Scientists unveil Ebola virus's 'replication machine'

In September this year, the Ebola outbreak reappeared in Uganda, Africa, which means that the "Sword of Damocles" of the Ebola virus is still hanging over our heads, threatening people's health at all times.

The scientific community has been trying to develop a low-cost, easy-to-promote broad-spectrum anti-Ebola virus small molecule drug, but lacks theoretical guidance. The analysis of the three-dimensional structure of the Ebola virus polymerase is a global problem.

Now, the team of Academician Gao Fu and the team of Researcher Shi Yi from the Institute of Microbiology, Chinese Academy of Sciences, have collaborated to analyze the three-dimensional structure of the Ebola virus polymerase complex for the first time, laying a key theoretical foundation for understanding the Ebola virus replication mechanism at the molecular level. They also analyzed the molecular mechanism by which the century-old drug suramin can effectively inhibit the activity of Ebola virus polymerase, providing new targets and directions for the development of anti-Ebola virus drugs. The related research was published in Nature on September 28.

Find the "Copy Machine"

Ebola virus disease is a severe, acute infectious disease caused by the Ebola virus, which is mainly transmitted between humans or primates. Since it was first discovered in Zaire (now the Democratic Republic of the Congo) and Sudan in Africa in 1976, the Ebola virus has been raging in Africa for nearly 50 years, with more than 30 outbreaks, causing tens of thousands of deaths, with a mortality rate as high as 90%.

Currently, there are two Zaire Ebola virus antibody drugs on the market. "Antibody drugs are specific and cannot be used to treat other types of Ebola viruses and filamentous viruses such as Marburg virus. They are also prone to immune escape, which may cause the antibody drug to become less effective or even ineffective," Shi Yi, the corresponding author of the paper, told China Science Daily.

In addition, he said that the high manufacturing cost of antibody drugs and the need for low-temperature storage are not conducive to their popularization and use in Africa. The development of effective and safe broad-spectrum antiviral small molecule drugs is an important research direction for responding to different types of Ebola virus and other filovirus infections.

Scientists have discovered that the transcription and replication of the Ebola virus genome is completed by a complex formed by the viral polymerase L protein and other auxiliary proteins. The polymerase complex is an ideal target for the development of broad-spectrum antiviral drugs.

Previous studies have also found that the two drugs, remdesivir and favipiravir, have good in vitro antiviral activity, but the clinical effects are not ideal. Currently, there are no small molecule drugs approved for the treatment of Ebola virus infection in clinical practice.

So, how to optimize the structure of existing drugs targeting polymerases? Can new drugs be developed to treat Ebola virus disease by targeting conserved sites of polymerases? To answer these questions, researchers need to figure out the replication mechanism of Ebola virus.

Ebola virus belongs to the Filoviridae family. In the past 10 years, scientists have gained some understanding of the complex structure of this virus, such as its ribonucleic acid (RNA) genome is wrapped by nucleoprotein (NP) to form a ribonucleoprotein complex (RNP), which further combines with polymerase protein (L), viral accessory protein (VP35), transcription activator protein (VP30) and nucleocapsid-associated protein (VP24) to form a helical nucleocapsid structure, which is surrounded by matrix protein (VP40) and further combines with the viral surface spike glycoprotein (GP) to form a complete virus particle.

Resolving the three-dimensional structure of the Ebola virus polymerase is a challenge faced by virologists around the world. After years of trial and error, Shi Yi and Gao Fu's team obtained the Ebola virus polymerase complex protein (L-VP35 complex) and used cryo-electron microscopy to resolve its high-resolution three-dimensional structure. They found that the Ebola virus polymerase L protein forms a stable complex with the VP35 protein tetramer to replicate and transcribe the viral genome.

Uncovering the “Dancing Together” Mechanism

Through in-depth observation, the research team gained a microscopic understanding of the dynamic conformational changes of the polymerase complex.

As the core of the virus's "replication machine", the polymerase involves multiple conformational changes in the process of generating progeny RNA, thereby promoting the smooth synthesis of the product. The most important conformational change is the transition from the starting state to the extension state.

Based on previous research, researchers captured the fine structure of the Ebola virus polymerase in an extended state by changing the sample preparation conditions of the cryo-electron microscope.

How does VP35 protein "dance" with L protein? Studies have found that during viral replication, VP35 protein acts as a "bridge" to connect L protein and RNP. "When L protein replicates the viral genome, it uses the spiral RNP as a template instead of the naked RNA. At this time, VP35 protein mainly performs the function of a molecular chaperone, mediating the replication of L protein in RNP units," said Shi Yi.

He explained that in addition to the oligomerization domain in the middle, the VP35 protein tetramer has four N-terminal (amino-terminal) domains and four C-terminal (carboxyl-terminal) domains at both ends.

One of the C-termini binds to the L protein, further stabilizing the binding of the L protein to the VP35 protein tetramer. At the same time, the other seven ends act like "octopus tentacles" to help the L protein slide on the RNP structure and bind to the RNP in the monomeric state, preventing it from non-specifically interacting with the host RNA and ensuring that the monomeric NP can be used for the generation of daughter RNPs.

"Without VP35 protein, L protein cannot carry out genome replication and transcription." Shi Yi said that if the combination of the two is blocked, the virus will not be able to replicate.

Guiding drug design and optimization

The development of small molecule drugs that can effectively inhibit the Ebola virus has always been an international hot topic, but also a difficult point.

Shi Yi said that understanding the molecular details of the interaction interface of the L-VP35 complex provides new targets and important guiding information for the further development of drugs targeting polymerases.

It is worth noting that the researchers pointed out that the N-terminal domain of the Ebola virus polymerase has an insertion domain unique to filamentous viruses, which is essential for its activity and can become a potential target for the development of antiviral drugs.

The research team also explored the molecular mechanism of the century-old drug suramin's anti-Ebola virus activity in vitro.

Suramin was first isolated by German chemist Paul Ehrlich in the early 20th century and has been widely used to treat parasitic diseases such as African sleeping sickness and onchocerciasis since the early 1920s. In recent years, scientists have discovered that suramin has anti-COVID-19 and cancer activity. Preliminary studies have suggested that suramin also has anti-Ebola virus activity, but its mechanism of action is not very clear.

Through in vitro enzyme activity and cell replicon experiments, the researchers found that suramin can effectively inhibit the activity of Ebola virus polymerase, and further used cryo-electron microscopy to analyze the complex structure of Ebola virus polymerase and suramin, revealing that suramin exerts its inhibitory effect by binding to the ribonucleotide triphosphate entry channel of the polymerase and blocking the substrate from entering the enzyme activity center. The molecular details of the interaction between suramin and L protein provide key reference information for further modification and optimization of suramin drugs.

A peer reviewer said that the structure of the Ebola virus L protein has been a recognized gap in the field, and this study provides important information that will help promote structure-based antiviral drug design.

Related paper information

https://doi.org/10.1038/s41586-022-05271-2

(Originally published in China Science Daily, 2022-09-30, Page 1)