Nonproductive exposure of PBMCs to SARS-CoV-2 induces cell-intrinsic innate immune responses.

Cell-intrinsic responses mounted in PBMCs during mild and severe COVID-19 differ quantitatively and qualitatively. Whether they are triggered by signals emitted by productively infected cells of the respiratory tract or result from physical interaction with virus particles remains unclear. Here, we analyzed susceptibility and expression profiles of PBMCs from healthy donors upon ex vivo exposure to SARS-CoV and SARS-CoV-2. In line with the absence of detectable ACE2 receptor expression, human PBMCs were refractory to productive infection. RT-PCR experiments and single-cell RNA sequencing revealed JAK/STAT-dependent induction of interferon-stimulated genes (ISGs) but not proinflammatory cytokines. This SARS-CoV-2-specific response was most pronounced in monocytes. SARS-CoV-2-RNA-positive monocytes displayed a lower ISG signature as compared to bystander cells of the identical culture. This suggests a preferential invasion of cells with a low ISG baseline profile or delivery of a SARS-CoV-2-specific sensing antagonist upon efficient particle internalization. Together, nonproductive physical interaction of PBMCs with SARS-CoV-2- and, to a much lesser extent, SARS-CoV particles stimulate JAK/STAT-dependent, monocyte-accentuated innate immune responses that resemble those detected in vivo in patients with mild COVID-19.

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.

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.

Ultra-deep profiling of alternatively spliced Drosophila Dscam isoforms by circularization-assisted multi-segment sequencing.

The Drosophila melanogaster gene Dscam (Down syndrome cell adhesion molecule) can generate thousands of different ectodomains via mutual exclusive splicing of three large exon clusters. The isoform diversity plays a profound role in both neuronal wiring and pathogen recognition. However, the isoform expression pattern at the global level remained unexplored. Here, we developed a novel method that allows for direct quantification of the alternatively spliced exon combinations from over hundreds of millions of Dscam transcripts in one sequencing run. With unprecedented sequencing depth, we detected a total of 18,496 isoforms, out of 19,008 theoretically possible combinations. Importantly, we demonstrated that alternative splicing between different clusters is independent. Moreover, the isoforms were expressed across a broad dynamic range, with significant bias in cell/tissue and developmental stage-specific patterns. Hitherto underappreciated, such bias can dramatically reduce the ability of neurons to display unique surface receptor codes. Therefore, the seemingly excessive diversity encoded in the Dscam locus might nevertheless be essential for a robust self and non-self discrimination in neurons.