Difference between revisions of "Part:BBa K404011"

Revision as of 16:47, 30 October 2010

pCMV_[AAV2]-VP2

pCMV_AAV2-VP2
BioBrick Nr.	BBa_K404011
RFC standard	RFC 10
Requirement	pSB1C3_001
Source	pAAV_RC from Stratagene
Submitted by	[http://2010.igem.org/Team:Freiburg_Bioware FreiGEM 2010]

The viral capsid is formed by the three structural proteins VP1, VP2 and VP3 which are encoded by the cap gene in an overlapping reading frame. They form an icosahedral symmetry arranged in a stoichiometric ratio of 1:1:10. The mRNA encoding for the cap proteins is transcribed from p40 and alternative spliced to minor and major products. Sharing a common C terminus and stop codon, the VP proteins begin with a different start codon. The translation of VP2 from the major spliced mRNA is not as efficient as of VP1 and VP3 because it initiates at a Thr codon (ACG). The N terminus of VP2 has an extension of about 70 amino acids and similar to VP1, it contains nuclear localization signals (BR)(+). These elements are conserved almost in all parvoviruses. The PLA2 domain is involved in successful infection because it needs to be presented on the surface for endosomal release. VP2 is expendable and fusion of larger motifs to its N-terminus does not affect viral assembly and genome packaging.
CMV promoter is derived from human Cytomegalovirus, which belongs to Herpesvirus group. All family members share the ability to remain in latent stage in the human body. CMV is located upstream of immediate-early gene. However, CMV promoter is an example of widely used promoters and is present in mammalian expression vectors. The advantage of CMV is the high-level constitutive expression in mostly all human tissues [Fitzsimons et al., 2002].

References

DiPrimio, Asokan, Govindasamy, Agbandje-McKenna, & Samulski, June 2008. Surface loop dynamics in adeno-associated virus capsid assembly. Journal of virology, 167(1), 5178–5189
Fitzsimons, H.L., Bland, R.J. & During, M.J. (2002). Promoters and regulatory elements that improve adeno-associated virus transgene expression in the brain. Methods San Diego Calif, 28(2), pp.227-236. Available at: http://www.ncbi.nlm.nih.gov/pubmed/12413421.
Warrington, K. H., Gorbatyuk, O. S., Harrison, J. K., Opie, S. R., Zolotukhin, S., Muzyczka, N., et al. (2004). Adeno-associated virus type 2 VP2 capsid protein is nonessential and can tolerate large peptide insertions at its N terminus. Journal of virology, 78(12), 6595-609. doi: 10.1128/JVI.78.12.6595-6609.2004.

Figure 1: The VP proteins are encoded in an overlapping open reading frame. .

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
COMPATIBLE WITH RFC[21]
23
COMPATIBLE WITH RFC[23]
25
INCOMPATIBLE WITH RFC[25]
Illegal NgoMIV site found at 665
1000
INCOMPATIBLE WITH RFC[1000]
Illegal SapI site found at 1425

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-The viral capsid is formed by the three structural proteins VP1, VP2 and VP3 which are encoded by the cap gene in an overlapping reading frame. They form an icosahedral symmetry arranged in a stoichiometric ratio of 1:1:10. The mRNA encoding for the cap proteins is transcribed from p40 and alternative spliced to minor and major products. Sharing a common C terminus and stop codon, the VP proteins begin with a different start codon. The translation of VP2 from the major spliced mRNA is not as efficient as of VP1 and VP3 because it initiates at a Thr codon (ACG). The N terminus of VP2 has an extension of about 70 amino acids and similar to VP1, it contains two functional elements: a phospholipase A2 (PLA2) domain and nuclear localization signals (BR)(+). These elements are conserved almost in all parvoviruses. The PLA2 domain is involved in successful infection because it needs to be presented on the surface for endosomal release. VP2 is expendable and fusion of larger motifs to its N-terminus does not affect viral assembly and genome packaging.
+The viral capsid is formed by the three structural proteins VP1, VP2 and VP3 which are encoded by the cap gene in an overlapping reading frame. They form an icosahedral symmetry arranged in a stoichiometric ratio of 1:1:10. The mRNA encoding for the cap proteins is transcribed from p40 and alternative spliced to minor and major products. Sharing a common C terminus and stop codon, the VP proteins begin with a different start codon. The translation of VP2 from the major spliced mRNA is not as efficient as of VP1 and VP3 because it initiates at a Thr codon (ACG). The N terminus of VP2 has an extension of about 70 amino acids and similar to VP1, it contains nuclear localization signals (BR)(+). These elements are conserved almost in all parvoviruses. The PLA2 domain is involved in successful infection because it needs to be presented on the surface for endosomal release. VP2 is expendable and fusion of larger motifs to its N-terminus does not affect viral assembly and genome packaging.
 <br/>
 CMV promoter is derived from human Cytomegalovirus, which belongs to Herpesvirus group. All family members share the ability to remain in latent stage in the human body. CMV is located upstream of immediate-early gene. However, CMV promoter is an example of widely used promoters and is present in mammalian expression vectors. The advantage of CMV is the high-level constitutive expression in mostly all human tissues [Fitzsimons et al., 2002].