Part:BBa_K404153

[AAV2]-NLS

[AAV2-NLS]
BioBrick Nr.	BBa_K404153
RFC standard	RFC 25
Requirement	pSB1C3
Source	pAAV_MCS: provided by Stratagene
Submitted by	[http://2010.igem.org/Team:Freiburg_Bioware FreiGEM 2010]

General informations

The AAV capsid consists of 60 capsid protein subunits composed of the three cap proteins VP1, VP2, and VP3, which 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 the VP proteins. VP1, VP2 and VP3 share a common C terminus and stop codon, but begin with a different start codon. The N termini of VP1 and VP2 play important roles in infection and contain motifs that are highly homologous to a phospholipase A2 (PLA2) domain and nuclear localization signals (NLS, see basic regions). These elements are conserved in almost all parvoviruses. (Johnson et al., 2010a).

Whereas VP1 is translated from the minor spliced mRNA, while 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 motif of about 70 amino acids that is highly homologous to phospholipase A2 (PLA2) domain. Furthermore, there are nuclear localization sequences (BR)(+), which are supposed to be necessary for endosomal escape and nuclear entry. (Bleker, Pawlita, & Kleinschmidt, 2006), (DiPrimio, Asokan, Govindasamy, Agbandje-McKenna, & Samulski, 2008), (Johnson et al., 2010a).

The translation of VP2 from the major spliced mRNA is less efficient compared to the translation of VP3 because it initiates at Thr codon (ACG). VP2 and VP1 have an extension at the N terminus that remains internal when exposing the capsid to experimental conditions like low pH or heat. The exact role of VP2 remains unknown, although the protein is thought to be nonessential for viral assembly and infectivity. (DiPrimio, Asokan, Govindasamy, Agbandje-McKenna, & Samulski, 2008), (Johnson et al., 2010b)

NLS

NLS are located in basic regions on the N terminus of VP2 (35 aa) and VP1 (172 aa) and mediate genome delivery into the nucleus and transduction [Hoque et al.,1999; Grieger et al., 2006]. Nuclear localisation sequence is hydrophilic and contains ß-turn and coil regions [Kalderon, et al, 1984]. It was also described in CPV and MVM viruses. Compared to CPV, MVM virus contains several NLS within the capsid, which are activated at different infection stages [Lombardo et al., 2000; Lombardo et al., 2002]

Characterization

Specific targeting of tumor cells was, besides producing recombinant Adeno-associated virus 2 (AAV2) particles for therapeutical applications, one intention of the Virus Construction Kit provided by the iGEM team Freiburg_Bioware 2010.

Besides fusing larger motifs to the N-terminus of the viral protein 2 (VP2) open reading frame (ORF), we designed a method for inserting into VP1. It is expected that these peptides become located on the virus surface either by transit through the pores or by exposure during capsid assembly.

This part was created in order to be fused to the desired surface exposed motif before cloning into VP1, promising better infectivity of the AAV2 therapeutic vectors (Grieger et al., 2007).

 

Nuclear Localization by Fluorescence Microscopy

The iGEM team Freiburg_Bioware 2010 tested the efficacy of this part by cloning it together with mVenus into VP1.

AAV-293 cells were transfected with a 50:50 ratio of the Rep/Cap(VP1KO) to the CMV_VP1_NLS_mVenus_VP2/3 plasmid. We packaged mCherry, driven by the CMV promoter, into the virus capsids and followed protein expression via fluorescence microscopy. 30 hours post transfection mCherry fluorescence was detectable in the whole cytosol of the successfully transfected cells, demonstrating that DNA located between the AAV2 ITRs is already transcribed in the producer cell line. In contrast to that mVenus fluorescence signal could be observed only in the nuclei (Fig. 1). This indicated that the nuclear localization sequence targets the single VP1_NLS_mVenus_VP2/3 proteins efficiently to the cell nucleus, where assembly and packaging of the virus particles takes place. 

Figure 1: Fluorescence Microscopy. A) Brightfiled picture. B) Excitation at 555 nm showed mCherry signal in the cytosol. C) Excitation at 505 nm revealed mVenus fluorescence in the nuclei, indicating functionality of the nuclear localization sequence inserted – together with mVenus – into VP1. D) Merged image.

  

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
  COMPATIBLE WITH RFC[1000]

References

Bleker, S., Pawlita, M., & Kleinschmidt, J., 2006. IImpact of capsid conformation and Rep-capsid interactions on adeno-associated virus type 2 genome packaging. Journal of virology, 80(2), 810-820. doi: 10.1128/JVI.80.2.810.
DiPrimio, N., Asokan, A., Govindasamy, L., Agbandje-McKenna, M., & Samulski, R. J. , 2008. Surface loop dynamics in adeno-associated virus capsid assembly. Journal of virology, 82(11), 5178-89. doi: 10.1128/JVI.02721-07.
Grieger et al., (2007). Surface-exposed adeno-associated virus Vp1-NLS capsid fusion protein rescues infectivity of noninfectious wild-type Vp2/Vp3 and Vp3-only capsids but not that of fivefold pore mutant virions. J Virol. 2007 Aug;81(15):7833-43. Epub 2007 May 16.
Hoque, 1999. Nuclear transport of the major capsid protein is essential for adeno-associated virus capsid formation. Journal of Virology, 73(9), pp.7912-7915.
Kalderon, D, 1984. Sequence requirements for nuclear location of simian virus 40 large-T antigen. Nature, 311(5981), pp.33-38
Johnson, J.S., 2010. Mutagenesis of adeno-associated virus type 2 capsid protein VP1 uncovers new roles for basic amino acids in trafficking and cell-specific transduction. Journal of virology, 84(17), 8888-902. doi: 10.1128/JVI.00687-10.
Lombardo, E, 2000. A beta-stranded motif drives capsid protein oligomers of the parvovirus minute virus of mice into the nucleus for viral assembly. Journal of Virology, 74(8), pp.3804-3814
Lombardo, E , 2002. Complementary roles of multiple nuclear targeting signals in the capsid proteins of the parvovirus minute virus of mice during assembly and onset of infection. Journal of Virology, 76(14), pp.7049-7059