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

Abstract

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.

Increased temperature‑related adeno‑associated virus vectors transduction of ovarian cancer cells ‑ essential signatures of AAV receptor and heat shock proteins

“…The safety of rAAV vectors as well as their presence in medicine result in a number of studies revealing critical points in the pathway of gene transfer and intracellular events involving rAAV (1,4). The role of the miRNA signature (5) and the expression of AAV membrane receptors (6–8) in the AAV cellular transmission are in the course of documentation. Discovering cellular mechanisms of rAAV transduction helps to understand the AAV biology and makes it possible to design new vectors-synthetic AAV mosaic serotypes (9), vectors with dsDNA (10), AAV chemo-conjugates (11).…”

Preventing packaging of translatable P5-associated DNA contaminants in recombinant AAV vector preps

“…Previous studies of wild-type AAV have revealed bidirectional P5 promoter activity, driving expression of small upstream RNAs. 21 , 22 Transfection of a plasmid with GFP-encoding DNA positioned directly upstream of P5 resulted in GFP expression, notably greater than expression generated when the GFP-encoding DNA was placed upstream of ITR sequences ( Figure 3 A). To determine whether transcriptional activity from P5 was driving RNA and protein expression of its associated rAAV contaminants, we constructed a fluorescence-based system where reporter sequences were positioned upstream of P5 and thus packaged as contaminants in rAAV ( Figure S1 ).…”

A Comprehensive RNA-seq Analysis of Human Bocavirus 1 Transcripts in Infected Human Airway Epithelium

“…This technology is a powerful tool for the systematic and unbiased profiling of viral transcription/expression. Both mRNA-seq and small RNA-seq have been applied to profile gene expression of AAV2 in the presence or absence of coinfection of helper viruses, which identified novel AAV2 mRNA transcripts from both the positive and negative stands of the AAV2 genome, as well as viral miRNAs [30]. In the present study, we utilized mRNA-seq and small RNA-seq of RNA samples isolated from HBoV1-infected HAE-ALI cultures to study the transcription profile of HBoV1.…”