Multi-omics reveals principles of gene regulation and pervasive non-productive transcription in the human cytomegalovirus genome

Jürges, Christopher; Lodha, Manivel; Le‐Trilling, Vu Thuy Khanh; Bhandare, Pranjali; Wolf, Elmar; Zimmermann, Albert; Trilling, Mirko; Prusty, Bhupesh K.; Dölken, Lars; Erhard, Florian

doi:10.1101/2022.01.07.472583

Cited by 9 publications

(14 citation statements)

References 59 publications

(92 reference statements)

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“…Importantly, the PRO-Cap approach not only detects stable transcripts but also transcription of highly unstable transcripts including promoterand enhancer-derived RNAs. Interestingly, dRNA-seq and STRIPE-seq analysis on HCMV-infected fibroblasts, which both only detect stable transcripts, only confirmed ≈1,700 of the >7,000 TSRs but indicated extensive non-productive (pervasive) transcription of the HCMV genome [45]. Our data for MCMV are consistent with our findings for HCMV showing that a large fraction of the >7,000 TSRs reported for HCMV presumably do not correspond to stable viral transcripts.…”

Section: Discussionsupporting

confidence: 87%

“…Tr1 and Tr3 promoters were associated with distinct TATA and TATT-box elements, respectively, readily explaining the expression of early and late genes as shown for various herpesviruses. Similar to HCMV [45], the TATT-motif in Tr3 promoters that is recognized by the viral LTF complex tended to be located by about 2 nt further upstream of the TiSS in comparison to the canonical TATA-box motif in the promoters of Tr1 and cellular genes. Tr1 gene expression peaked at 4 hpi following massive repression of transcriptional activity consistent with previous findings [46] describing a similar suppression for MCMV early genes peaking at 3 hpi including the m169 (MAT i.e., Most Abundant Transcript) and m152 genes (>500-fold downregulation) [46].…”

Section: Discussionmentioning

confidence: 99%

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Decoding murine cytomegalovirus

Lodha

Muchsin

Jürges

et al. 2022

Preprint

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The genomes of both human cytomegalovirus (HCMV) and murine cytomegalovirus (MCMV) were first sequenced over 20 years ago. Similar to HCMV, the MCMV genome had initially been proposed to harbor ≈170 open reading frames (ORFs). More recently, omics approaches revealed HCMV gene expression to be substantially more complex comprising several hundreds of translated ORFs. Here, we provide a state-of-the art reannotation of lytic MCMV gene expression based on integrative analysis of a large set of omics data. Our data reveal 363 viral transcription start sites (TiSS) that give rise to 380 and 454 viral transcripts and ORFs, respectively. The latter include >200 small ORFs, some of which represented the most highly expressed viral gene products. By combining TiSS profiling with metabolic RNA labelling and chemical nucleotide conversion sequencing (dSLAM-seq), we provide a detailed picture of the expression kinetics of viral transcription. This not only resulted in the identification of a novel MCMV immediate early transcript encoding the m166.5 ORF, which we termed ie4, but also revealed a group of well-expressed viral transcripts that are induced later than canonical true late genes and contain an initiator element (Inr) but no TATA- or TATT-box in their core promoters. We show that viral uORFs tune gene expression of longer viral ORFs expressed in cis at translational level. Finally, we identify a truncated isoform of the viral NK-cell immune evasin m145 arising from a viral TiSS downstream of the canonical m145 mRNA. Despite being ≈5-fold more abundantly expressed than the canonical m145 protein it was not required for downregulating the NK cell ligand, MULT-I. In summary, our work will pave the way for future mechanistic studies on previously unknown cytomegalovirus gene products in an important virus animal model.

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Section: Discussionsupporting

confidence: 87%

Section: Discussionmentioning

confidence: 99%

Decoding murine cytomegalovirus

Lodha

Muchsin

Jürges

et al. 2022

Preprint

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“…A major advantage of the nucleotide conversion approach, aside from lower requirements of starting material and a simplified experimental workflow, is that it can be combined with more specialized protocols, e.g. to profile transcription start sides [9] or ribosome occupancy [10]. Furthermore, we and others have combined 4sU conversion with single cell RNA-seq to study the heterogeneity of gene regulation [11][12][13].…”

Section: Introductionmentioning

confidence: 99%

grandR: a comprehensive package for nucleotide conversion sequencing data analysis

Rummel¹,

Sakellaridi²,

Erhard³

2022

Preprint

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Metabolic labeling of RNA is a powerful technique for studying the temporal dynamics of gene expression. Nucleotide conversion approaches greatly facilitate the generation of data but introduce challenges for their analysis. We here present grandR, a comprehensive package for quality control, differential gene expression analysis, kinetic modeling, and visualization of such data. We compare several existing methods for inference of RNA synthesis rates and half-lives using progressive labeling time courses. We demonstrate the need for recalibration of effective labeling times and introduce a Bayesian approach to study the temporal dynamics of RNA using snapshot experiments.

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“…Functional genomics data for the US10/11 locus were extracted from our recent integrative metaanalysis (Jürges et al, 2022). Briefly, primary human foreskin fibroblasts (HFFα) were infected with…”

Section: Transcriptional Analysis Of Us10 and Us11mentioning

confidence: 99%

Multimodal HLA-I genotype regulation by human cytomegalovirus US10 and resulting surface patterning

Gerke

Bauersfeld

Oberhardt

et al. 2023

Preprint

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To control human cytomegalovirus (HCMV) infection, NK cells and CD8+ T-cells are crucial. HLA class I (HLA-I) molecules play a central role for both NK and T-cell responses and are targets of multifaceted HCMV-encoded immunoevasins. A so far insufficiently studied HLA-I immunoevasin is the glycoprotein US10. It was shown that US10 targets HLA-G, but it is unknown whether US10 contributes also to escape from classical HLA-I antigen presentation. Our biochemical analysis revealed that early during maturation, all investigated HLA-I (HLA-A/B/C/E/G) heavy chains are recognized and bound by US10. Remarkably, the consequences of this initial binding strongly depended on both the HLA-I geno- and allotypes: i) HLA-A molecules escaped down-regulation by US10, ii) tapasin-dependent HLA-B molecules exhibited impaired recruitment to the peptide loading complex and maturation, iii) HLA-C and HLA-G, but not HLA-A/B/E, strongly bound US10 also in their β2m-assembled form. Thus, US10 senses geno- and allotypic differences in a so far unparalleled and multimodal manner, suggestive of adaptation to HLA-I genotype differences. At a further level of complexity, in HCMV-infected fibroblasts inhibition of overlapping US10 and US11 transcription revealed an additional HLA-I specificity, suggesting targeting of HLA-I in a synergistically arranged manner. Our study unveils the exceptional HLA-I selectivity of HCMV-encoded US10 and underlines its contribution to immune escape.

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Multi-omics reveals principles of gene regulation and pervasive non-productive transcription in the human cytomegalovirus genome

Cited by 9 publications

References 59 publications

Decoding murine cytomegalovirus

Decoding murine cytomegalovirus

grandR: a comprehensive package for nucleotide conversion sequencing data analysis

Multimodal HLA-I genotype regulation by human cytomegalovirus US10 and resulting surface patterning

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