Abstract
Vaccinia virus is capable of replicating in a wide range of vertebrate animal cells. However, deletion of the two virus host range genes, E3L and K3L, would render replication of the virus abortive in all the cell types tested, except the cells with defective PKR and RNase L activity. By expressing different poxvirus K3 ortholog proteins in the E3L and K3L double knockout vaccinia virus, we can construct a mutant vaccinia virus with modified host range. Here, using poxvirus K3 ortholog as a positive selection marker, we developed a proof-of-concept protocol to construct recombinant vaccinia viruses expressing foreign proteins. Such a recombinant virus has a modified host range in comparison to wild-type vaccinia virus. This protocol offers the following advantages:➢Cheap: no additional selection reagent is required.➢Highly efficient: nearly all recombinant virus plaques picked would be the stable recombinant virus expressing the protein of interest.➢Rapid: starting from transfection with the recombinant DNA vector, a purified recombinant virus expressing the protein of interest could be obtained within a week.
Highlights
We used the construction of the recombinant VACV expressing a highly immunogenic human herpesvirus 8 (HHV8) glycoprotein K8.1 (Genbank accession number GU233125) as an example for validating the method
The tagged HHV8 K8.1 gene was cloned under the control of the synthetic early/late promoter as shown in the Fig. 1. 1 μg of the HHV8 K8.1 shuttle vector was transfected into A549/PKR+RNaseL ko cells, which had been infected with the parental virus VACV E3L K3L at a m.o.i. of 0.1. 24 h post transfection/infection, the virus was collected and a 5-fold serial dilution of the virus was added to BHK21 cells in a 12-well tissue culture plate
Following 1 h incubation, the virus inoculum was removed, cells were washed once with PBS and overlaid with MEM containing 2% fetal calf serum (FCS), 1x of penicillin-streptomycinglutamine, and 1% Low melting point (LMP). 48 h post infection, the infected cells were examined under a fluorescence microscope and the plaques expressing only green fluorescent protein were marked, picked with a glass Pasteur pipette and transferred to a 1.5 ml screw-capped sterile tube
Summary
By expressing different poxvirus K3 ortholog proteins in the E3L and K3L double knockout vaccinia virus, we can construct a mutant vaccinia virus with modified host range. Using poxvirus K3 ortholog as a positive selection marker, we developed a proof-of-concept protocol to construct recombinant vaccinia viruses expressing foreign proteins. Such a recombinant virus has a modified host range in comparison to wild-type vaccinia virus. Method name: Novel method making recombinant vaccinia viruses Keywords: Poxvirus, Vaccinia, Recombination, Host range, k3L Article history: Received 23 January 2020; Accepted 5 May 2020; Available online 15 May 2020. Subject area: More specific subject area: Method name: Name and reference of original methods: Resources availability: Immunology and Microbiology Virology/poxviruses/vaccinia Novel method making recombinant vaccinia viruses N/A N/A
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