Adeno-associated virus type 2 small RNA-Seq analysis

Most DNA viruses express small regulatory RNAs, which interfere with viral or cellulargene expression. For adeno-associated virus (AAV), a small ssDNA virus with acomplex biphasic life cycle miRNAs or other small regulatory RNAs have not yet beendescribed. This is the first comprehensive Illumina-based RNA-Seq analysis of smallRNAs 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 withinthe AAV-ITR and apparently transcribed from the newly identified anti-p5 promoter. Anadditional small RNA hotspot was located downstream of the p40 promoter, fromwhere transcription of non-coding RNAs associated with the inhibition of adenovirusreplication were recently described. Parallel detection of known Ad and HSV miRNAsindirectly validated the newly identified small AAV RNA species. The predominantsmall RNAs were analyzed on Northern blots and by human argonaute proteinmediatedco-immunoprecipitation. None of the small AAV RNAs showedcharacteristics of bona fide miRNAs, but characteristics of alternative RNA processingindicative of differentially regulated AAV promoter-associated small RNAs.Furthermore, the AAV-induced regulation of cellular miRNA levels was analyzed atdifferent time points post infection. In contrast to other virus groups AAV infection hadvirtually no effect on the expression of cellular miRNA, which underscores the longestablishedconcept that wild-type AAV infection is apathogenic.