TY - INPR A1 - Hennig, Thomas A1 - Prusty, Archana B. A1 - Kaufer, Benedikt A1 - Whisnant, Adam W. A1 - Lodha, Manivel A1 - Enders, Antje A1 - Thomas, Julius A1 - Kasimir, Francesca A1 - Grothey, Arnhild A1 - Herb, Stefanie A1 - Jürges, Christopher A1 - Meister, Gunter A1 - Erhard, Florian A1 - Dölken, Lars A1 - Prusty, Bhupesh K. T1 - Selective inhibition of microRNA processing by a herpesvirus-encoded microRNA triggers virus reactivation from latency N2 - Herpesviruses have mastered host cell modulation and immune evasion to augment productive infection, life-long latency and reactivation thereof 1,2. A long appreciated, yet elusively defined relationship exists between the lytic-latent switch and viral non-coding RNAs 3,4. Here, we identify miRNA-mediated inhibition of miRNA processing as a novel cellular mechanism that human herpesvirus 6A (HHV-6A) exploits to disrupt mitochondrial architecture, evade intrinsic host defense and drive the latent-lytic switch. We demonstrate that virus-encoded miR-aU14 selectively inhibits the processing of multiple miR-30 family members by direct interaction with the respective pri-miRNA hairpin loops. Subsequent loss of miR-30 and activation of miR-30/p53/Drp1 axis triggers a profound disruption of mitochondrial architecture, which impairs induction of type I interferons and is necessary for both productive infection and virus reactivation. Ectopic expression of miR-aU14 was sufficient to trigger virus reactivation from latency thereby identifying it as a readily drugable master regulator of the herpesvirus latent-lytic switch. Our results show that miRNA-mediated inhibition of miRNA processing represents a generalized cellular mechanism that can be exploited to selectively target individual members of miRNA families. We anticipate that targeting miR-aU14 provides exciting therapeutic options for preventing herpesvirus reactivations in HHV-6-associated disorders like myalgic encephalitis/chronic fatigue syndrome (ME/CFS) and Long-COVID. KW - Herpesvirus KW - HHV-6 KW - miRNA processing KW - miR-30 KW - mitochondria KW - fusion and fission KW - type I interferon KW - latency KW - virus reactivation Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-267858 UR - https://doi.org/10.21203/rs.3.rs-820696/v1 ET - submitted version ER - TY - THES A1 - Herb, Stefanie Maria T1 - Regulation of MCMV immediate early gene expression by virally encoded miRNAs T1 - Regulation der MCMV immediate early Genexpression durch viral kodierte miRNAs N2 - Gene expression in eukaryotic cells is regulated by the combinatorial action of numerous gene-regulatory factors, among which microRNAs (miRNAs) play a fundamental role at the post-transcriptional level. miRNAs are single-stranded, small non-coding RNA molecules that emerge in a cascade-like fashion via the generation of primary and precursor miRNAs. Mature miRNAs become functional when incorporated into the RNA induced silencing complex (RISC). miRNAs guide RISCs to target mRNAs in a sequence-specific fashion. To this end, base-pairs are usually formed between the miRNA seed region, spanning nucleotide positions 2 to 8 (from the 5' end) and the 3'UTR of the target mRNA. Once miRNA-mRNA interaction is established, RISC represses translation and occasionally induces direct or indirect target mRNA degradation. Interestingly, miRNAs are expressed not only in every multicellular organism but are also encoded by several viruses, predominately by herpesviruses. By controlling both, cellular as well as viral mRNA transcripts, virus-encoded miRNAs confer many beneficial effects on viral growth and persistence. Murine cytomegalovirus (MCMV) is a ß-herpesvirus and so far, 29 mature MCMV-encoded miRNAs have been identified during lytic