Disease relevance of rep

• Cell lines stably expressing rep/cap are important tools for studying adeno-associated virus (AAV) biology and producing AAV vectors [1].
• Infection of K209 cells with adenovirus leads to a 1000-fold amplification of the rep/cap gene with high-yield production of AAV vectors [1].
• It can not only be combined with other AAV packaging systems, including rep-containing cell lines and herpes simplex virus hybrid packaging methods, but also be used in other vector systems as well [2].
• The acquisition of rep by HHV-6 could be due to natural transfer of genetic information between DNA viruses of eukaryotes and is likely to have important consequences for the life-cycle of HHV-6 and for the host CD4 cell [3].
• In addition, the experiments revealed a transcribed sequence element located within the TAR-coding sequence termed AHHH (AAV-HIV homology element derived from HIV-1) which is involved in rep-mediated inhibition [4].

High impact information on rep

• These results demonstrate that the large AAV Rep proteins have a direct role in AAV DNA replication; namely, they provide the activities required for the resolution of covalently joined AAV termini [5].
• At the Rep binding site, five Rep monomers bind five tetranucleotide direct repeats; each repeat is recognized by two Rep monomers from opposing faces of the DNA [6].
• This Rep recognition sequence is a GCTC repeating motif very similar to sequences within the inverted terminal repeats of the AAV genome which are also bound by Rep78 and Rep68 [7].
• This human genomic DNA can be complexed with AAV DNA by Rep proteins as demonstrated by a dual-label (32P/biotin) assay [7].
• We also demonstrate that Rep proteins, the major AAV regulatory proteins, bind to E1A, the immediate early gene of Ad responsible for hyperphosphorylation and dissociation of pRB-E2F complex [8].

Chemical compound and disease context of rep

• Mutation of a consensus purine nucleotide binding site in the adeno-associated virus rep gene generates a dominant negative phenotype for DNA replication [9].
• An adeno-associated virus (AAV) genome with a Lys-to-His (K340H) mutation in the consensus nucleotide triphosphate binding site of the rep gene has a dominant-negative DNA replication phenotype in vivo [10].
• Pulse-chase experiments with radiolabeled methionine suggest that Rep protein remains associated with the virus particle for up to 8 hr after labeling in infected cells [11].
• Induction of apoptosis by cadmium and the adeno-associated virus Rep proteins [12].

Biological context of rep

• The left-hand open reading frame (ORF) and both inverted terminal repeats (ITRs) have the highest homology with the rep ORF and ITRs of AAV serotype 5 (AAV-5) (89 and 96%, respectively) [13].
• Here we describe a non-HeLa-derived rep/cap cell line called K209, generated by stable transfection of A549 cells with a plasmid construct containing the P5 rep/cap cassette from AAV2 [1].
• In this study, we have used DISC-HSV as a helper for rAAV replication, and have simulated to some extent the amplication of the rep and cap genomes seen in wtAAV infection by incorporating both these and vector sequences in HSV amplicons [14].
• In the current study, a novel strategy was designed to both optimize AAV rep gene expression and increase vector yield, as well as simultaneously to diminish the potential of generating rcAAV particles from the helper plasmid [2].
• Subsequently, the AAV Rep and Cap genes and an AAV vector containing a green fluorescent protein (GFP) reporter gene were stably introduced into the E1A-E1B cell line, generating inducible AAV-GFP packaging cell lines [15].

Anatomical context of rep

• Several rep/cap cell lines have been isolated, each of which is based on HeLa cells [1].
• We recently characterized a stable HeLa rep-cap cell clone (HeRC32) and demonstrated that upon vector transfection and adenovirus infection, efficient rAAV assembly correlated with a 100-fold amplification of the integrated rep-cap sequence with the inverted terminal repeats (ITRs) deleted [16].
• In contrast, AAV-2 rep inhibited HIV LTR activity in both fibroblast and T-cell lines [17].
• In situ hybridization revealed DNA of AAV to be present in the villous moiety (trophoblast) of the placenta but not in the embryo or decidua. in the same cells, AAV proteins (including the replication-associated rep proteins) were detected by immunofluorescence analysis [18].
• K562 cells were cotransfected with a rep plasmid and a plasmid containing a neo gene flanked by the ITRs [19].

Associations of rep with chemical compounds

• We have developed a simplified method for generating rAAV by establishing neomycin-resistant cell lines containing copies of the AAV rep-cap genes and a rAAV vector [20].
• Infection of 293 cells with Bac-AAV virus expressing the rep gene results in a 10- to 50-fold increase in the number of Hygr stable cell clones [21].
• This feature was incorporated into the transducing particle by conjugating a rep expression plasmid to the hybrid virus through a polylysine bridge [22].
• We investigated the expression kinetics of the rep gene using cycloheximide (CHX) and phosphonoformic acid (PFA), which are inhibitors of protein synthesis and viral DNA synthesis, respectively [23].
• An AAV genome having a Lys340 to His (K340H) mutation in the consensus purine nucleotide binding site of the rep gene protein exhibited a dominant-negative phenotype for DNA replication [24].

Analytical, diagnostic and therapeutic context of rep

• A stable cell line carrying adenovirus-inducible rep and cap genes allows for infectivity titration of adeno-associated virus vectors [25].
• Additionally, rep expression determined the localization of the transgene cassette in the aavs1 site in approximately 41% of cases as detected by both Southern blotting and fluorescent in situ hybridization analysis [21].
• In the majority of these clones, the transgene cassette was integrated at AAVS1, and no sequences outside the ITR cassette, rep in particular, were present as determined by PCR, ITR rescue/replication assays, and Southern analysis [26].
• Finally, immunofluorescence analysis showed that HeRC32 cells expressing the DBP also simultaneously expressed the Rep proteins, suggesting a possible involvement of the latter in rep-cap amplification [16].
• Analysis by pulsed-field gel electrophoresis of adenovirus-infected HeRC32 cells indicated that amplified rep-cap sequences were found in an extrachromosomal form [16].

References