Background

With the development of synthetic biology, engineered viruses have been illuminating their adaptability and versatility with a diverse spectrum of applications, such as gene transfer methods, genetic engineering, immunotherapy and drug delivery systems. Additionally, virus-based nanoparticles have emerged as a beacon of vaccine innovation, gastric therapeutics, greatly broadening the scope of viral engineering applications [1]. Nevertheless, compared to other microbial chassis, the application of viruses is significantly limited by the lack of convenient and safe techniques for viral engineering, mostly due to the high diversity and obligatory parasitism of viruses.

Vaccinia virus (VACV) is a commonly used chassis virus which can be engineered genetically to contain and express foreign DNA without impairing the ability of the virus to replicate [2]. With such foreign DNA, recombinant VACV can be loaded with antigen or ligand molecules on its surface, to induce protection against infectious agents or achieve directed delivery to specific targets. However, loading molecules to VACV surface via genetic engineering requires special expertise and facilities which can be hardly fulfilled in most situations, hindering the application of engineered VACV.

Aim

We, Team HunanU, are going to develop Click Virus, an engineered VACV that can be easily ligated with any customized molecules on its surface via click reactions, to provide an easy-to-use viral chassis for various applications.

Principles and methods

The general approach of Click Virus is to introduce azido groups as bioorthogonal sites onto the membrane proteins of VACV, which allows covalent ligation of molecules via alkyne or Dibenzocyclooctyne (DBCO) click reactions (Figure 1). Since no natural amino acid contains azido groups, the introduction of azido groups to the VACV protein will be achieved by artificial translational mechanism.

Briefly, we start with genetically modifying the coding region of VACV protein A27L in which we replace the codons encoding phenyalanines with the stop codon UAG via virus-specific DNA recombination. Then, 4-azido-L-phenylalanine and its artificial tRNA holding the anticodon of CUA are added when the modified VACV is packed, so that the virus produced will carry mutant A27L with 4-azido-L-phenylalanines. Finally, any customized molecules, as long as conjugated with alkyne or DBCO, can be ligated to the mutant A27L via click reactions, generating modified VACV for specific applications [3]. In this process, any genetically modified VACV not translating 4-azido-L-phenylalanine will carry truncated A27L that will impair the remote transmission of the virus, which improves the quality and safety of the engineered viruses.

Perspectives

Click Virus provides a handy and powerful tool for the application of VACV in a variety of fields, including basic research and medical practice. With Click Virus, one can easily prepare customized VACV particles without any genetic engineering, which significantly lower the threshold for the use of engineered VACV.

For instance, when conjugated with tumor-specific ligands, Click Virus can act as an oncolytic virus specifically targeting tumor cells. In this project, we are planning to add folate to Click Virus to verify its ability to target cancer cells highly expressing folate receptor.

Click Virus can be also used to develop enhanced vaccines for various pathogens. Once loaded with vaccine peptides and introduced into the body, the Click Virus can stimulate both B-cell and T-cell immunity targeting the vaccine epitope, which may enhance the protection of the vaccine. Especially, the progeny viruses lacking functional A27L will bear low infectivity, which will be safer than classic virus-carrying vaccines.

Additionally, by clicking on specific fluorescent molecules to Click Virus, Single-Virus Tracking (SVT) technology can be applied for virological research. SVT technology enables quantitative analysis of the virus's entry, transport, fusion, genome release, and assembly and release processes, allowing scientists to track the behavior of individual viruses within host cells in real time. It directly observes the interactions between the virus and cellular receptors and the utilization of cellular structures by the virus, providing a powerful tool for understanding the mechanisms of viral infection, evaluating the effectiveness of antiviral drugs, and developing new therapeutic strategies [4].

References

• [1] Wen, AM and Steinmetz, NF. Design of virus-based nanomaterials for medicine, biotechnology, and energy. Chem Soc Rev. 2016;45(15): 4074–4126.
• [2] Chi, H, Wang, X, et al. Engineering and modification of microbial chassis for systems and synthetic biology. Synth Syst Biotechnol. 2018;4(1):25-33.
• [3] Pokorski, JK and Steinmetz NF. The Art of Engineering Viral Nanoparticles. Mol Pharm. 2011;8(1):29-43.
• [4] Liu, SL, Wang, ZG, et al. Single-Virus Tracking: From Imaging Methodologies to Virological Applications. Chem Rev. 2020;120(3):1936-1979.