The adeno-associated virus (AAV) Rep protein acts as both a repressor and an activator to regulate AAV transcription during a productive infection

Abstract

Adeno-associated virus (AAV) uses three promoters, p5, p19, and p40, to regulate viral gene expression. The p5 and p19 promoters direct the synthesis of the viral regulatory proteins, Rep78 and -68 and Rep52 and -40, respectively. The p5 Rep proteins bind a linear 22-bp sequence, the Rep binding element (RBE), that is within both the terminal repeat (TR) and the p5 promoter. In the absence of helper virus, all four Rep proteins have been shown to reduce transcription from the viral p5 and p19 promoters. In this report, we focus on the roles of these proteins and the RBEs in controlling transcription during a productive infection, that is, in the presence of adenovirus. We find that in the presence of adenovirus, the p5 RBE represses p5 transcription while the RBE in the TR activates p5. However, both the TR RBE and the p5 RBE transactivate the p19 and p40 promoters. The fact that the p5 RBE-Rep complex can transactivate p19 and p40 while repressing p5 suggests that Rep78/68 is both a repressor and a transactivator. Rep repression of p5 is specific for the p5 RBE, as other p5 promoter elements do not support this activity. We also demonstrate that in the presence of adenovirus, the p19 Rep proteins, which do not bind to the RBE, can eliminate repression of the p5 promoter by Rep78 and Rep68. This may occur by the association of Rep52 with Rep78 or Rep68 to produce a Rep78/68-Rep52 protein complex which can be detected in vivo by immunoprecipitation. Finally, two Rep mutants that were deficient in RBE binding and transactivation but positive for p5 repression were identified. These mutants may define interaction domains involved in making contacts with other proteins that facilitate repression. These observations suggest a mechanism for controlling the p5 and p19 mRNA levels during a productive AAV infection.

Efficient Production of Adeno‐associated Virus Vectors Using Split‐type Helper Plasmids

“…The Rep78/68 expression plasmid (pR78/68) was derived from pRep with a mutation of the first ATG of Rep52/40 to GGA as described previously. 12,13) Since it has been demonstrated that the p5 promoter sequence has the ability to enhance both the p19 and p40 promoter activities during productive infection, 14) we inserted the p5 promoter sequence at the 3′ end of the polyadenylation sequence of pCap and pR52/40Cap. To construct the Cap expression plasmid (pCap) and Rep52/ 40 plus Cap expression plasmid (pR52/40Cap), a BamHI-XbaI fragment (nt 1498-4488) and SfiI-XbaI fragment (nt 537-4488) of pRepCap were cloned into pUC18, respectively.…”

“…Rep52/ 40 are required for the accumulation of ssDNA forms, 13) and it has been reported that Rep52/40 attenuate the repression of the p5 promoter by Rep78/68 in the presence of helper virus. 14) Transcription from the p40 promoter generates two alternatively spliced forms, the minor transcript encoding VP1 and the major transcript for VP2 and VP3. 15,16) One of the limiting factors for AAV vector-mediated gene therapy is the difficulty in producing high-titer AAV vector stocks.…”

The Use of Heterologous Promoters for Adeno‐Associated Virus (AAV) Protein Expression in AAV Vector Production

“…In the absence of a helper virus, Rep78/68 repress both p5 and p19 transcription (18). In the presence of a helper virus, the AAV promoters, particularly p5, are transactivated by the adenovirus ElA and host YY1 proteins, and Rep78/68 positively regulate the p19 and p40 promoters (19,21,28). Transcription from p19 promoter generates spliced and unspliced tran-*Address correspondence to Dr .…”

“…Transcription from p19 promoter generates spliced and unspliced tran-*Address correspondence to Dr . Keiya (11,18,28). Transcription from p40 promoter generates two alternatively spliced transcripts; the minor encodes VP1 and the major encodes VP2 and VP3 (1,13).…”

“…Since Rep78/68 proteins are required for replication from an AAV origin within the ITR and transactivate both p19 and p40 promoters during productive infection in the presence of adenovirus (21,28,34), a change in the expression level of Rep78/68 would affect the expression of other AAV proteins and thereby alter the efficiency of AAV vector production. In this study, we constructed various helper plasmids in which p5 promoter was replaced with heterologous promoters [human polypeptide chain elongation factor 1-oc (EF) (24), cytomegalovirus immediate-early (CMV-LE) (36), simian virus 40 early (SV40), B19 parvovirus p6 (B 19p6) (27) and chicken P-actin promoter plus cytomegalovirus enhancer (CAG) (25) promoters] to examine the effect of different amounts of Rep78/68 on the expression of Rep52/40 and Cap, rAAV DNA replication and the efficiency of AAV vector production.…”