AAV Rep Proteins

Rep Gene Location and Structure

The AAV genome is a linear, single-stranded (ssDNA) molecule containing 4680 nucleotides and a 145-base terminal repeat sequence (ITR) at its end. The ITR sequence folds into a hairpin structure and serves as the only known cis -acting element required for the initiation of DNA replication and packaging of recombinant AAV genomes into infectious virions. The ITR element is located on either side of two translated open reading frames (ORFs). The left ORF (or rep gene) encodes four nonstructural Rep proteins, which are critical for viral DNA replication and packaging. There are 2 promoters on the left side of the AAV genome, namely p5 and p19, which can produce 2 different lengths of mRNA, each containing an alternatively spliced intron, so 4 different mRNAs containing overlapping sequences can be produced. The Rep proteins, namely Rep78, Rep68, Rep52, and Rep40, encode the Rep proteins required for AAV replication, packaging, and genome integration. Rep78 and Rep68 proteins can specifically bind to the hairpin structure formed by ITR and cleave at specific sites. All Rep proteins have ATP-binding activity and helicase activity, which can up-regulate the transcription of p40 promoter and down-regulate the activities of p5 and p19 promoters. The ORF (or cap gene) on the right encodes three structural proteins, VP1, VP2, and VP3. VP1 is translated from a 2.6 kb mRNA, while VP2 and VP3 are translated from a 2.3 kb mRNA. The start codons of VP1, VP2 and VP3 are AUG (2 203), ACG(2 614) and AUG(2 809) respectively. ACG(2 614) is a start codon unique to eukaryotic cells. Its Starting capability is lower than AUG. If ACG (2 614) in the cap gene is mutated to AUG (2 614), VP2 expression can be increased, but VP3 expression will be reduced.

Anti-tumor Effect of Rep Protein

Studies have found that the non-structural protein Rep78 of AAV2 can inhibit tumor transformation, with three main mechanisms: a. Promoting the function of tumor suppressor genes. At the protein level, Rep78 can interact with E2F-1 and stabilize the pRb-E2F-1 complex; At the DNA level, Rep78 can bind to the E2F-1 promoter and down-regulate the adenovirus-mediated transcription of E2F-1. This dual role of Rep protein reduces the free state of E2F-1 in the cell, thereby making it have the ability to suppress tumors. b. inhibit the expression of proto-oncogenes. Rep78 protein can down-regulate the expression of human c-fos and c-myc proto-oncogene promoters. c. Inhibiting viral oncogenes, Rep78 protein can inhibit the activity of the P97 promoter of HPV-16, thereby inhibiting the occurrence of cervical cancer. Other studies have found that overexpression of Rep78 protein can inhibit DNA synthesis in S phase. Rep78 can completely arrest cells in S phase, which rarely occurs after cellular DNA damage. The Rep78 protein inhibits the activity of Cdc25A by binding to two proteins that stabilize cell cycle regulatory phosphatase (Cdc25A), making it unable to activate the downstream substrates-cyclin-dependent kinases Cdk1 and Cdk2, while Cdk1 and Cdk2 are required during DNA replication. But this alone cannot completely arrest cells in S phase. The Rep78 protein nicks the cell's chromosomes, thereby introducing a cell cycle checkpoint kinase (ATM)-mediated DNA damage response. Activated ATM regulates the cell cycle by phosphorylating its downstream corresponding protein Chk2, ultimately causing the cell cycle to stay in the G1/G2 phase. Mutation analysis demonstrates that the zinc finger structure and nuclease activity of Rep78 are required for S phase arrest. The above factors work together to completely inhibit host DNA synthesis.

In short, compared with other viral vectors, AAV has unique advantages, which makes it have broad prospects in gene therapy, tumor prevention and treatment, etc. AAV itself also has a specific killing effect on some special tumor cells. By producing Rep protein, it interferes with the cell cycle and proliferation, and initiates programmed apoptosis of tumor cells. At the same time, it does not have any adverse effects on normal cells.

Anti-AAV Antibody ELISA Kit
AAV Antibodies and Titration ELISA