Friedl, M.-S. | bioRxiv Science

bioRxiv2021

HSV-1 and influenza infection induce linear and circular splicing of the long NEAT1 isoform

The herpes simplex virus 1 (HSV-1) virion host shut-off (vhs) protein cleaves both cellular and viral mRNAs by a translation-initiation-dependent mechanism. Here, we show that vhs-mediated degradation of mRNAs leads to an accumulation of circular RNAs (circRNAs) relative to linear mRNAs during HSV-1 infection. Strikingly, we found that circular splicing of the long isoform (NEAT1_2) of the nuclear paraspeckle assembly transcript 1 (NEAT1) was massively induced during HSV-1 infection. In contrast to other circRNAs, induction of the NEAT1_2 circRNA was independent of vhs and occurred while NEAT1_2 was still bound to the chromatin. This was associated with induction of linear splicing of NEAT1_2 both within and downstream of the circRNA. NEAT1_2 splicing was absent in uninfected cells but can be induced by ectopic co-expression of the HSV-1 immediate-early proteins ICP22 and ICP27. Interestingly, NEAT1_2 circular and linear splicing was also up-regulated in influenza A virus (IAV) infection but absent in stress conditions, which disrupt transcription termination similar to but not by the same mechanisms as HSV-1 and IAV infection. Large-scale analysis of published RNA-seq data uncovered induction of NEAT1_2 splicing in cancer cells upon inhibition or knockdown of cyclin-dependent kinase 7 (CDK7) or the MED1 subunit of the Mediator complex phosphorylated by CDK7. Finally, CDK7 inhibition also disrupted transcription termination, highlighting a possible link between disruption of transcription termination and NEAT1_2 splicing.

bioinformatics↗

bioRxiv2020

Dissecting HSV-1-induced host shut-off at RNA level

Herpes simplex virus 1 (HSV-1) installs a profound host shut-off during lytic infection. The virion host shut-off (vhs) protein plays a key role in this process by efficiently cleaving both host and viral mRNAs in a translation-initiation-dependent manner. Furthermore, the onset of viral DNA replication is accompanied by a rapid decline in transcriptional activity of the host genome. Both mechanisms have tremendous impact on the RNA expression profile of the infected cells. To dissect their relative contributions and elucidate gene-specific host transcriptional responses throughout the first 8h of lytic HSV-1 infection, we here employed RNA-seq of total, newly transcribed (4sU-labelled) and chromatin-associated RNA in wild-type (WT) and {Delta}vhs infection of primary human fibroblasts. Following virus entry, vhs activity rapidly plateaued at an elimination rate of around 30% of cellular mRNAs per hour until 8h p.i. In parallel, host transcriptional activity dropped down to 10-20%. While the combined effects of both phenomena dominated infection-induced changes in total RNA, extensive gene-specific transcriptional regulation was observable in chromatin-associated RNA. This was surprisingly concordant between WT HSV-1 and its {Delta}vhs mutant and at least in parts mediated by the embryonic transcription factor DUX4. Furthermore, both WT and {Delta}vhs infection induced strong transcriptional up-regulation of a small subset of genes. Most of these were either poorly or not at all expressed prior to infection but already primed by H3K4me3 histone marks at their promoters. Most interestingly, analysis of chromatin-associated RNA revealed vhs-nuclease-activity-dependent transcriptional down-regulation of at least 150 cellular genes, in particular of many genes encoding integrin adhesome and extracellular matrix components. This was accompanied by a vhs-dependent reduction in protein levels by 8h p.i. for many of these genes. In summary, our study provides a comprehensive picture of the molecular mechanisms that govern cellular RNA metabolism during the first 8h of lytic HSV-1 infection. Author SummaryThe HSV-1 virion host shut-off (vhs) protein efficiently cleaves both host and viral mRNAs in a translation-dependent manner. In this study, we model and quantify changes in vhs activity as well as the virus-induced global loss of host transcriptional activity during productive HSV-1 infection. In general, HSV-1-induced alterations in total RNA levels were found to be predominantly shaped by these two global processes rather than gene-specific regulation. In contrast, chromatin-associated RNA depicted gene-specific transcriptional changes. This revealed highly concordant transcriptional changes in WT and{Delta} vhs infection, confirmed DUX4 as a key transcriptional regulator in HSV-1 infection and depicted vhs-dependent, transcriptional down-regulation of the integrin adhesome and extracellular matrix. The latter explained some of the gene-specific effects previously attributed to vhs-mediated mRNA degradation and resulted in a concordant loss in protein levels by 8h p.i. for many of the respective genes.

microbiology↗