Part:BBa_K3504014

nSp2 Protease-Semliki forest virus

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

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.

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.

Improvements

Using information in literature we were able to increase the replicon cloning and functional ability by adding G110G, G763R Mutation to NSP2

Figure 3. Nsp2 G110G Amino acid mutation.

Figure 4. Nsp2 G763R 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 EcoRI site found at 487
  Illegal EcoRI site found at 2065
  Illegal PstI site found at 1099
  Illegal PstI site found at 2113
  
• 12
  INCOMPATIBLE 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
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 487
  Illegal EcoRI site found at 2065
  
• 23
  INCOMPATIBLE 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
  
• 25
  INCOMPATIBLE 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
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 836
  Illegal BsaI.rc site found at 2319