Part:BBa_K3504014
nSp2 Protease-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
Nsp2 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 ∼90kDa alpha-virus nsP2 protein also have several functions in viral infection.nsP2 was initially described as consisting of two domains, an N-terminal helicase domain, which also exhibits nucleoside triphosphatase (NTPase) activity and a C-terminal protease domain. The first putative N-terminal domain has exhibited cofactor-like properties regarding the activity of the nsP2 protease domain. The second putative domain appears to function in promoter selection, as this domain has been the site of suppressor-mutations developed in response to promoter site mutations. In the context of viral replication, nsP2 exhibits three important functions, acting as a helicase, a triphosphatase and a protease. In addition to these roles, nsP2 is intimately involved in the shutoff of host macro-molecular synthesis.nsP2 functions as a helicase to unwind RNA secondary structures formed during viral RNA replication. The helicase activity of nsP2 is dependent on the NTPase activity of the N-terminal domain, as mutations in the Walker A motif ablated helicase activity in both recombinant and tissue culture models.Recent data has indicated that the helicase activity of nsP2 likely acts in coordination with the polymerase activity of nsP4 and is dependent on the full-length protein, and that a severable helicase domain is not present.Collectively, these data indicate that the helicase activity of nsP2 is essential for viability, presumably due to its function during viral replication The RTPase activity of nsP2 is responsible for the removal of the γ-phosphate from the 5′ end of nascent positive-sense RNAs to yield a diphosphate moietyat the 5′ terminus, enabling the RNA to act as a substrate for the nsP1-mediated capping reaction.The C-terminal domain of nsP2 was genetically identified as the protease responsible for the processing of the non-structural polyprotein nsP2 may also perform functions in RNA synthesis beyond its roles as protease, RTPase, and helicase. It has been proposed that nsP2 acts as a transcription factor for subgenome synthesis by binding to the subgenomic promoter.
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.
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
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 487
Illegal EcoRI site found at 2065
Illegal PstI site found at 1099
Illegal PstI site found at 2113 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 487
Illegal EcoRI site found at 2065
Illegal NheI site found at 786
Illegal PstI site found at 1099
Illegal PstI site found at 2113 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 487
Illegal EcoRI site found at 2065 - 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 487
Illegal EcoRI site found at 2065
Illegal PstI site found at 1099
Illegal PstI site found at 2113 - 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 487
Illegal EcoRI site found at 2065
Illegal PstI site found at 1099
Illegal PstI site found at 2113
Illegal NgoMIV site found at 169
Illegal NgoMIV site found at 1279
Illegal NgoMIV site found at 2016
Illegal NgoMIV site found at 2026 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 836
Illegal BsaI.rc site found at 2319
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