Background
In 1918, the Spanish flu outbreak claimed at least 50 million lives worldwide. In 2002, SARS emerged, with 8,096 cases reported across 29 countries and regions by 2004, resulting in 774 deaths and a mortality rate of 9.6%. In December 2019, COVID-19 erupted globally, infecting over 760 million people and resulting in 6.9 million deaths, though the actual numbers are believed to be higher. Emerging viruses have continually appeared throughout human history, cumulatively causing millions of deaths. Currently, climate change and globalization have created more favorable conditions for virus transmission, suggesting that future outbreaks of emerging viruses may occur more frequently.
Therefore, the development of antiviral drugs to address future sudden outbreaks and the challenges posed by viral mutations has become a key focus for researchers and industry professionals.
Currently, although there are various types of antiviral medications available, they face challenges such as drug resistance, side effects, and rapid viral mutations leading to drug failure. For instance, neuraminidase inhibitors like Oseltamivir (Tamiflu) inhibit the viral neuraminidase to prevent replication and spread. However, the high variability of influenza viruses may lead to drug ineffectiveness, and their efficacy against certain strains may be limited[2]. Possible side effects include nausea, vomiting, headaches, and psychiatric symptoms, such as hallucinations and abnormal behavior[3].
In response to the rapid mutation of viruses, many broad-spectrum antiviral drugs are under development. These include nucleoside analogs, protease inhibitors, peptide inhibitors, small molecules, and virus fusion inhibitors, designed based on conserved aspects of the viral life cycle and highly conserved targets shared among different viruses[4]. While these drugs can act on multiple viruses or various strains of the same virus, they still present risks of resistance and side effects. For example, Lamivudine (LAM), one of the first approved antiviral drugs for hepatitis B, has seen increasing resistance due to the virus's propensity for genetic mutations[5]. Some patients undergoing HIV treatment with protease inhibitors have also experienced abnormal fat distribution[6].
Can we develop antiviral drugs with low resistance and minimal side effects to tackle the rapid mutations of viruses?
Viruses within the same genus often share several highly conserved epitopes. For enveloped viruses, entry into host cells requires the fusion of the viral membrane with the cellular membrane. This process begins with virus-receptor recognition, followed by membrane fusion or endocytosis. Viral surface proteins (SP), which include receptor-binding subunits, membrane fusion subunits, cellular receptors, and host cell proteases necessary for SP cleavage, are common antiviral targets[7]. For instance, HIV subtypes bind to specific receptors or co-receptors on CD4+ T cells via the viral envelope protein (Env), initiating membrane fusion and uncoating to facilitate infection[8].
Figure 1: Membrane fusion mechanism of enveloped viruses, taking HIV as example[9]
Our objective is to develop broad-spectrum antiviral drugs that target the viral membrane fusion process, a critical step for enveloped viruses. These drugs are designed to target conserved sites involved in membrane fusion process of multiple viral subtypes, which reduces the likelihood of resistance developing[10]. Such drugs could potentially serve as a crucial first line of defense against future viral pandemics.
NanoDisguiser: Immune nanodisc fighting against the virus
This year, the iGEMers from Beijing Normal University have turned their attention to a promising structural biology tool—nanodiscs. Nanodiscs are disc-shaped phospholipid bilayers self-assembled from phospholipids and membrane scaffold proteins (MSP), playing an important role in membrane protein purification and research, drug delivery, and other areas[11]. We have modified them to have the ability to resist infectious viruses. Our goal is to develop a new drug with lower immunogenicity and resistance, providing new ideas for the treatment of infectious viruses. We hope our attempt can offer new solutions for the prevention and control of more infectious diseases.
Figure 2: Schematic diagram of the nanodisc structure
Some viruses infect cells through membrane fusion and uncoating process. For example, HIV infects cells by binding the viral surface Env protein to specific receptors or coreceptors on CD4+T cells, initiating the membrane fusion and uncoating process[12]. Through literature review, we found that nanodiscs modified with the poliovirus receptor (PVR) can mediate the fusion of the poliovirus membrane and the removal of capsid proteins, releasing RNA and thereby losing the ability to infect other cells[13]. Similar methods have also been successfully applied to influenza viruses and others[14]. This approach has inspired our project, where we aim to modify virus receptors on nanodiscs to induce membrane fusion, thereby rendering the virus incapable of infecting other cells.
Figure 3: The process of nanodisc-mediated poliovirus membrane fusion[13]
Introduce NanoDisguiser! We designed a nanodisc capable of mimicking virus host cells in the human body. In the nanodisc, the phospholipid bilayer encapsulating viral receptor proteins is surrounded and stabilized by two MSP molecules in a belt-like manner to maintain the disc-shaped structure of the nanodisc.
Figure 4: Structure of NanoDisguiser
We anticipate that the viral receptor proteins on the surface of NanoDisguiser can bind to the surface proteins of virus, thereby triggering the irreversible uncoating process of virus and rendering it non-infectious to other cells. We hope our attempt will make more researchers aware of the immense potential of nanodiscs, enabling them to play a larger role in the field of infectious disease prevention and control in the future.
Make NanoDisguiser more efficient
We further explored the design of synthetic biology and protein engineering to enhance the antiviral capability of NanoDisguiser.
We aimed for NanoDisguiser to precisely accommodate host cell receptor and the corresponding coreceptors, thus leveraging the split-GFP (sGFP) two-component technology to construct membrane protein dimers in nanodiscs[15]. We designed sGFP1-10 tether and sGFP11 tether fusion proteins. The sGFP fragments self-assemble to form GFP, which can serve as a specific purification handle, while the interaction between biotin and streptavidin is used to link membrane proteins. Meanwhile, the use of sGFP allows controlling the stoichiometry and orientation of membrane proteins, with the sGFP part anchoring to one side of the protein, forcing parallel orientation when the complex inserts into the nanodisc and ensuring a 1:1 ratio of the two proteins.
Figure 5: Construction of membrane protein dimers by split-GFP two-component technology
We made extensive designs in the circularization of MSP to further engineer NanoDisguiser. Inspired by the de novo design of protein logic gates and MSP's implementation of self-circularization through SpyCatcher-Spytag system, we hope to use protein linkers to connect MSP end-to-end to realize controlled multi-polymerization. Our concept provides a new method for efficient regulation of nanodisc area, aiming to promote the engineering production of large nanodiscs.
Our optimization of MSP circularization involves several aspects:
- We selected different protein linkers similar to SpyCatcher-SpyTag to avoid intramolecular circularization, accurately achieving 100% multi-polymerization and improving yield.
- The inspiration for controlled circularization comes from the "AND gate" in logic gates, where only when all proteins are expressed will the corresponding size MSP be output.
- One of our linkers forms a fusion protein with split-mCherry , which can serve as a marker for successful circularization.
- We plan to use the SpyCatcher-SpyTag protein tags along with multiple promoters to control the expression of different types of MSP in the same engineered bacterium.
Figure 6: Controlled multi-polymerization of MSP
Additionally, we supposed that the NanoDisguiser circularized with split-mCherry may have other magical functions—monitoring virus binding. When NanoDisguiser interacts with other proteins, the curvature of the disc changes, probably altering the binding of split-mCherry on MSP proteins, and hopefully causing detectable changes in fluorescence intensity[16].
Figure 7: Split-mCherry cyclized nanodisc
References
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