Lytic herpes simplex virus 1 (HSV-1) infection triggers disruption of transcription termination (DoTT) of most cellular genes, resulting in extensive intergenic transcription. Similarly, cellular ...stress responses lead to gene-specific transcription downstream of genes (DoG). In this study, we performed a detailed comparison of DoTT/DoG transcription between HSV-1 infection, salt and heat stress in primary human fibroblasts using 4sU-seq and ATAC-seq. Although DoTT at late times of HSV-1 infection was substantially more prominent than DoG transcription in salt and heat stress, poly(A) read-through due to DoTT/DoG transcription and affected genes were significantly correlated between all three conditions, in particular at earlier times of infection. We speculate that HSV-1 either directly usurps a cellular stress response or disrupts the transcription termination machinery in other ways but with similar consequences. In contrast to previous reports, we found that inhibition of Ca2+ signaling by BAPTA-AM did not specifically inhibit DoG transcription but globally impaired transcription. Most importantly, HSV-1-induced DoTT, but not stress-induced DoG transcription, was accompanied by a strong increase in open chromatin downstream of the affected poly(A) sites. In its extent and kinetics, downstream open chromatin essentially matched the poly(A) read-through transcription. We show that this does not cause but rather requires DoTT as well as high levels of transcription into the genomic regions downstream of genes. This raises intriguing new questions regarding the role of histone repositioning in the wake of RNA Polymerase II passage downstream of impaired poly(A) site recognition.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
High-risk human papillomaviruses (HPVs), including HPV-16 and HPV-18, are the causative agents of cervical carcinomas and are linked to several other tumors of the anogenital and oropharyngeal ...regions. The majority of HPV-induced tumors contain integrated copies of the normally episomal HPV genome that invariably retain intact forms of the two HPV oncogenes E6 and E7. E6 induces degradation of the cellular tumor suppressor p53, while E7 destabilizes the retinoblastoma (Rb) protein. Previous work has shown that loss of E6 function in cervical cancer cells induces p53 expression as well as downstream effectors that induce apoptosis and cell cycle arrest. Similarly, loss of E7 allows increased Rb expression, leading to cell cycle arrest and senescence. Here, we demonstrate that expression of a bacterial Cas9 RNA-guided endonuclease, together with single guide RNAs (sgRNAs) specific for E6 or E7, is able to induce cleavage of the HPV genome, resulting in the introduction of inactivating deletion and insertion mutations into the E6 or E7 gene. This results in the induction of p53 or Rb, leading to cell cycle arrest and eventual cell death. Both HPV-16- and HPV-18-transformed cells were found to be responsive to targeted HPV genome-specific DNA cleavage. These data provide a proof of principle for the idea that vector-delivered Cas9/sgRNA combinations could represent effective treatment modalities for HPV-induced cancers. Importance: Human papillomaviruses (HPVs) are the causative agents of almost all cervical carcinomas and many other tumors, including many head and neck cancers. In these cancer cells, the HPV DNA genome is integrated into the cellular genome, where it expresses high levels of two viral oncogenes, called E6 and E7, that are required for cancer cell growth and viability. Here, we demonstrate that the recently described bacterial CRISPR/Cas RNA-guided endonuclease can be reprogrammed to target and destroy the E6 or E7 gene in cervical carcinoma cells transformed by HPV, resulting in cell cycle arrest, leading to cancer cell death. We propose that viral vectors designed to deliver E6- and/or E7-specific CRISPR/Cas to tumor cells could represent a novel and highly effective tool to treat and eliminate HPV-induced cancers.
