Part:BBa_K4862000
NS3-HEL
Basic Part: BBa_K4862000 (NS3-HEL)
Profile
- Name: NS3-HEL
- Base Pairs: 1344 bp
- Origin: Dengue virus 2, Synthetic
- Properties: The helicase domain of NS3 (NS3 hel) participates in viral RNA replication and is essential for genome propagation.
Usage and Biology
The helicase domain of NS3 (NS3 hel), together with NS5, an RNA-dependent RNA polymerase, participates in viral RNA replication and it is essential for genome propagation. It has been demonstrated that the interaction between DENV NS3 hel and NS4B triggers the dissociation of the helicase from single-stranded RNA thereby modulating viral replication[1]. The enzymatic activities and role of NS3 proteins in viral replication and polyprotein processing have been studied for several members of the Flaviviridae family [2–4], but only a few studies have identified point mutations in NS3 modulating viral pathogenesis.
NS3 protein is one of the most highly conserved proteins in flaviviruses. This multifunctional protein has at least three different activities [5]. It has a serine protease domain that catalyzes the cleavage of several viral proteins, an RNA helicase domain, and an RNA triphosphatase domain, which promotes dephosphorylation of the 59UTR region during capping activities [6].
RNA helicase is responsible for almost all metabolism of RNA; in the Dengue virus, NS3 protein contains the activity of multiple enzymes, including helicase. Inserting the NS3 sequence is for expressing the protein we need and testing whether the medicine finally injected would be effective. The other structure of the pET28a plasmid would not be changed.
While these two plasmids are constructed, they would be transplanted into BL21 bacteria, which can express the NS3 protein we need. After growing for a few hours, the solution (these two plasmids and the liquid medium) would be ultrasonically crushed, destroying the bacteria's basic structure. The answer, consisting of the proteins and bacteria fragments, would be purified for extracting the NS3 proteins.
The NS3 proteins have the ability as a Helicase, which would activate the helicase to work. The protein would be added to a fluorescence DNA vector to observe whether the helicase works. If so, the fluorescence would disappear; if not, the fluorescence would be observed, which also means the medicine for restraining the replication of DNA is working. As NS3 proteins are the main protein active helicase in the Dengue virus, if the ability is restricted, the duplication of the RNA of the bacteria would be stopped, which means the spread of the virus would be halted.
References
- de Borba L, Strottmann DM, de Noronha L, Mason PW, Dos Santos CN. Synergistic interactions between the NS3(hel) and E proteins contribute to the virulence of dengue virus type 1. PLoS Negl Trop Dis. 2012;6(4):e1624. doi: 10.1371/journal.pntd.0001624. Epub 2012 Apr 17. PMID: 22530074; PMCID: PMC3328427.
- Kapoor M, Zhang L, Ramachandra M, Kusukawa J, Enber KE, et al. (1995). The association between NS3 and NS5 proteins of DENV2 in the putative RNA replicase is linked to differential phosphorylation of NS5. J Biol Chem 270:19100–19106.
- Westaway EG, Mackenzie JM, Khromykh AA (2003) Kunjin RNA replication and applications of Kunjin replicons. Adv Virus Res 59: 99–140.
- Umareddy I, Chao A, Sampath A, Gu F, Vasudevan SG (2006) Dengue virus NS4B interacts with NS3 and dissociates it from single-stranded RNA. J Gen Virol 87: 2605–2614.
- Assenberg R, Mastrangelo E, Walter TS, Verma A, Milani M, et al. (2009). Crystal structure of a novel conformational state of the flavivirus NS3 protein: implications for polyprotein processing and viral replication. J Virol 83: 12895–12906.
- Bollati M, Alvarez K, Assenberg R, Baronti C, Canard B, et al. (2010) Structure and functionality in flavivirus NS-proteins: Perspectives for drug design. Antiviral Res 87: 125–148.
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