Part:BBa_K404092

[AAV2]-Rep68ex

[AAV2-Rep68ex]
BioBrick Nr.	BBa_K404092
RFC standard	RFC 10
Requirement	pSB1C3
Source	pAAV_MCS provided by Stratagene
Submitted by	[http://2010.igem.org/Team:Freiburg_Bioware FreiGEM 2010]

 

Rep proteins

Overview

The Adeno-associated virus (AAV) consists of two open reading frames (ORF), rep and cap ORF. The four non-structural rep genes are driven by two promoters located at map units 5 (p5 promoter) and 19 (p19 promoter). Rep proteins are involved in genome encapsidation (reference), regulation of gene expression (reference) and replication of the viral genome (reference).

Figure 1: Genomic organization of the AAV-2 genome. The rep gene codes for four non-structural proteins – Rep40, Rep52, Rep68 and Rep78 – which are involved in gene regulation, genime encapsiation and viral DNA integration.

 

The two larger proteins Rep78/68 play an essential role in viral genome integration and regulation of AAV gene expression, whereas the smaller Rep proteins are involved in viral genome encapsidation. Rep proteins act both as repressors and activators of AAV transcription in respect to the absence and presence of helper viruses such as adenoviruses (Ad) or herpes simplex viruses (HSV) by interacting with several cellular proteins (Nash, Chen, Salganik, & Muzyczka, 2009).

Furthermore, in the absence of Rep proteins, as it is the case in recombinant AAVs, integration of the viral genome into the human genome is rare and random. There are several hotspots for integration of wtAAV genomes such as the human chromosome 19q13.42, known as the AAVSI site, but as well some other accessible chromatin regions for preferred integration have been found (5p13.3 and 3p24.3). Integration into the human genome is mediated by the two regulatory proteins Rep68 and Rep78 driven by the AAV p5 promoter. The proteins bind to the Rep binding site (RBS) which is located within the inverted terminal repeats (ITRs). The minimal consensus Rep binding site (RBS) GAGT GAGC is found within the ITRs and in the p5 integration-efficient element (p5IEE) of the p5 promoter (Hüser et al., 2010). Rep78/68 proteins possess DNA-binding (reference), helicase (reference) and site-specific endonuclease activity located within the first 200 amino acids (Davis, Wu, & Owens, 2000). Since the N-terminal region is unique to the larger Rep proteins, the two smaller Rep proteins possess other biological functions. Rep52/40 gene expression is driven by the p19 promoter which is located within rep ORF and the proteins are involved in encapsidating the viral genome into the preformed capsids. Gene expression of these proteins is suppressed in absence of adenovirus infection by binding of Rep78/68 to the p5 promoter. Gene expression of p19 and p40 is transacvtivated by the Rep proteins Rep78/68 during coinfection.

Rep 68

Rep68 in some seconds

68 kDa

Endonuclease activity

ATPase and helicase activity

Regulate gene expression

Involved in genome integration into human chromosome

Rep68 is a regulatory protein driven by the p5 promoter with an apparent molecular weight of 68 kDa lacking 92 amino acids from the carboxy terminus due to splicing of mRNA coding for the two larger Rep proteins.

The non-structural protein Rep68 belongs to the superfamily 3 (SF3) helicase found in other small DNA and RNA viruses such as simian virus 40 (SV40) and bovine papillomavirus (Mansilla-Soto et al., 2009). Formation of oligomeric complexes of Rep proteins provides the basis for the functional versatility of the two larger regulatory proteins. The AAA⁺ motor domain is known to function as an initiator for oligomerization of the Rep proteins. The cooperative effect of both domains appears to be further regulated by ATP binding as well as different DNA substrates such as dsDNA and ssDNA. Assembly of different nucleoprotein structures suggest that viral replication and genome integration is regulated and controlled by distinct Rep complexes which means that in presence of dsDNA Rep68 assembles to smaller complexes than in presence of ssDNA resulting in octamers.

 

 

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 725
  Illegal XhoI site found at 1592
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]