The Cellular Mirna, Mir-190, is Upregulated in Type I Ebv Latency by Ebers and Modulates Cellular Mrnas involved in Cell Survival and Viral Reactivation

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Doctor of Philosophy (PhD)
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Cell & Molecular Biology
Type I latency
Cell Biology
Molecular Biology
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Cramer, Elizabeth Mary

Epstein-Barr Virus (EBV) is a highly prevalent human pathogen infecting over 90% of the population. Much of the success of the virus is attributed to its ability to maintain latency through different programs in host cells. MicroRNAs (miRNA) are small, non-coding RNAs capable of post-transcriptionally regulating mRNA expression. A microarray comparison of EBV type I latency and type III latency infected cells yielded evidence of differential cellular microRNA expression. I hypothesized that one of these differentially upregulated type I latency miRNAs, miR-190, is important in maintenance of latency I, and miR-190 upregulation is due to viral gene expression. Lentiviral overexpression systems were used to overexpress miR-190 and a microarray of gene expression revealed candidate miR-190 targets, including: TP53INP1 and NR4A3. The modulation of these targets by miR-190 was confirmed through evaluating mRNA and protein level changes in the presence or absence of miR-190. In the case of TP53INP1, a 3’UTR target site was identified through mutagenesis. The effect of miR-190 expression was evaluated for markers of cell cycle and cell death by flow cytometry, western blot and RT-PCR. Measures of viral reactivation were lowered in the presence of miR-190 after induction by anti-IgG stimulation. I also observed upregulation of miR-190/Talin2 promoter activity or miR-190 expression in the presence of EBERs, Epstein-Barr encoded RNAs. Interestingly, a panel of type I latency cell lines had higher EBER1 expression compared to their type III latency counterparts. Work by others has indicated that EBERs activate the double-stranded RNA (dsRNA) sensor, retinoic acid-inducible gene 1 (RIG-I). Transiently expressed, constitutively activated RIG-I induced miR-190 expression and promoter activity. Knockdown of RIG-I in the type I latency cells yielded lowered miR-190 expression levels. To investigate how miR-190 is upregulated I generated miR-190/Talin2 promoter reporters that lacked YinYang1 (YY1) and Nuclear factor-κB (NF-kB) binding motifs. In the presence of EBERs, promoters with these deleted binding motifs had lowered activation compared to the full miR-190/TLN2 promoter. This work describes a mechanism by which EBERs upregulate a cellular miRNA, miR-190, which aids in type I latency preservation by preventing apoptosis, promoting cell cycle and maintaining virus in its latent state.

Yan Yuan
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