infection. Computational analysis of previously conducted photoactivated ribonucleotide-enhanced individual nucleotide resolution crosslinking immunoprecipitation (PAR-iCLIP) experiments identified a read cluster within the 3' untranslated region (3'UTR) of the immediate early 3 (IE3) transcript in MCMV. Based on miRNA target predictions, two highly abundant MCMV miRNAs, namely miR-m01-2-3p and miR-M23-2-3p were found to potentially bind to two closely positioned target sites within the IE3 PAR-iCLIP peak. To confirm this hypothesis, we performed luciferase assays and showed that activity values of a luciferase fused with the 3'UTR of IE3 were downregulated in the presence of miR-m01- 2 and miR-M23-2. In a second step, we investigated the effect of pre-expression of miR-m01-2 and miR-M23-2 on the induction of virus replication. After optimizing the transfection procedure by comparing different reagents and conditions, plaque formation was monitored. We could demonstrate that the replication cycle of the wild-type but not of our MCMV mutant that harbored point mutations in both miRNA binding sites within the IE3-3'UTR, was significantly delayed in the presence of miR-m01-2 and miR-M23-2. This confirmed that miR-m01-2 and miR-M23-2 functionally target the major transcription factor IE3 which acts as an indispensable regulator of viral gene expression during MCMV lytic infection. Repression of the major immediate early genes by viral miRNAs is a conserved feature of cytomegaloviruses. The functional role of this type of regulation can now be studied in the MCMV mouse model. N2 - In eukaryotischen Zellen wird die Expression von Genen durch das Zusammenspiel vieler verschiedener biologischer Regulatoren, wie microRNAs (miRNAs), kontrolliert. MiRNAs sind einzelsträngige, kurze, nicht-kodierende RNA-Moleküle, die aus sogenannten primären miRNAs und Vorläufer-miRNAs entstehen und die Genexpression auf Ebene der Posttranskription beeinflussen. Um ihre Funktion ausüben zu können, werden reife miRNAs in RNA-induzierte Silencing-Komplexe (RISCs) eingebaut und zu ihren Ziel-mRNAs geführt. Durch Wechselwirkungen zwischen der miRNA "seed-Region , die die Nukleotide 2 bis 8 vom 5'-Ende überspannt und der 3'UTR (3' untranslatierte Region) der Ziel-mRNA, unterdrückt RISC die Translation der Ziel-mRNA und kann deren Abbau durch direkte sowie indirekte Mechanismen induzieren. Die Expression von miRNAs wurde nicht nur in multizellulären Organismen, sondern in bereits zahlreichen Viren, insbesondere in der Virusfamilie der Herpesviridae, nachgew- iesen. Viruskodierte miRNAs kontrollieren dabei zelluläre wie auch virale mRNA-Transkripte und verleihen dem Virus einen Selektionsvorteil bzgl. Wachstum und Persistenz. Das mur- ine Cytomegalievirus (MCMV) ist ein β-Herpesvirus, das nach aktuellem Wissensstand 29 reife miRNAs kodiert, die allesamt während der lytischen Infektion identifziert wurden. Bioinformatische Analysen eines vor dieser Arbeit durchgeführten PAR-iCLIP-Experiments (photoactivated ribonucleotide-enhanced individual nucleotide resolution crosslinking and immunoprecipitation), zeigten einen PAR-iCLIP Peak in der 3'UTR (3' untranslatierte Region) des immediate early 3-Transkripts (IE3) von MCMV. Unter Verwendung von RNAhbybrid, einem miRNA target prediction tool, fanden sich zwei virale miRNAs, näm- lich miR-m01-2-3p und miR-M23-2-3p mit potentiellen Bindestellen innerhalb der 3'UTR des MCMV IE3 Transkripts. Unsere konsekutiv durchgeführten Luciferase-Assays be- stätigten, dass sowohl miR-m01-2 als auch miR-M23-2 an die 3'UTR von IE3 binden. Beide viralen miRNAs führten zu einer verminderten Luciferaseaktivität unter Verwendung von Reportern, in denen die 3'UTR des IE3-Gens mit dem Luciferase-Transkript