Difference between revisions of "Part:BBa K3504000"

Latest revision as of 08:36, 21 October 2021

nSP1capping-Eastern equine encephalitis 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

Nsp1 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

The ∼60kDa alpha-virus nsP1 protein has two primary functions in the alpha-virus replication process. nsP1 contains the N-terminal domain which has Rossman-like methyletransferase (MTase) motifies which results in the capping reaction eventually. Following the N-terminal domain are tandem features that confer association of the nsP1 protein to host membranes. An amphipathic helix and palmitoylation both act to anchor the nsP1 protein, and nsP1-containing non-structural polyproteins, to the host membrane. The membrane association isn’t necessary for nsP1 enzymatic function but on the contrary this function requires lipids. The addition of the 5’ cap to the viral RNA genome and subgenome is clearly the role of nsP1 but that occurs after the triphosphatase activity of nsP2. Also this indicated that the nsP1 has MTase and GTase-like activities that made the capping of the positive-sense viral RNA possible. The GTase reaction is catalyzed by the nsP1 and this distinguishes it from the typical eukaryotic GTase reaction. To link to the GTP it requires S-adenosylmethionine and acid hydrolysis of nsP1-GMP yielded 7MeGMP and not GMP. This GT activity relays on a successful MTase activity to occur and in addition to the mutational evidence this lead to the conclusion that the MTase activity of nsP1 occurs prior to the transfer to the 5′ end of the substrate RNA where it is a complete contrast to the eukaryotic capping. So it was observed the the nsP1 mutants that do not have the GTaseor MTase activity are non-viable.

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.(1)

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.

Structural Characterization by AFCM-Egypt 2021

Our attention was directed towards using Non-structural proteins as they direct the replication of our vector into millions of copies, which further augments the rate of vaccine delivery into target cells. NSPs also play a major role in enhancing the stability, activity and regulation of vector expression. Four NSPs were chosen for our alphaviral vector as its replication is a delicate process, requiring specific protein-protein interactions among the NSPs and host factors.

Figure 5.Structural Characterization of NSP1

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. Sequence and Features