Part:BBa_K3504002

nSP3-Eastern equine encephalitis virus

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.Method for in vitro replicon evolution: Jurkat cells were transfected with replicon RNA encoding mCherry under the SGP and grown in cell culture. The top 20% of the mCherry+ population were sorted for approximately every 10 days during serial passaging as indicated by the flow cytometry histograms, leading to an enrichment in cells expressing high levels of the reporter gene. Cells from the 5th sort were isolated for replicon sequencing.

Figure 2.Identification of mutations: Total RNA from mCherry positive cells was extracted and reverse transcribed to cDNA. Then, nsP1–4 and the subgenomic promoter were amplified by seven pairs of specific primers and amplicons from Loci 1–7 were engineered into plasmid DNA and transformed into E. coli for amplication. Six clones from each locus were randomly picked for Sanger Sequencing. Schematic at bottom left shows the approximate locations in nsP2 and nsP3 where point mutations were identified in 5 mutant alleles with c2 harboring two linked mutations.

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.

References

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.

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