Differential Contribution of Adeno-Associated Virus Type 2 Rep Protein Expression and Nucleic Acid Elements to Inhibition of Adenoviral Replication in cis and in trans

Abstract

The helper-dependent adeno-associated virus type 2 (AAV-2) exhibits complex interactions with its helper adenovirus. Whereas AAV-2 is dependent on adenoviral functions for productive replication, it conversely inhibits adenoviral replication, both when its genome is present in trans after coinfection with both viruses and when it is present in cis, as in the production of recombinant adenovirus (

A Regulatory Element Near the 3′ End of the Adeno-Associated Virus rep Gene Inhibits Adenovirus Replication in cis by Means of p40 Promoter-Associated Short Transcripts

“…In our recent study (10), we could confirm that this 3= rep sequence, which we termed the Rep inhibition sequence for adenoviral replication (RIS-Ad), and not Rep protein expression is the major hurdle to the propagation of rAd/AAV hybrid vectors. RIS-Ad, which comprises the AAV-2 p40 promoter and the intron, seems to function at least partly in cis by a yet unknown mechanism (10).…”

“…Rep78/Rep68 strongly suppress early adenoviral gene expression and genome replication (7) and to a lesser extent suppress HSV genome replication (8,9). In addition to the large Rep proteins, AAV-mediated inhibition of adenoviral replication in trans critically depends on the presence of the AAV inverted terminal repeats (ITRs) (10). These represent the only AAV sequences required in cis for AAV genome replication and packaging.…”

“…rAd genomic particles in a small aliquot of freeze-thaw supernatants were quantified by use of a Light-Cycler-based real-time PCR with a QuantiTect SYBR green PCR kit (Qiagen) as described recently (10). Primers E4-Q1 and E4-Q2 for amplification of a 202-bp region from the Ad5 E4 region have also been described previously (10).…”

“…Extraction of viral DNA by a modified Hirt procedure and quantification of newly replicated adenoviral genomes were performed essentially as described previously (10).…”

“…rAd genomic particles in a small aliquot of freeze-thaw supernatants were quantified by use of a Light-Cycler-based real-time PCR with a QuantiTect SYBR green PCR kit (Qiagen) as described recently (10). Primers E4-Q1 and E4-Q2 for amplification of a 202-bp region from the Ad5 E4 region have also been described previously (10). Primers GFP-Q1 (5=-GAG CTG AAG GGC ATT GAC TT-3=) and GFP-Q2 (5=-GCC GAT TGG AGT GTT CTG TT-3=) were used for amplification of a 198-bp region from the green fluorescent protein (GFP) ORF (nt 370 to 567), while primers RIS-Q1 (5=-AAG GGT GGA GCC AAG AAA AG-3=) and RIS-Q2 (5=-GAT GCA GTC ATC CAA ATC CA-3=) amplified a 393-bp sequence of the 3= AAV-2 rep region.…”