Design

Overview

In order to enhance the antiviral efficacy of NanoDisguiser, we have designed additional modifications based on actual needs, focusing on the engineering of MSP and the assembly of multivalent receptors, as larger nanodiscs and a greater number of receptor proteins are necessary for some specific viruses. We ultimately hope to perform functional validation of NanoDisguiser, using virus-like particles (VLPs) to test NanoDisguiser's ability to reduce viral infection of cells.

Part 1 Engineering Modification of MSP

MSP consists of a set of amphipathic α-helices derived from apolipoprotein A-1 (ApoA-1). In nanodiscs, engineered MSPs can stabilize target membrane proteins in nanoscale lipid bilayers.

In current methods, the diameter of nanodiscs is determined by the length of MSP. However, due to increased heterogeneity, generating large nanodiscs for membrane protein complexes is challenging^[1]. In our project, we hope to prepare large nanodiscs to carry multiple membrane proteins and better achieve the function of inducing viral membrane fusion. Therefore, we aim to design multi-polymerized MSP to create larger NanoDisguisers.

1.1 Mono-MSP Self-Circularization — SpyCatcher-SpyTag Technology

SpyCatcher-SpyTag is a commonly used specific covalent bonding system in protein engineering. The cysteine residue's thiol group (-SH) on the SpyTag linker can undergo a Michael addition reaction with a specific site on the SpyCatcher linker, forming a stable thioether bond and achieving highly specific binding between SpyCatcher and SpyTag.

We selected different sizes of MSPs, specifically MSP1D1, NW15, and NW50, and fused SpyCatcher and SpyTag to the N- and C-terminus of the MSPs, respectively, forming spMSP. By utilizing the specific covalent binding of the SpyCatcher and SpyTag linkers, we aim to prepare nanodiscs of different diameters^[1].

1.2 MSP Multi-Polymerization — Various Linkers and mCherry-Circularized Large Nanodiscs

However, self-circularized Mono-MSPs encounter numerous challenges in production: The only feasible way to produce MSPs of varying lengths is by adjusting the length of the DNA reading frame. Furthermore, when MSP proteins are too short, they are prone to forming inclusion bodies or being degraded, thus complicating protein purification. Conversely, when MSP proteins are too long, the production strain experiences excessive stress, making it difficult to achieve high-level protein expression. Due to these reasons, MSP production often suffers from issues such as high sequence repetitiveness, regulatory difficulties, and low production efficiency, placing the production of large antiviral nanodiscs in a predicament.

Therefore, we proposed the concept of multi-polymerized MSPs. Similar to the self-circularization of Mono-MSPs using SpyCatcher-SpyTag technology, we aim to create larger diameter circularized MSPs by linking multiple linkers end-to-end, thereby preparing larger nanodiscs.

We selected three linkers (Spy/Sdy/Snoop) and integrated them into the N- terminus and C-terminus of MSP in a certain order, connecting them through the formation of covalent bonds. We used mCherry[1-10] and [11] parts to emit fluorescence at the final linker connection to verify the success of multi-polymerized MSP circularization. The fluorescence intensity also indicates the relative amount of successful multi-polymerized MSP circularization. As for MSP, we have chosen NW15, which is easy to produce. Furthermore, through modeling, we found that when mCherry and Tag/Catcher components are directly spliced together, cross-linking interference may occur between two systems. Therefore, we have added flexible GS linkers to achieve the connections involved in the process.

1.3 Regulating the production of different types of MSPs in the same engineered strain.

In order to simplify the complexity of pathway design, improve production efficiency and realize artificial control of the production of self-cyclization Mono-MSP and multi-polymerized MSP of different lengths in the same engineered bacteria, we further optimized the pathway. Below we will use dimeric MSP as an example to explain how our pathway works.

By adding homology arms H1 and H2 to both sides of the basal pathway, the basal pathway was homologous recombined into the genome of E. coli using the λ-Red homologous recombination system. The λ-Red recombination system enables the manipulation of target genes by linear fragment homologous recombination, and has been widely used in gene knockout, gene replacement, and addition of genomic gene tag sequences in Escherichia coli. This system is derived from λphage, which is mainly encoded by three genes, exo, beta, and gam. Exo protein functions as a 5 '-3' exonuclease, degrading DNA from the 5 'end to the 3' end of the DNA duplex. Beta is a single-stranded DNA-binding protein that mediates single-stranded DNA annealing. Gam protein binds to RecBCD exonuclease and inhibits its degradation of foreign DNA ^[2].

First, cells were transfected with plasmid pKD46 carrying three genes, exo, beta and gam, which are controlled by the L-arabinose promoter and can be induced to express Exo, Beta and Gam. pKD46 carried a temperature-sensitive replicon, which was gradually lost when incubated at 37℃.

Then the plasmid pMAK705 was used as a template to amplify the pathway in Figure 4, and the obtained fragment was introduced into Escherichia coli transformed with pKD46. Under the action of recombinase (Exo, Beta and Gam), homologous recombination occurred, resulting in homologous recombination of the pathway into Escherichia coli. Then, using the thermosensitive characteristics of pKD46 and pMAK705, the introduced pKD46 and pMAK705 were eliminated by increasing the temperature, and the recombinant strains were screened by kanamycin resistance.

Expression produces cyclized MSP when promoter 2 transcription is regulated and dimeric MSP when promoter 1 transcription is regulated. By increasing the number of promoter-Spy-Tag-MSP repeats in the basal pathway using different promoters, we can achieve more diverse regulation of MSP production in the same engineering strain, which provides a new strategy for more efficient manufacturing of large nanodiscs.

