Difference between revisions of "Part:BBa K404009"

Revision as of 21:01, 27 October 2010

pCMV_[AAV2]-VP1

pCMV_AAV2-VP1
BioBrick Nr.	BBa_K404009
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. Whereas VP1 is translated from the minor spliced mRNA, VP2 and VP3 are translated from the major spliced mRNA. The minor spliced product is approximately 10-fold less abundant than the major spliced mRNA. Thus, there is much less VP1 than VP2 and VP3 resulting in a capsid stoichiometric ratio of 1:1:10. The N terminus of VP1 has an extension of 65 amino acids including an additional extension of 138 N-terminal amino acids forming the unique portion of VP1. It contains a motiv of about 70 amino acids that is highly homologous to a phospholipase A2 (PLA2) domain. Furthermore, there are nuclear localization sequences (NLS) which are supposed to be necessary for endosomal escape and nuclear entry.

References

Bleker, Pawlita, & Kleinschmidt, 2006. Impact of capsid conformation and rep-capsid interactions on adeno-associated virus type 2 genome packaging. Journal of Virology, 810–820

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
INCOMPATIBLE WITH RFC[21]
Illegal XhoI site found at 701
Illegal XhoI site found at 887
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 1836

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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 contains a motiv of about 70 amino acids that is highly homologous to a phospholipase A2 (PLA2) domain.  Furthermore, there are nuclear localization sequences (BR)(+) which are supposed to be necessary for endosomal escape and nuclear entry. The exact role of VP2 remains unknown, although the protein is thought to be nonessential for viral assembly and infectivity.
+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. Whereas VP1 is translated from the minor spliced mRNA, VP2 and VP3 are translated from the major spliced mRNA. The minor spliced product is approximately 10-fold less abundant than the major spliced mRNA. Thus, there is much less VP1 than VP2 and VP3 resulting in a capsid  stoichiometric ratio of 1:1:10. The N terminus of VP1 has an extension of 65 amino acids including an additional extension of 138 N-terminal amino acids forming the unique portion of VP1. It contains a motiv of about 70 amino acids that is highly homologous to a phospholipase A2 (PLA2) domain.  Furthermore, there are nuclear localization sequences (NLS) which are supposed to be necessary for endosomal escape and nuclear entry.
- (Quelle)
-<h3>Characterization</h3>
 <h3>References</h3>
+<b>Bleker, Pawlita, & Kleinschmidt</b>, 2006. Impact of capsid conformation and rep-capsid interactions on adeno-associated virus type 2 genome packaging. Journal of Virology, 810–820 <br />
 <center><img src="https://static.igem.org/mediawiki/parts/a/a7/Freiburg10_Cap_proteins_VP1_2%263.png" width="600"
 height="auto" margin: 10px 10px 10px 10px/></center>