Part:BBa_K404151

[AAV2]-VP23 (ViralBrick-587KO-Empty)

Capsid

The AAV capsid consists of 60 capsid protein subunits. The three cap proteins VP1, VP2, and VP3 are encoded in an overlapping reading frame. Arranged in a stoichiometric ratio of 1:1:10, they form an icosahedral symmetry. The mRNA encoding for the cap proteins is transcribed from p40 and alternative spliced to minor and major products. Alternative splicing and translation initiation of VP2 at a nonconventional ACG initiation codon promote the expression of VP1, VP2 and VP3. The VP proteins share a common C terminus and stop codon, but begin with a different start codon. The N-terminus of VP1 plays an important role in infection and contains a motif highly homologous to a phospholipase A2 (PLA2) domain and nuclear localization signals (BR)(+). VP 2 contains basic regions, too.

This part is used for cotranfection with parts containing VP1up (BBa_K404164-BBa_K404166)

ViralBrick 587-KO empty

The primary receptor of AAV-2 is the heparan sulfate proteoglycan (HSPG) receptor (Perabo et al. 2006). Its binding motif consists of five amino-acids located on the capsid surface: R484/R487, K532, R585/587. (Trepel et al. 2009). The positively charged arginine residues interact with the HSPGs' negatively charged acid residues. Opie et al. have shown that two point mutations (R585A and R588A) are sufficient to eliminate the heparin binding affinity in AAV2. (Opie et al. 2003). This ViralBrick has been created to introduce this knockout into other constructs. The BioBricks which contain this knockout are annotated with „587-KO“.

Usage

One aim of the iGEM team Freiburg_Bioware 2010's research is on the one hand to knock down the natural tropism of the Adeno-associates virus 2 (AAV2) particles and on the other hand to specifically target tumor cells. This is achieved by genetic engineering of the virus surface. For this purpose, two different strategies were developed, including insertion of motifs into surface-exposed loops or fusion to the N-terminus of this part.

The resulting protein can be expressed in trans to the other structural and regulatory elements of the virus – the RepCap plasmid – , replacing 100 % of VP2 by start codon mutation (BBa_K404004, [AAV2]-Rep-VP13(ViralBrick-587KO-empty)_p5-TATAless). This allows titration and control of the amount of the VP2-targeting-subunit that becomes incorporated into the virus capsid.

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
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 1841
  Illegal SapI site found at 752