Part:BBa_K3504002

nSP3-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

Nsp3 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

∼60kDa nsP3 : The functional role of the nsP3 hasn’t been clear throughout history. It still has a primary part in the RNA synthesis as mutations in nsP3 exhibited defects in the start of minus-strand synthesis or subgenomic RNA synthesis. The alphavirus nsP3 protein has three recognized domains: the macrodomain, the alphavirus unique domain (AUD) and the hypervariable region. The macrodomains of CHIKV and VEEV were found to bind to DNA, RNA and polyADP-ribose in addition to exhibiting adenosine diphosphoribose 1′-phosphate phosphatase activity. Genetic manipulations within the AUD have resulted in defects in minus-strand and subgenomic RNA synthesis, polyprotein processing, and neurovirulence where the exact mechanism is not identified up till now. The C-terminal domain of nsP3 is characterized as being hypervariable and this hypervariable domain is shown to be responsible for the formation of virus-species specific complexes in infected cells. At this time it is not clear that the identified interactions of nsP3 with host factors influence viral RNA synthesis or whether they are indicative of another nsP3 function that regulates the host cell environment.

Characterization

Figure 1.in vitro replicon enhancement method: Using transfected Jurkat cells with replicon RNA which was encoded for mCherry and grown in cell culture under the SGP. Then during serial passage as shown in the flow cytometry histograms, the top 20 percent of mCherry were sorted around every 10 days. this has resulted that cells expressing higher levels of repoter gene were enriched. finally the 5th sort cells were seperated from the rest for replicon sequencing.(1)

Figure 2.Mutation identification:Positive mCherry cells was reverse transcribed to cDNA,after that Nsp1 to 4 and the SGP were magnified by seven pairs of primers and amplicons and later on engineered into DNA of the plasmid and changed into E.coli to be amplified. The lower left part schematic illustrates roughly the locations of point mutations in the 5th sort in NSP2 & NSP3.(1)

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 3. 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.(2)

Figure 4. 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 5. 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 6. 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.

Improvements

Using information in literature we were able to increase the replicon cloning and functional ability by adding K94E, S243G,E255D,V305M Mutation to NSP3

Figure 1. Nsp3 K94E Amino acid mutation.

Figure 2. Nsp3 S243G Amino acid mutation.

Figure 3. Nsp3 E255D Amino acid mutation.

Figure 4. Nsp3 V305M Amino acid mutation.

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 1.Structural Characterization of NSP3

References

1-Li, Y., Teague, B., Zhang, Y., Su, Z., Porter, E., Dobosh, B., ... & Weiss, R. (2019). In vitro evolution of enhanced RNA replicons for immunotherapy. Scientific reports, 9(1), 1-10.

2-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), 349-354.1-10.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal PstI site found at 426
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 1297
  Illegal PstI site found at 426
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal XhoI site found at 1461
  Illegal XhoI site found at 1522
  Illegal XhoI site found at 1563
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal PstI site found at 426
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal PstI site found at 426
  Illegal AgeI site found at 1002
  Illegal AgeI site found at 1371
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 1560
  Illegal BsaI site found at 1578
  Illegal BsaI.rc site found at 1087