Scientists headed by teams at La Jolla Institute for Immunology (LJI) and Purdue University have published the results of a study that they claim represents the first-ever look at a key stage in the life cycles of two paramyxoviruses, measles and Nipah virus. Results from the research offer new insights into viral replication that could help inform future drug discovery efforts.
“This work solves a long-standing mystery: how viruses assemble themselves,” said LJI professor Erica Ollmann Saphire, PhD, who served as study co-senior author with professor Robert Stahelin, PhD, of Purdue University. “We know that a virus’s many pieces come together at the cell membrane, but we didn’t know what the trigger was that starts that irreversible assembly process.” Stahelin added, “This study succeeds by identifying how paramyxoviruses are able to utilize a host cell lipid for viral spread. This work will inform future drug discovery endeavors.”
Saphire, Stahelin, and colleagues reported their findings in Science Advances, in a paper titled, “Measles and Nipah virus assembly: Specific lipid binding drives matrix polymerization.”
The paramyxovirus family of viruses includes measles and Nipah virus, as well as mumps, Newcastle disease, and canine distemper. The researchers suggest that this family of viruses has the potential to trigger a devastating pandemic. “Paramyxoviruses represent a major risk to human and animal health, and it is vital that efforts continue to focus on new targets within the virus replication cycle,” they wrote.
First author Michael Norris, PhD, a former postdoctoral associate at LJI and current assistant professor at the University of Toronto, added, “The infectiousness of measles is unmatched by any known virus. If one person with measles coughs in a room with 100 unvaccinated people, around 90 would become infected. Nipah virus is not as contagious, but it is incredibly lethal, with between 40% and 90% of infections causing death. Just imagine if a paramyxovirus emerged that was as contagious as measles and as deadly as Nipah.”
The results of the team’s new study reveal how future therapies might stop these viruses in their tracks. For their work the researchers used several imaging techniques, including x-ray crystallography and electron microscopy, to capture viral assembly. During viral assembly, key proteins and genetic material are recruited to specific areas on infected host cell membranes. Viral matrix (M) proteins come together to form a lattice against the inside of the cell membrane. These matrix proteins are the drivers of the virus assembly process. Norris calls them the “field marshals” that gather and guide the other proteins needed to form a new virus. Matrix proteins also give a virus its shape.
As viral assembly continues, the lattice of matrix proteins begins to push the membrane outward to form a “bud” and recruits other viral proteins to this site. Once the bud has all the needed components in place, it splits away from the parent cell to form a new virus that can then infect a new host cell.
Researchers hope by understanding viral assembly better, they can design therapies that interrupt the process. This approach holds promise; the drug Lenacapavir targets the assembly process of HIV and is in clinical trials right now, and Norris suggests there’s potential to use the same strategy to stop paramyxoviruses. “This HIV therapy is a proof of principle that targeting viral assembly is a viable strategy for drug development,” he said.
Scientists just need a clear view of the paramyxovirus assembly process. The challenge for researchers is trying to see the matrix proteins in action. As the authors noted, “Paramyxoviruses bud from the plasma membranes (PMs) of infected cells. Viral matrix (M) proteins coordinate budding of new virions by marshaling other viral structural components, including surface glycoproteins and viral replication complexes at assembly sites along the host PM … However, the nature of M protein binding to membranes, why virus assembly happens largely at the PM, what triggers viral matrix polymerization, and whether interaction of M with lipid membranes alone is sufficient to form outward protrusions in the PM remain unclear.”
Norris and his colleagues examined viral assembly in both the measles virus and Nipah virus. The results showed how two matrix proteins come together in a sort of hug to form a two-sided “dimer” structure. The researchers demonstrated that blocking formation of this dimer also stops viral assembly. That was no surprise, and what the researchers really needed to know was how these dimers interact with other structures during the budding process. Their study showed that the dimer hug floats toward the inside of the cell membrane to collide with the membrane.
From work spearheaded by the Stahelin Lab at Purdue University, the team found that the matrix proteins specifically bind to a lipid molecule in the host cell membrane called phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2]. This interaction anchors matrix proteins to the host cell membrane surface and provides meeting points for virus assembly at specific sites along the membrane. Norris and his colleagues captured this interaction in detail using x-ray crystallography.
Then came a big structural surprise. The team found that Nipah virus matrix proteins actually change their structure to open up a lipid-binding pocket for PI(4,5)P2. “This was an extremely exciting aspect of the study,” stated Norris. He emphasizes that this pocket doesn’t exist prior to membrane binding—and the pocket would not have been spotted without the structure captured in this study. Discovery of the pocket revealed a new potential target for developing inhibitors of the assembly process.
PI(4,5)P2 also turned out to be the secret ingredient that triggers matrix protein dimers to bind to one another to form a lattice on the inner surface of the host cell membrane. When the matrix proteins change their structure to open the PI(4,5)P2 pocket, they also adopt a shape that drives lattice assembly.
This change in the matrix protein structure then puts a bend in the cell membrane. Before PI(4,5)P2 binding, the space between two matrix proteins in a hug has a concave bowl shape with angled sides. After PI(4,5)P2 binding to the matrix proteins, these angled sides flatten to transform the bowl shape to a plate shape and force the membrane to curve upward. The soft curve of the cell membrane then pops up to start forming a new bud that will eventually make up a new virus. The scientists learned a lot from these structures. “We didn’t know how much the molecules would change as the process was triggered or what the structure would look like as they zip together,” noted Saphire. The team summarized, “Using x-ray crystallography, electron microscopy, and molecular dynamics, we demonstrate that PI(4,5)P2 binding induces conformational and electrostatic changes in the M protein surface that trigger membrane deformation, matrix layer polymerization, and virion assembly.”
Measles is still a major killer around the world. India and Bangladesh deal with yearly Nipah outbreaks. These viruses aren’t going away, and effective therapies are needed to stop outbreaks. A broad paramyxovirus therapy could also protect livestock from disease and ensure food security. Newcastle disease in poultry is caused by paramyxovirus infections that can wipe out entire flocks before symptoms are even detected. An outbreak of Newcastle disease in 2018–2020 in California caused the culling of 1.2 million birds.
The new study shows the potential for a pan-paramyxovirus therapy that targets viral assembly in multiple viruses. Norris pointed out that while the genomes of measles and Nipah viruses are very different, the measles and Nipah matrix proteins look almost identical.
“Because these matrix protein structures are highly conserved, we could potentially target one virus and have an inhibitor that could target all the rest of the viruses in this family,” Norris stated. The authors further wrote in their paper, “Although future investigations are needed to determine how M proteins, lipids, and host proteins work in concert to form virions, illuminating structures and membrane interactions of M can explain how they direct virion formation and will provide 3D templates for the design of agents to inhibit paramyxovirus assembly and egress.”
Norris was able to start looking for matrix protein inhibitors thanks to funding from LJI’s Tullie and Rickey Families SPARK Award for Innovation in Immunology. This funding allowed him to rapidly narrow down a list of over 7.4 million drug candidates to identify around 100 that will go into further testing.
The next steps are to better understand the molecular interactions that make up the matrix lattice and to better understand how matrix proteins recruit and interact with the other viral proteins during the assembly process. “We’re looking at leveraging this work to design broad-spectrum inhibitors of viral assembly,” said Norris. In their paper, the team concluded, “… structure-guided development of small-molecule inhibitors that target the conserved PI(4,5)P2 binding site on paramyxovirus M proteins will be important for development of broad-spectrum antivirals against paramyxovirus infections.”
