Adeno-associated virus (AAV) is recognized for its bipartite life cycle with productive replication dependent on coinfection with adenovirus (Ad) and AAV latency being established in the absence of a helper virus. The shift from latent to Ad-dependent AAV replication is mostly regulated at the transcriptional level. The current AAV transcription map displays highly expressed transcripts as found upon coinfection with Ad. So far, AAV transcripts have only been characterized on the plus strand of the AAV single-stranded DNA genome. The AAV minus strand is assumed not to be transcribed. Here, we apply Illumina-based RNA sequencing (RNA-Seq) to characterize the entire AAV2 transcriptome in the absence or presence of Ad. We find known and identify novel AAV transcripts, including additional splice variants, the most abundant of which leads to expression of a novel 18-kDa Rep/VP fusion protein. Furthermore, we identify for the first time transcription on the AAV minus strand with clustered reads upstream of the p5 promoter, confirmed by 5' rapid amplification of cDNA ends and RNase protection assays. The p5 promoter displays considerable activity in both directions, a finding indicative of divergent transcription. Upon infection with AAV alone, low-level transcription of both AAV strands is detectable and is strongly stimulated upon coinfection with Ad. A deno-associated viruses (AAV) are helper-dependent members of the parvovirus group that require coinfection with an unrelated helper virus, particularly adenovirus (Ad) for productive replication (1). Despite of several identified AAV serotypes most research has been done with prototype AAV type 2. The AAV2 genome consists of a linear, single-stranded DNA of 4.7 kb. Both ends carry identical inverted terminal repeats (ITR) of 145 bp (2), which flank the two major open reading frames (ORF), called rep and cap. The rep ORF codes for four nonstructural proteins, Rep78 and a C-terminally spliced variant, Rep68. In addition, N-terminally truncated versions thereof are expressed, called Rep52 and Rep40, respectively. The Rep proteins are required as regulators for various steps of the AAV life cycle. AAV cap harbors the ORF for the capsid proteins (VP1 to -3) and a separate ORF for the assembly-activating protein (AAP) (3).The current knowledge of AAV transcription dates back to early research in the 1980s when the three AAV2 promoters, p5, p19, or p40 and major transcripts derived thereof were characterized (4, 5). All AAV transcripts identified since then map to only one DNA plus strand (6), which led to the assumption that the complementary AAV minus strand was not transcribed. Furthermore, spliced AAV transcripts were identified displaying a single, shared intron located in the center of the AAV2 genome (4, 7). Initially, a single splice donor site at AAV nucleotide 1906 and a single splice acceptor site located at nucleotide (nt) 2228 were described. Later, an alternative splice acceptor site at nt 2201 was detected (8), allowing the expression of VP1 protein ...
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...
Most DNA viruses express small regulatory RNAs, which interfere with viral or cellular gene expression. For adeno-associated virus (AAV), a small ssDNA virus with a complex biphasic life cycle miRNAs or other small regulatory RNAs have not yet been described. This is the first comprehensive Illumina-based RNA-Seq analysis of small RNAs expressed by AAV alone or upon co-infection with helper adenovirus or HSV. Several hotspots of AAV-specific small RNAs were detected mostly close to or within the AAV-ITR and apparently transcribed from the newly identified anti-p5 promoter. An additional small RNA hotspot was located downstream of the p40 promoter, from where transcription of non-coding RNAs associated with the inhibition of adenovirus replication were recently described. Parallel detection of known Ad and HSV miRNAs indirectly validated the newly identified small AAV RNA species. The predominant small RNAs were analyzed on Northern blots and by human argonaute protein-mediated co-immunoprecipitation. None of the small AAV RNAs showed characteristics of bona fide miRNAs, but characteristics of alternative RNA processing indicative of differentially regulated AAV promoter-associated small RNAs. Furthermore, the AAV-induced regulation of cellular miRNA levels was analyzed at different time points post infection. In contrast to other virus groups AAV infection had virtually no effect on the expression of cellular miRNA, which underscores the long-established concept that wild-type AAV infection is apathogenic.
Adeno-associated virus (AAV) type 5 represents the genetically most distant AAV serotype and the only one isolated directly from human tissue. Seroepidemiological evidence suggests herpes simplex virus (HSV) as a helper virus for human AAV5 infections, underlining the in vivo relevance of the AAV-herpesvirus relationship. In this study we analysed, for the first time, HSV helper functions for productive AAV5 replication, and compared these to AAV2. Using a combination of HSV strains and plasmids for individual genes, the previously defined HSV helper functions for AAV2 replication were shown to induce AAV5 gene expression, DNA replication and production of infectious progeny. The helper functions comprise the replication genes for ICP8 (UL29), helicase-primase (UL5/8/52), and DNA polymerase (UL30/42). HSV immediate-early genes for ICP0 and ICP4 further enhanced AAV5 replication, mainly by induction of rep gene expression. In the presence of HSV helper functions, AAV5 Rep co-localized with ICP8 in nuclear replication compartments, and HSV alkaline exonuclease (UL12) enhanced AAV5 replication, similarly to AAV2. UL12, in combination with ICP8, was shown to induce DNA strand exchange on partially double-stranded templates to resolve and repair concatemeric HSV replication intermediates. Similarly, concatemeric AAV replication intermediates appeared to be processed to yield AAV unit-length molecules, ready for AAV packaging. Taken together, our findings show that productive AAV5 replication is promoted by the same combination of HSV helper functions as AAV2.
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