@phdthesis{Herb2023,
  author    = {Herb, Stefanie Maria},
  title     = {Regulation of MCMV immediate early gene expression by virally encoded miRNAs},
  doi       = {10.25972/OPUS-32331},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-323314},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2023},
  abstract  = {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.},
  subject      = {miRNS},
  language  = {en}
}