Part:BBa_K5142048

Click Virus

This part is a genetically engineered vaccinia virus (VACV) with modified A27L protein containing three 4-azido-L-phenylalanine residues close to its N-terminus. The azido groups serve as the sites for the click reaction of azido and dibenzocyclooctyne (DBCO) groups, which is the reason why this part is named “Click Virus”. Since A27L distributes on the surface of VACV and azido-DBCO click reaction can occur under physiological conditions without any catalyst, it is quite easy to link any customized DBCO-conjugated element onto the surface of Click Virus covalently via click reaction. Depending on the molecules linked, the modified Click Virus can be applied to a wide variety of biological and medical fields, such as targeting delivery of genes, oncolytic virus and vaccine carrier. In summary, Click Virus is a pluripotent chassis allowing rapid development of customized viral particles without further genetical engineering.

Source

This part is derived from Orthopoxvirus vaccinia Tian Tan (AF095689.1) by mutating 3 codons in A27L gene to amber suppressor codons (TAG) and translating them to azidophenylalanine using an artificial translation system.

Safety consideration

We chose to mutate A27L to reduce the biosafety risk of Click Virus in use. Since the amber suppressor mutations are close to the N terminus of A27L, premature termination of A27L translation will occur if the artificial translation system is not provided. Therefore, all the progeny of Click Virus after the first round of infection will be A27L deficiency virus which will be trapped in the target cells due to impaired ability of dissemination.

Usage

To link a customized element to Click Virus, the following experimental procedures are suggested.
1.Conjugate the customized element (CE) with DBCO by chemical synthesis. A flexible linker, such as a polyethylene glycol (PEG, MW 1000), is recommended to bridge the target element and DBCO to avoid unexpected interactions between them. Obtain CE-PEG-DBCO.
2.Add CE-PEG-DBCO directly to Click Virus (106 pfu/mL) kept in DMEM. A final CE-PEG-DBCO concentration of 10 μM is recommended in the initial trial. Incubate the mixture in room temperature for 1 h. CE-PEG-DBCO concentration and incubation time can be optimized according to the CE.
3.Test the CE-linked Click Virus or keep it in -80 °C for storage.
Please note that all the experiments involving the virus should be conducted in a BSL-2 facility.

Verification

We verified the reliability of Click Virus by linking it with folate-PEG-DBCO and testing its specificity in infecting to 293T cells overexpressing FOLR2 (folate receptor 2). In this experiment, viral infection was indicated by GFP expressed by the virus, and FOLR2-overexpressed cells were indicated by mCherry co-expressed with FOLR2 via T2A-mediated cleavage. As the result shown in Figure 1, the ratios of specific infection to FOLR2-overexpressed cells increase in a dose-dependent pattern according to the reactive concentration of folate-PEG-DBCO, reaching the highest ratio of 80% under 10 μM. This result prove the correct function of Click Virus.

Figure 1. fluorescence images of click virus (green) and cells overexpressing FOlR2(red) under different reactive concentrations of folate-DBCO. The yelLow regions in the merge figures indicate the FOLR2-overexpressed cells infected by click virus. The composition column shows the ratios of different colors in each treatment.

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Functional Parameters