Difference between revisions of "Part:BBa K3504016"

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nSP4-RNA-directed RNA Polymerase-Semliki forest virus

NOTICE: Parts in our range for this season have been created as a part of our Phase I design of our project. These parts HAVE NOT been tested or characterized in the lab due to COVID-19-related precautionary measures. We have enriched our new parts pages with data from literature and results from our modeling and simulations. If you are intending on using this part or others in our range, please keep in mind these limitations and update these parts with data from your experimentation. Feel free to reach us at: igem.afcm@gmail.com for further inquiries.

Part Description

Nsp4 is one of four non structural proteins that together forms the main complex responsible for the synthesis positive-sense viral RNAs, results in the synthesis of both the genomic and subgenomic RNAs, of which the subgenomic RNA is produced in excess of the viral genome. Which allows the virus to self-replicate into millions of copies of the virus.

Usage

∼70kDa nsP4 protein : All the previous nonstructural proteins shared work in the synthesis of the RNA but the nsP4 is especially responsible for the RNA synthetic properties of the viral replicase complex. The nsP4 protein contains the core RdRp domain and motifs. Many alphavirus species produce significantly less nsP4 than the other non-structural proteins. N-terminal truncation mutants of nsP4 have demonstrated terminal adenylyltransferase (TATase) activity, indicating a potential role in polyadenylation, but were ultimately found to lack de-novo copying activity.Full-length recombinant nsP4 exhibited TATase activity and was capable of de novo RNA synthetic activity only after the addition of the other viral non-structural proteins supplied from mammalian cell membrane fractions. Despite nsP4 being the sole viral protein with RdRp activity, viral replication is the sum of coordinated non-structural protein activity. Some mutational analyses have indicated that determinants for RNA synthesis exist within nsP4. Genetic evidence suggests nsP4 has absolutely conserved N-terminal tyrosine interacts with nsP1 for minus-strand synthesis. Stollar and colleagues have determined several determinants of promoter binding in nsP4 as residues of nsP4 were found to contact the subgenomic and genomic promoters as determined by RNA cross-linking experiments.

Characterization

We have made simulations using mathematical modelling techniques to characterize the increase in expression when using replicons over traditional methods while also providing simulations that Characterize the function of replicon by eliciting an increased response in both Dendritic Cell population and T-Helper Population.

We also provide Functional characterization of replicons from literature. As This figure shows HIVA-specific T-cell responses after a single immunization with clinical-grade plasmid DNA vaccines between DREP.HIVA and pTHr.HIVA in individual mice immunized by 10 μg of them all of which complies with our mathematical modelling & simulations

Figure 1. Functional characterization of replicons from literature. This figure shows HIVA-specific T-cell responses after a single immunization with clinical-grade plasmid DNA vaccines between DREP.HIVA and pTHr.HIVA in individual mice immunized by 10 μg of them.

Figure 2. Mathematical modelling simulation of Number of positive strand RNA in traditional vaccination presented by the graph to the left vs with the use of self amplifying replicon on the right.

Figure 3. Mathematical modelling simulation of T-helper cells population response according to logfc in response to DREP vaccine on the left vs traditional DNA vaccine on the right.

Figure 4. Mathematical modelling simulation of Dendritic Cells population response according to logfc in response to DREP vaccine on the left vs traditional DNA vaccine on the right.

References

1-Nordström, E. K., Forsell, M. N., Barnfield, C., Bonin, E., Hanke, T., Sundström, M., ... & Liljeström, P. (2005). Enhanced immunogenicity using an alphavirus replicon DNA vaccine against human immunodeficiency virus type 1. Journal of general virology, 86(2), 349-354.

2-Innate Immune System. (n.d.). Retrieved October 26, 2020, from https://www.sciencedirect.com/topics/immunology-and-microbiology/innate-immune-system

3-Rupp, J., Sokoloski, K., Gebhart, N., & Hardy, R. (2015, September). Alphavirus RNA synthesis and non-structural protein functions. Retrieved October 26, 2020, from https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4635493/

Sequence and Features Sequence and Features