Part:BBa_K3504013
nSP1capping-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
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
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
- 10INCOMPATIBLE WITH RFC[10]Illegal PstI site found at 358
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 564
Illegal PstI site found at 358 - 21COMPATIBLE WITH RFC[21]
- 23INCOMPATIBLE WITH RFC[23]Illegal PstI site found at 358
- 25INCOMPATIBLE WITH RFC[25]Illegal PstI site found at 358
Illegal NgoMIV site found at 1551 - 1000COMPATIBLE WITH RFC[1000]
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