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Bluetongue Virus Particles as Nanoreactors for Enzyme Delivery and Cancer Therapy.

ABSTRACT: The side effects of chemotherapy can be reduced by targeting tumor cells with an enzyme (or the corresponding gene) that converts a nontoxic prodrug into a toxic drug inside the tumor cells, also killing the surrounding tumor cells via the bystander effect. Viruses are the most efficient gene delivery vehicles because they have evolved to transfer their own nucleic acids into cells, but their efficiency must be balanced against the risks of infection, the immunogenicity of nucleic acids, and the potential for genomic integration. We therefore tested the effectiveness of genome-free virus-like particles (VLPs) for the delivery of Herpes simplex virus 1 thymidine kinase (HSV1-TK), the most common enzyme used in prodrug conversion therapy. HSV1-TK is typically delivered as a gene, but in the context of VLPs, it must be delivered as a protein. We constructed VLPs and smaller core-like particles (CLPs) based on Bluetongue virus, with HSV1-TK fused to the inner capsid protein VP3. TK-CLPs and TK-VLPs could be produced in large quantities in plants. The TK-VLPs killed human glioblastoma cells efficiently in the presence of ganciclovir, with an IC₅₀ value of 14.8 μM. Conversely, CLPs were ineffective because they remained trapped in the endosomal compartment, in common with many synthetic nanoparticles. VLPs are advantageous because they can escape from endosomes and therefore allow HSV1-TK to access the cytosolic adenosine triphosphate (ATP) required for the phosphorylation of ganciclovir. The VLP delivery strategy of TK protein therefore offers a promising new modality for the treatment of cancer with systemic prodrugs such as ganciclovir.

SUBMITTER: Thuenemann EC

PROVIDER: S-EPMC8086382 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:UnlabelledBluetongue virus (BTV), a member of the Orbivirus genus in the Reoviridae family, is a double-capsid insect-borne virus enclosing a genome of 10 double-stranded RNA segments. Like those of other members of the family, BTV virions are nonenveloped particles containing two architecturally complex capsids. The two proteins of the outer capsid, VP2 and VP5, are involved in BTV entry and in the delivery of the transcriptionally active core to the cell cytoplasm. Although the importance of the endocytic pathway in BTV entry has been reported, detailed analyses of entry and the role of each protein in virus trafficking have not been possible due to the lack of availability of a tagged virus. Here, for the first time, we report on the successful manipulation of a segmented genome of a nonenveloped capsid virus by the introduction of tags that were subsequently fluorescently visualized in infected cells. The genetically engineered fluorescent BTV particles were observed to enter live cells immediately after virus adsorption. Further, we showed the separation of VP2 from VP5 during virus entry and confirmed that while VP2 is shed from virions in early endosomes, virus particles still consisting of VP5 were trafficked sequentially from early to late endosomes. Since BTV infects both mammalian and insect cells, the generation of tagged viruses will allow visualization of the trafficking of BTV farther downstream in different host cells. In addition, the tagging technology has potential for transferable application to other nonenveloped complex viruses.ImportanceLive-virus trafficking in host cells has been highly informative on the interactions between virus and host cells. Although the insertion of fluorescent markers into viral genomes has made it possible to study the trafficking of enveloped viruses, the physical constraints of architecturally complex capsid viruses have imposed practical limitations. In this study, we have successfully genetically engineered the segmented RNA genome of bluetongue virus (BTV), a complex nonenveloped virus belonging to the Reoviridae family. The resulting fluorescent virus particles could be visualized in virus entry studies of both live and fixed cells. This is the first time a structurally complex capsid virus has been successfully genetically manipulated to generate virus particles that could be visualized in infected cells.

Project description:Bluetongue virus (BTV) and epizootic haemorrhagic disease virus (EHDV) are related orbiviruses, transmitted between their ruminant hosts primarily by certain haematophagous midge vectors (Culicoides spp.). The larger of the BTV outer-capsid proteins, 'VP2', can be cleaved by proteases (including trypsin or chymotrypsin), forming infectious subviral particles (ISVP) which have enhanced infectivity for adult Culicoides, or KC cells (a cell-line derived from C. sonorensis). We demonstrate that VP2 present on purified virus particles from 3 different BTV strains can also be cleaved by treatment with saliva from adult Culicoides. The saliva proteins from C. sonorensis (a competent BTV vector), cleaved BTV-VP2 more efficiently than those from C. nubeculosus (a less competent/non-vector species). Electrophoresis and mass spectrometry identified a trypsin-like protease in C. sonorensis saliva, which was significantly reduced or absent from C. nubeculosus saliva. Incubating purified BTV-1 with C. sonorensis saliva proteins also increased their infectivity for KC cells ?10 fold, while infectivity for BHK cells was reduced by 2-6 fold. Treatment of an 'eastern' strain of EHDV-2 with saliva proteins of either C. sonorensis or C. nubeculosus cleaved VP2, but a 'western' strain of EHDV-2 remained unmodified. These results indicate that temperature, strain of virus and protein composition of Culicoides saliva (particularly its protease content which is dependent upon vector species), can all play a significant role in the efficiency of VP2 cleavage, influencing virus infectivity. Saliva of several other arthropod species has previously been shown to increase transmission, infectivity and virulence of certain arboviruses, by modulating and/or suppressing the mammalian immune response. The findings presented here, however, demonstrate a novel mechanism by which proteases in Culicoides saliva can also directly modify the orbivirus particle structure, leading to increased infectivity specifically for Culicoides cells and, in turn, efficiency of transmission to the insect vector.

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Bluetongue Virus Particles as Nanoreactors for Enzyme Delivery and Cancer Therapy.

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