Dissecting Herpes Simplex Virus 1-Induced Host Shutoff at the RNA Level

Abstract

Herpes simplex virus 1 (HSV-1) induces 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 host and viral mRNAs. Furthermore, the onset of viral DNA replication is accompanied by a rapid decline in host transcriptional activity. To dissect relative contributions of both mechanisms and elucidate gene-specific host transcriptional responses throughout the first 8h of lytic HSV-1 infection, we employed RNA-seq of total, newly transcribed (4sU-labelled) and chromatin-associated RNA in wild-type (WT) and Δvhs 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 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 and was surprisingly concordant between WT and Δvhs infection. Both induced strong transcriptional up-regulation of a small subset of genes that were poorly 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 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. IMPORTANCE The 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 virus-induced global loss of host transcriptional activity during productive HSV-1 infection. In general, HSV-1-induced alterations in total RNA levels were dominated by these two global effects. In contrast, chromatin-associated RNA depicted gene-specific transcriptional changes. This revealed highly concordant transcriptional changes in WT and Δvhs 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 components. The latter explained seemingly 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.

The herpes simplex virus 1 protein ICP4 acts as both an activator and repressor of host genome transcription during infection

“…We found that within 4h of HSV-1 infection of human cells there are dramatic changes to the host cell mRNA transcriptional program, with hundreds of genes losing Pol II occupancy and hundreds of others gaining Pol II. This is largely consistent with the transcriptional reprogramming observed in prior studies that also studied the effect of HSV-1 infection on host cell transcription in human cells looking at nascent mRNA production via PRO-seq or 4SU-seq rather than using Pol II ChIP-seq [10,11,13,39,40]. The effect of HSV-1 infection on Pol II occupancy in human cells is significantly different from that seen in mouse cells where only the reduction of Pol II occupancy was observed after 4h of infection with little to no activation [12], suggesting that the activation of Pol II transcription is a signature specific to the host species.…”

Herpesviral induction of germline transcription factor DUX4 is critical for viral gene expression

“…In a recent study, we observed that the upregulation of the cellular protein Tripartite-motif 43 (TRIM43) upon HSV-1 infection is dependent on the embryonic transcription factor DUX4 7 , which was recently confirmed by Friedel et al 8 . DUX4 is a germline transcription factor exclusively expressed from the 4-cell to the 8-cell state, a short phase during human embryonic development [9][10][11] .…”

A Review of the Multipronged Attack of Herpes Simplex Virus 1 on the Host Transcriptional Machinery

“…While recent studies have identified many interesting findings from total RNA analysis [131][132][133][134][135], general conclusions at the level of transcription are complicated by the varying technical methods and biological contexts utilized. Interestingly, nuclease activity of VHS globally reduced Pol II transcription of host genes, as was observed before with the SOX nuclease from murine gamma-herpesvirus 68 (MHV68) [111,136]. A proper discussion of host mRNA accumulation, its translation, and resulting outcomes on infection, particularly regarding innate immunity, is outside the scope of this review.…”

“…Such techniques include those using chemical labeling (e.g., 4sU-Seq), long-read sequencing, or minimally normalization of polyadenylated RNA to chromatin-associated rather than total/total nuclear RNA. Chromatin-associated RNA closely matches nascent transcriptomes determined by chemical labeling in HSV infection [111], and is currently the most cost-effective and procedurally simple approach to differentiate co-from post-transcriptional events directly compatible with established HSV infection protocols for total RNA.…”

“…While microarray studies indicated the seeming induction of several host genes [128][129][130], particularly antiviral responses, viral and cellular profiles induced are highly variable between individual cells and depend on various factors that include virus dose, virus stock quality, viral genetic variability, the status of the cell and cell type tested. Notably, RNA-Seq data has to be carefully analyzed to avoid misinterpretations as many transcripts are affected by termination failure and, in turn, are atypically spliced, might extend into downstream genes, and, more importantly, fail to get exported to the cytoplasm [81,82,111]. The post-transcriptional stability and, thereby, viral and cellular RNA levels are regulated by the nuclease activity of the aptly named virion host shutoff (VHS) protein, further complicating conclusions of transcriptional regulation from the level of total, cytoplasmic, or polyadenylated RNA.…”