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, which should spare circular RNAs ...(circRNAs). Here, we show that vhs-mediated degradation of linear mRNAs leads to an enrichment of circRNAs relative to linear mRNAs during HSV-1 infection. This was also observed in influenza A virus (IAV) infection, likely due to degradation of linear host mRNAs mediated by the IAV PA-X protein and cap-snatching RNA-dependent RNA polymerase. For most circRNAs, enrichment was not due to increased circRNA synthesis but due to a general loss of linear RNAs. In contrast, biogenesis of a circRNA originating from the long isoform (NEAT1_2) of the nuclear paraspeckle assembly transcript 1 (NEAT1) was induced both in HSV-1 infection-in a vhs-independent manner-and in IAV infection. This was associated with induction of novel linear splicing of NEAT1_2 both within and downstream of the circRNA. NEAT1_2 forms a scaffold for paraspeckles, nuclear bodies located in the interchromatin space, must likely remain unspliced for paraspeckle assembly and is up-regulated in HSV-1 and IAV infection. We show that NEAT1_2 splicing and up-regulation can be induced by ectopic co-expression of the HSV-1 immediate-early proteins ICP22 and ICP27, potentially linking increased expression and splicing of NEAT1_2. To identify other conditions with NEAT1_2 splicing, we performed a large-scale screen of published RNA-seq data. This uncovered both induction of NEAT1_2 splicing and poly(A) read-through similar to HSV-1 and IAV infection in cancer cells upon inhibition or knockdown of CDK7 or the MED1 subunit of the Mediator complex phosphorylated by CDK7. In summary, our study reveals induction of novel circular and linear NEAT1_2 splicing isoforms as a common characteristic of HSV-1 and IAV infection and highlights a potential role of CDK7 in HSV-1 or IAV infection.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Infection by viruses, including herpes simplex virus-1 (HSV-1), and cellular stresses cause widespread disruption of transcription termination (DoTT) of RNA polymerase II (RNAPII) in host genes. ...However, the underlying mechanisms remain unclear. Here, we demonstrate that the HSV-1 immediate early protein ICP27 induces DoTT by directly binding to the essential mRNA 3' processing factor CPSF. It thereby induces the assembly of a dead-end 3' processing complex, blocking mRNA 3' cleavage. Remarkably, ICP27 also acts as a sequence-dependent activator of mRNA 3' processing for viral and a subset of host transcripts. Our results unravel a bimodal activity of ICP27 that plays a key role in HSV-1-induced host shutoff and identify CPSF as an important factor that mediates regulation of transcription termination. These findings have broad implications for understanding the regulation of transcription termination by other viruses, cellular stress and cancer.
Eukaryotic gene expression is extensively regulated by cellular stress and pathogen infections. We have previously shown that herpes simplex virus 1 (HSV-1) and several cellular stresses cause ...widespread disruption of transcription termination (DoTT) of RNA polymerase II (RNAPII) in host genes and that the viral immediate early factor ICP27 plays an important role in HSV-1-induced DoTT. Here, we show that HSV-1 infection also leads to widespread changes in alternative polyadenylation (APA) of host mRNAs. In the majority of cases, polyadenylation shifts to upstream poly(A) sites (PAS), including many intronic PAS. Mechanistically, ICP27 contributes to HSV-1-mediated APA regulation. HSV-1- and ICP27-induced activation of intronic PAS is sequence-dependent and does not involve general inhibition of U1 snRNP. HSV1-induced intronic polyadenylation is accompanied by early termination of RNAPII. HSV-1-induced mRNAs polyadenylated at intronic PAS (IPA) are exported into the cytoplasm while APA isoforms with extended 3' UTRs are sequestered in the nuclei, both preventing the expression of the full-length gene products. Finally we provide evidence that HSV-induced IPA isoforms are translated. Together with other recent studies, our results suggest that viral infection and cellular stresses induce a multi-faceted host response that includes DoTT and changes in APA profiles.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The issue of whether viruses are subject to restriction by endogenous microRNAs (miRNAs) and/or by virus-induced small interfering RNAs (siRNAs) in infected human somatic cells has been ...controversial. Here, we address this question in two ways. First, using deep sequencing, we demonstrate that infection of human cells by the RNA virus dengue virus (DENV) or West Nile virus (WNV) does not result in the production of any virus-derived siRNAs or viral miRNAs. Second, to more globally assess the potential of small regulatory RNAs to inhibit virus replication, we used gene editing to derive human cell lines that lack a functional Dicer enzyme and that therefore are unable to produce miRNAs or siRNAs. Infection of these cells with a wide range of viruses, including DENV, WNV, yellow fever virus, Sindbis virus, Venezuelan equine encephalitis virus, measles virus, influenza A virus, reovirus, vesicular stomatitis virus, human immunodeficiency virus type 1, or herpes simplex virus 1 (HSV-1), failed to reveal any enhancement in the replication of any of these viruses, although HSV-1, which encodes at least eight Dicer-dependent viral miRNAs, did replicate somewhat more slowly in the absence of Dicer. We conclude that most, and perhaps all, human viruses have evolved to be resistant to inhibition by endogenous human miRNAs during productive replication and that dependence on a cellular miRNA, as seen with hepatitis C virus, is rare. How viruses have evolved to avoid inhibition by endogenous cellular miRNAs, which are generally highly conserved during metazoan evolution, remains to be determined. Importance: Eukaryotic cells express a wide range of small regulatory RNAs, including miRNAs, that have the potential to inhibit the expression of mRNAs that show sequence complementarity. Indeed, previous work has suggested that endogenous miRNAs have the potential to inhibit viral gene expression and replication. Here, we demonstrate that the replication of a wide range of pathogenic viruses is not enhanced in human cells engineered to be unable to produce miRNAs, indicating that viruses have evolved to be resistant to inhibition by miRNAs. This result is important, as it implies that manipulation of miRNA levels is not likely to prove useful in inhibiting virus replication. It also focuses attention on the question of how viruses have evolved to resist inhibition by miRNAs and whether virus mutants that have lost this resistance might prove useful, for example, in the development of attenuated virus vaccines.
