates COPI binding. These signals can be selectively utilized under different circumstances and these characteristics may ensure the adaptation of viruses to different intracellular contexts.
Through collaboration with virologists Drs. Hongyu Deng and Yu Chen, the team uses virus-like particles and a safe replicon system to examine whether blocking M transportation or M topology would affect viral assembly. They found under both circumstances, viral infection is dramatically reduced.
“The proposed assembly mechanism applies to other betacoronaviruses and suggests that interference with M topology and trafficking can be a general strategy for preventing coronavirus in the future,” said Dr. Hu.