Part 2 Membrane Protein Polymerization

Vesicular stomatitis virus (VSV) is an enveloped, negative-strand RNA virus that belongs to the Vesiculovirus genus of the Rhabdovirus family. Low-density lipoprotein receptor (LDL-R) serves as a major entry receptor for VSV. The viral particles interact with CR2 and CR3 on LDL-R through the G protein, thereby mediating the fusion of the virus with the host cell membrane^[4].

Based on the key process of VSV entry into cells mediated by VSV-G, we selected the receptor LDL-R as the key protein to be inserted into the nanodisks.

To assemble LDL-R in the correct orientation and quantity on the nanodiscs, we utilized the split-GFP dual-component technology (sGFP) for receptor coupling. sGFP consists of a large GFP[1-10] β-strand and a small GFP[11] β-strand, neither of which fluoresce independently. When the two separate fragments come into close proximity, they rapidly self-assemble to form a complete GFP structure, restoring fluorescence. Based on this, we fused the C-terminus of the receptors with split-GFP, designing LDL-R – GFP[1-10] and LDL-R – GFP[11] fusion proteins.

After expressing and purifying the two fusion proteins, the split-GFP fragments reassemble, linking the two receptors together. The assembled sGFP is highly stable, so we later use it as a specific purification handle for complex purification. The split-GFP simultaneously anchors the C-terminus of the proteins, ensuring that when the complex is inserted into the nanodiscs, the N-termini of both receptors are on the same side of the nanodiscs, enforcing parallel orientation and ensuring a 1:1 ratio of the inserted receptors.

To minimize any impact on the function of the membrane proteins already assembled into the nanodiscs, we inserted a 3C protease cleavage site between the receptor proteins and sGFP^[3], allowing for the removal of sGFP after nanodisc assembly.

After one iteration and inspired by BNU-China 2023, we ultimately decided to express sGFP fused with monomeric streptavidin (mSA) as the sGFP linker. By leveraging the interaction between streptavidin and biotin, we can link sGFP to biotinylated membrane proteins. The pathway also includes a His tag, which we can use to purify and isolate recombinant proteins via metal ion affinity chromatography.

Part 3 Functional Verification: Novel SEND System

We designed a clever assay to verify that NanoDisguiser can exert antiviral effects in vitro while avoiding the safety risks associated with using viruses or pseudoviruses. We used a virus-like particle carrying GFP mRNA to simulate the virus, and the expression level of green fluorescent protein in cells can reflect the destructive effect of the nanodisc on the virus.

To build a VLP simulating membrane fusion virus, we modified the SEND system constructed by Zhang Feng's team^[6]. Our improved SEND system is composed of PEG10 protein, vesicular stomatitis virus envelope protein VSVg and GFP mRNA. PEG10 is an LTR retrotransposon-derived protein, which can form VLP capsid, bind and transfer RNA. The transportation of GFP mRNA depends on the packaging of PEG10 UTR. We connected PEG10 5'UTR and PEG10 3'UTR upstream and downstream of GFP mRNA respectively to achieve mRNA transport by PEG10 VLP (this plasmid is named cargoRNA). The exosomes formed by VSVg protein have a membrane fusion mechanism similar to that of vesicular stomatitis virus, and can be loaded with PEG10 VLP and transfected into cells.

We co-transfected the three plasmids, capsid, cargoRNA, and fusogen, into HEK293FT cells to collect exosomes that mimic the virus. Then the HEK293T cells are infected with these exosomes, and GFP mRNA is injected into the cells, where the expression of green fluorescent protein indicates that the cells have been infected. When NanoDisguiser is added together with the exosome-mediated action on 293T cells, the diminished or disappearance of the fluorescence signal can be detected to verify the blockage of virus invasion by NanoDisguiser.

References

[1] Zhang S, Ren Q, Novick SJ, Strutzenberg TS, Griffin PR, Bao H. One-step construction of circularized nanodiscs using SpyCatcher-SpyTag. Nature Communications. 2021;12(1):5451. doi: 10.1038/s41467-021-25737-7.

[2] ZHANG Shu Ya, LI Duan You, LIU Ying Li, HE Tian Tian, NIE Pin, XIE Hai Xia. EPITOPE TAGGING OF CHROMOSOMAL GENE IN EDWARDSIELLA PISCICIDA[J]. ACTA HYDROBIOLOGICA SINICA, 2023, 47(6): 895-902. DOI: 10.7541/2023.2022.0461

[3] CHAO SY, HU XJ. Application of gene editing technology in Escherichia coli. Chinese Journal of Biotechnology, 2022, 38(4): 1446-1461.

[4] Nikolic J, Belot L, Raux H, Legrand P, Gaudin Y, A Albertini A. Structural basis for the recognition of LDL-receptor family members by VSV glycoprotein. Nat Commun. 2018 Mar 12;9(1):1029. doi: 10.1038/s41467-018-03432-4. PMID: 29531262; PMCID: PMC5847621.

[5] Bruguera ES, Mahoney JP, Weis WI. Reconstitution of purified membrane protein dimers in lipid nanodiscs with defined stoichiometry and orientation using a split GFP tether. J Biol Chem. 2022;298(4):101628. Epub 2022/01/26. doi: 10.1016/j.jbc.2022.101628; PMID: 35074428; PMCID: PMC8980801.

[6] Segel, M., et al., Mammalian retrovirus-like protein PEG10 packages its own mRNA and can be pseudotyped for mRNA delivery. Science, 2021. 373(6557): p. 882-889.