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

Abstract

Adeno-associated virus (AAV) has long been known to inhibit helper adenovirus (Ad) replication independently of AAV Rep protein expression. More recently, replication of Ad serotype 5 (Ad5)/AAV serotype 2 (AAV-2) hybrid vectors was shown to be inhibited in cis by a sequence near the 3= end of AAV rep, termed the Rep inhibition sequence for adenoviral replication (RISAd). RIS-Ad functions independently of Rep protein expression. Here we demonstrate that inhibition of adenoviral replication by RIS-Ad requires an active AAV p40 promoter and the 5= half of the intron. In addition, Ad inhibition is critically dependent on the integrity of the p40 transcription start site (TSS) leading to short p40-associated transcripts. These do not give rise to effector molecules capable of inhibiting adenoviral replication in trans, like small polypeptides or microRNAs. Our data point to an inhibitory mechanism in which RNA polymerase II (Pol II) pauses directly downstream of the p40 promoter, leading to interference of the stalled Pol II transcription complex with the adenoviral replication machinery. Whereas inhibition by RIS-Ad is mediated exclusively in cis, it can be overcome by providing a replication-competent adenoviral genome in trans. Moreover, the inhibitory effect of RIS-Ad is not limited to AAV-2 but could also be shown for the corresponding regions of other AAV serotypes, including AAV-5. These findings have important implications for the future generation of Ad5/AAV hybrid vectors. IMPORTANCEInsertion of sequences from the 3= part of the rep gene of adeno-associated virus (AAV) into the genome of its helper adenovirus strongly reduces adenoviral genome replication. We could show that this inhibition is mediated exclusively in cis without the involvement of trans-acting regulatory RNAs or polypeptides but nevertheless requires an active AAV-2 p40 promoter and p40-associated short transcripts. Our results suggest a novel inhibitory mechanism that has so far not been described for AAV and that involves stalled RNA polymerase II complexes and their interference with adenoviral DNA replication. Such a mechanism would have important implications both for the generation of adenoviral vectors expressing the AAV rep and cap genes and for the regulation of AAV gene expression in the absence and presence of helper virus.A deno-associated virus (AAV) is characterized by a bipartite replication cycle, with productive infection being critically dependent upon the presence of a helper virus, such as adenovirus (Ad) (1) or herpes simplex virus (HSV) (2). In the absence of helper virus, AAV can establish latent infection by integration into the host genome (3, 4). During the productive replication cycle, AAV is able to inhibit the propagation of the coinfecting helper virus, especially evident in the case of adenovirus (5, 6). This inhibition has been attributed to the expression of the large regulatory (Rep) proteins Rep78 and Rep68. These promote the replication of the AAV genome and induce the expression of the AAV seroty...

The Interplay between Adeno-Associated Virus and Its Helper Viruses

“…AAV Rep52/78 proteins both comprise C-terminal PKA-specific inhibitor protein (PKI)-like motifs that directly interact with PKA and inhibit the cAMP/PKA signaling pathway and thus the AdV late gene expression [ 242 ]. Lastly, a short cis-regulatory element from the 3′-end of the rep gene, termed the Rep inhibition sequence for adenoviral replication (RISAd), has been identified to inhibit AdV replication [ 243 ]. This mechanism requires a functional AAV p40 promoter that gives rise to short p40 -associated transcripts in AdV vectors, which comprise the RISAd.…”

Comprehensive Small RNA-Seq of Adeno-Associated Virus (AAV)-Infected Human Cells Detects Patterns of Novel, Non-Coding AAV RNAs in the Absence of Cellular miRNA Regulation

“…Transcription of the small RNA sR-1862, located a few nucleotides downstream of the p40 TSS (nt 1853) could also lead to a secondary structure ( Fig 7D ). Recently, we have identified this region as cis -regulatory element for the inhibition of recombinant adenovirus replication [ 12 ]. Here we show by small RNA-Seq that p40-initiated transcripts although highly heterogeneous ( Fig 3A ), mostly represent sR-1862.…”

“…In addition, a heterogeneous group of small AAV p40 promoter-associated transcripts has been described [ 12 ]. A mechanism of RNA polymerase II pausing accompanied with the inhibition of recombinant adenoviral replication was postulated.…”

“…In a total RNA-Seq analysis, we have recently discovered transcription on the AAV negative (-) strand opposite to the p5 promoter, indicative of non-coding RNA species [ 11 ]. In addition, we have identified p40 promoter-associated short non-coding transcripts on the (+) strand relevant for the inhibition of adenovirus replication [ 12 ]. Apparently, non-coding RNA species are involved in the regulation of the AAV life cycle.…”

Improved Genome Packaging Efficiency of Adeno-associated Virus Vectors Using Rep Hybrids

“…To determine the p40 promoter activity the nt 1442 to 1878 (AAV1, SalI-KpnI fragment), nt 1428 to 1887 (AAV2, SalI-HindIII fragment), and nt 1340 to 1776 (AAV8, SalI-KpnI fragment) from the accession numbers listed above were cloned upstream of a luciferase gene. The p40 promoter regions were selected very generously to contain all previously described p40 promoter elements ( 37 , 46 ). Site-directed mutagenesis PCRs for the introduction of the stop codons to the 3′ ends of the AAV1 and AAV2 rep genes and for the correction of the AAV8 rep gene were done as previously described ( 45 ).…”