fusioniert war. xxiv Summary Das IE3 Protein gilt während des lytischen Zykluses als einer der wichtigsten Transkrip- tionsfaktoren von MCMV. Ebenfalls wurde der Einfluss der beiden viralen miRNAs auf die virale Reproduktion von uns untersucht. Hierfür wurden murine Zelllinien vor Infektion mit miR-m01-2 und miR- M23-2 transziert. Das Transfektionsverfahren optimierten wir zunächst durch Testung verschiedener Reagenzien und experimenteller Bedingungen. Schließlich zeigten wir mittels Plaqueassays, dass eine vor Infektion durchgeführte Transfektion mit miR-m01-2 und miR- M23-2 die Replikation von MCMV signifikant verzögerte. Unter Verwendung einer MCMV- Mutante, die durch Punktmutationen in beiden miRNA-Bindungsstellen innerhalb der IE3- 3'UTR charakterisiert war, ließ sich dieser Effekt aufheben. Unsere Experimente weisen somit stark darauf hin, dass miR-m01-2 und miR-M23-2 die Expression des IE3 Proteins regulieren und damit indirekt Einfluss auf die Genexpression während der lytischen Phase des Replikationszykluses von MCMV nehmen. Die miRNA-mediierte Repression der immediate early Genexpression stellt ein evolutionär konserviertes Merkmal von Zytomegalieviren dar. Für eine weitere Einordnung der Rolle dieser Genexpressionskontrolle bedarf es zukünftige Untersuchungen im MCMV-Tiermodell KW - miRNS KW - Cytomegalie-Virus KW - Herpes KW - Frühe Gene KW - PAR-CLIP KW - MCMV KW - miRNA KW - immediate early genes KW - lytic infection KW - IE3 KW - miRNA target KW - luciferase assay KW - CMV KW - HCMV KW - viral miRNAs Y1 - 2023 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-323314 ER - TY - INPR A1 - Hennig, Thomas A1 - Prusty, Archana B. A1 - Kaufer, Benedikt A1 - Whisnant, Adam W. A1 - Lodha, Manivel A1 - Enders, Antje A1 - Thomas, Julius A1 - Kasimir, Francesca A1 - Grothey, Arnhild A1 - Herb, Stefanie A1 - Jürges, Christopher A1 - Meister, Gunter A1 - Erhard, Florian A1 - Dölken, Lars A1 - Prusty, Bhupesh K. T1 - Selective inhibition of miRNA 1 processing by a herpesvirus encoded miRNA N2 - Herpesviruses have mastered host cell modulation and immune evasion to augment productive infection, life-long latency and reactivation thereof 1,2. A long appreciated, yet elusively defined relationship exists between the lytic-latent switch and viral non-coding RNAs 3,4. Here, we identify miRNA-mediated inhibition of miRNA processing as a thus far unknown cellular mechanism that human herpesvirus 6A (HHV-6A) exploits to disrupt mitochondrial architecture, evade intrinsic host defense and drive the lytic-latent switch. We demonstrate that virus-encoded miR-aU14 selectively inhibits the processing of multiple miR-30 family members by direct interaction with the respective pri-miRNA hairpin loops. Subsequent loss of miR-30 and activation of the miR-30/p53/Drp1 axis triggers a profound disruption of mitochondrial architecture. This impairs induction of type I interferons and is necessary for both productive infection and virus reactivation. Ectopic expression of miR-aU14 triggered virus reactivation from latency, identifying viral miR-aU14 as a readily drugable master regulator of the herpesvirus lytic-latent switch. Our results show that miRNA-mediated inhibition of miRNA processing represents a generalized cellular mechanism that can be exploited to selectively target individual members of miRNA families. We anticipate that targeting miR-aU14 provides exciting therapeutic options for preventing herpesvirus reactivations in HHV-6-associated disorders. KW - Herpesvirus KW - HHV-6A KW - miRNA processing KW - miR-30 KW - mitochondria KW - fusion and fission KW - type I interferon KW - latency KW - virus reactivation Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-267862 ET - accepted version ER -