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Target identification of MHV68 mirnas in lytic replication, latency and reactivation and characterization of a miRNA-antisense long noncoding RNA

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  1. Tez No: 403026
  2. Yazar: MEHMET KARA
  3. Danışmanlar: DR. SCOTT TIBBETTS
  4. Tez Türü: Doktora
  5. Konular: Mikrobiyoloji, Microbiology
  6. Anahtar Kelimeler: Belirtilmemiş.
  7. Yıl: 2016
  8. Dil: İngilizce
  9. Üniversite: University of Florida
  10. Enstitü: Yurtdışı Enstitü
  11. Ana Bilim Dalı: Belirtilmemiş.
  12. Bilim Dalı: Belirtilmemiş.
  13. Sayfa Sayısı: 151

Özet

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Özet (Çeviri)

Gammaherpesviruses, including the human pathogens Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV), are oncogenic viruses that establish lifelong infections in hosts and are associated with the development of lymphoproliferative diseases and lymphomas. Murine gammaherpesvirus 68 (MHV68) is a natural pathogen of rodents, and is genetically and pathogenically related to EBV and KSHV, providing a highly tractable model for studies of gammaherpesvirus biology and pathogenesis. miRNAs are ~20-22 nt long, noncoding RNA molecules that can inhibit gene expression by translational repression through selective binding of a target transcript. Several studies have been conducted to understand the role of miRNA in MHV68 during latency however, none of these miRNA targets have been identified. Here we utilized crosslinking immunoprecipitation and sequencing of hybrids (CLASH) technique to identify viral miRNA targets during latency, reactivation and lytic replication. Recent studies have shown that the majority of the mammalian genome is transcribed and gives rise to numerous long noncoding RNAs (lncRNAs). Likewise, it has recently been shown that the large double-stranded DNA virus genomes of herpesviruses undergo pervasive transcription, including the expression of many uncharacterized putative lncRNAs. Previous tiled microarray studies identified 30 novel“expressed genomic regions”(EGRs) of MHV68 transcription that were not predicted by previous canonical ORF-based annotation analyses. We sought to determine whether EGR1, which lies antisense to at least five MHV68-encoded miRNAs, encodes a bona fide lncRNA transcript. Using strand-specific northern blots, we identified a polyadenylated nuclear transcript that overlaps the important latency-associated genes M2 and M3. Knockdown of this transcript M3M2 in lytically infected cells using antisense oligonucleotides did not change viral growth kinetics and viral protein expression levels However, it might regulate M2 protein level through transcriptional interference. Furthermore, infection with a mutant virus lacking two M3M2-antisense pre-miRNA stem loops results in increased expression of M3M2, strongly suggesting its regulation by viral miRNAs. Thus, together these data demonstrate a tripartite relationship between lncRNA M3M2, an antisense miRNA, and the important latency gene M2. Based on the importance of M2 in latent infection, we hypothesize that this relationship may be a key control point for chronic infection and pathogenesis.

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