Sequences required for coordinate induction of adeno-associated virus p19 and p40 promoters by Rep protein

Abstract

A series of contiguous 30-bp deletions were introduced into the regions upstream of the pl9 and p40 promoters of adeno-associated virus (AAV), and the effects of these deletions on induction of AAV transcription by the rep gene products was evaluated. A novel complementation system was devised for supplying wild-type Rep protein when mutations disrupted the trans activation activity of the Rep protein. Transcription from the p40 promoter was eliminated upon deletion of the TATA sequence located between-4 and-33 from the cap site. Deletions which removed sequences from-34 to-123 bp from the p40 mRNA start site substantially reduced Rep induction of p40 transcription. p19 transcription was also undetectable when the p19 TATA sequence between-4 and-33 was deleted. In contrast to the p40 region, two types of cis-active sequences were found associated with the p19 promoter. Sequences between-4 and-63 bp relative to the p19 cap site were essential for Rep induction only from the p19 promoter. Deletions between-94 and-153 bp relative to the p19 cap site reduced Rep induction of both the p19 and p40 promoters coordinately. These two noncontiguous regions were separated by a 30-bp sequence that was not essential for transcription control. Further deletion analysis delineated a second cis-active element, associated with the p5 promoter (AAV nucleotides 191 to 320), which was also necessary for coordinate Rep activation of both the p19 and p40 promoters. Finally, the dependence of p40 transcription on the Rep-responsive elements within the p5 and p19 regions could be overcome by the presence of the AAV terminal repeats, suggesting that the terminal-repeats contained redundant Rep-responsive elements. These results implied an interdependence in cis between the three AAV promoters and suggested a novel mechanism for coordinate regulation of gene expression in response to the trans-activating Rep protein. Coordinate induction appeared to be the result of a simultaneous interaction between the Rep protein and sequence elements associated with two or all three of the AAV promoters.

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

“…A possible explanation for the decrease in Cap expression is that the amount of Rep78/68 may not be sufficient to enhance the Cap expression, because Rep78/68 is required to transactivate both the p19 and p40 promoters in the presence of adenovirus. 25) Since Rep78/68 is also required to replicate the vector DNA, the AAV vector yields may be reduced when the levels of Rep78/68 expression are very low. Thus, for AAV vector production it is important to express appropriate amounts of Rep78/68.…”

“…1, pIM45 contains the wild-type rep and cap genes at nt 145 to 4493 in a Bluescript M13+ vector. 25) To construct the Rep expression plasmid (pRep), a polylinker containing recognition sites for the enzymes 5′-SacI-ClaI-EcoRI-SmaI-BamHI-XbaI-HincII-PstI-EcoRV-HindIII-XhoI-KpnI-3′ was cloned into the BssHII site of pUC-MCS, 8) and then the following fragments were cloned into the plasmid; an AAV fragment containing the rep gene (nt 145-2252 in the AAV genome) into SpeI-PstI and the 135-bp HpaI-BamHI blunt-end modified SV40 early polyadenylation signal from pCMV-β plasmid (CLONETECH Laboratories, Palo Alto, CA) into the HpaI site. 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.…”

Adeno‐Associated Virus Rep78 Binds to E2‐Responsive Element 1 of Bovine Papillomavirus Type 1

“…Plasmids. The pCMVRep78 construct was made by inserting the XbaI fragment of pIM29 (31), containing the genomic DNA and promoters of AAV but lacking the 145-bp terminal repeats (TRs), 45 bp immediately 3 0 of the left TR, and 42 bp immediately 5 0 of the right TR, into the XbaI site of pRc/CMV (Invitrogen), which contains the human cytomegalovirus immediate-early promoter (hCMV IEP).…”

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 .…”

“…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.…”