Herpes simplex virus 1 (HSV-1) infection exerts a profound shutoff of host gene expression at multiple levels. Recently, HSV-1 infection was reported to also impact promoter-proximal RNA polymerase ...II (Pol II) pausing, a key step in the eukaryotic transcription cycle, with decreased and increased Pol II pausing observed for activated and repressed genes, respectively. Here, we demonstrate that HSV-1 infection induces more complex alterations in promoter-proximal pausing than previously suspected for the vast majority of cellular genes. While pausing is generally retained, it is shifted to more downstream and less well-positioned sites for most host genes. The downstream shift of Pol II pausing was established between 1.5 and 3 h of infection, remained stable until at least 6 hours postinfection, and was observed in the absence of ICP22. The shift in Pol II pausing does not result from alternative
transcription initiation at downstream sites or read-in transcription originating from disruption of transcription termination of upstream genes. The use of downstream secondary pause sites associated with +1 nucleosomes was previously observed upon negative elongation factor (NELF) depletion. However, downstream shifts of Pol II pausing in HSV-1 infection were much more pronounced than observed upon NELF depletion. Thus, our study reveals a novel aspect in which HSV-1 infection fundamentally reshapes host transcriptional processes, providing new insights into the regulation of promoter-proximal Pol II pausing in eukaryotic cells.
This study provides a genome-wide analysis of changes in promoter-proximal polymerase II (Pol II) pausing on host genes induced by HSV-1 infection. It shows that standard measures of pausing, i.e., pausing indices, do not properly capture the complex and unsuspected alterations in Pol II pausing occurring in HSV-1 infection. Instead of a reduction of pausing with increased elongation, as suggested by pausing index analysis, HSV-1 infection leads to a shift of pausing to downstream and less well-positioned sites than in uninfected cells for the majority of host genes. Thus, HSV-1 infection fundamentally reshapes a key regulatory step at the beginning of the host transcriptional cycle on a genome-wide scale.
Although RNA interference (RNAi) functions as a potent antiviral innate-immune response in plants and invertebrates, mammalian somatic cells appear incapable of mounting an RNAi response and few, if ...any, small interfering RNAs (siRNAs) can be detected. To examine why siRNA production is inefficient, we have generated double-knockout human cells lacking both Dicer and protein kinase RNA-activated. Using these cells, which tolerate double-stranded RNA expression, we show that a mutant form of human Dicer lacking the amino-terminal helicase domain can process double-stranded RNAs to produce high levels of siRNAs that are readily detectable by Northern blot, are loaded into RNA-induced silencing complexes, and can effectively and specifically inhibit the expression of cognate mRNAs. Remarkably, overexpression of this mutant Dicer, but not wild-type Dicer, also resulted in a partial inhibition of Influenza A virus—but not poliovirus—replication in human cells.
During lytic infection, herpes simplex virus (HSV) 1 induces a rapid shutoff of host RNA synthesis while redirecting transcriptional machinery to viral genes. In addition to being a major human ...pathogen, there is burgeoning clinical interest in HSV as a vector in gene delivery and oncolytic therapies, necessitating research into transcriptional control. This review summarizes the array of impacts that HSV has on RNA Polymerase (Pol) II, which transcribes all mRNA in infected cells. We discuss alterations in Pol II holoenzymes, post-translational modifications, and how viral proteins regulate specific activities such as promoter-proximal pausing, splicing, histone repositioning, and termination with respect to host genes. Recent technological innovations that have reshaped our understanding of previous observations are summarized in detail, along with specific research directions and technical considerations for future studies.
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