Identification of cellular gene targets of anti-viral miR-27
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Murine cytomegalovirus (MCMV) encodes a non-coding RNA, m169, that inhibits the cellular miRNA, miR-27. Previous studies have shown that the overexpression of miR-27 in vitro suppresses replication of MCMV and degradation of miR-27 by m169 is important for the viral replication during the lytic stage of infection in vivo. To understand why the virus specifically targets this cellular miRNA for degradation, this thesis focuses on identification of cellular target genes of miR-27 that are involved in viral growth in the lytic infection. Microarray analysis was conducted to globally examine cellular genes differentially expressed following miR-27 overexpression or repression during MCMV infection. Data obtained from the microarray analysis were analysed in order to select potential targets of miR-27 for functional screening. Functional screening involved siRNA knockdown of individual genes followed by infection with a GFP reporter virus (GFP-MCMV) to assess the effects on viral growth. Knockdown of 5 out of 55 genes (Rpl18a, Lyar, Itga5, Mapkapk3 and Pik3r1) led to a significant reduction in GFP expression. Based on luciferase reporter assays, Mapkapk3 was validated as a direct target of miR-27 with a seed site interaction in its 3’UTR. Mutation of this site in the mRNA was shown to eliminate miR-27-mediated repression. Analysis of MAPKAPK3 protein levels upon infection demonstrates that the protein levels are higher in cells infected with wild type MCMV versus the m169 deletion virus (MCMV Δm169). This is in line with the difference in miR-27 levels in the two infections showing a decrease of miR-27 in wild type MCMV and unaltered levels in MCMV Δm169 infection. Mapkapk3 is a direct downstream target of p38 mitogen-activated protein (MAP) kinase within the p38 MAP kinase pathway, which has previously been shown to be an essential pathway for CMV replication. Expression levels of substrates of MAPKAPK3 including HSP27 and ATF1 were examined during infection to evaluate whether they are regulated by miR-27. The level of phosphorylation of HSP27 was shown to correlate with the levels of MAPKAPK3 during infection and was higher in cells infected with wild type MCMV versus MCMV Δm169. This suggests that MAPKAPK3 and its substrate, HSP27, are regulated by miR-27 during MCMV infection. This work provides an important foundation for further functional studies on the role of Mapkapk3 and its substrates in MCMV infection and its capacity to be dynamically regulated by miR-27. Based on the microarray analysis upon miR-27 overexpression, it was shown that miR-27 has an impact on the cell cycle, consistent with previous studies. Functional analysis of miR-27 in the cell cycle using miR-27 mimics and inhibitors demonstrated that the mimics cause an increase of cells in S phase at early time points (12 and 14 h), whereas the inhibition of miR-27 results in a significant reduction in the S phase population and accumulation of cells in G1 phase. Luciferase reporter assays confirmed that two genes known to be associated with the cell cycle are direct targets of miR-27: polycomb ring finger oncogene 1 (Bmi1) and caveolin 1 (Cav1). Knockdown of Bmi1 and Cav1 leads to a significant decrease in the number of cells in S phase and accumulation of cells in the G1 phase; however, this is the opposite result to that observed with the miR-27 mimics. These results suggest that the increase in cells in the S phase induced by miR-27 mimics is unlikely to be associated with targeting of Bmi1 and Cav1. Furthermore, knockdown of Bmi1 and Cav1 does not affect viral replication in vitro. Since miR-27 induces the transition of cells from the G1 to S phase, further studies are required to identify the miR-27 targets involved in this function. To identify direct targets of miR-27 through biochemical methods, one chapter of this thesis was devoted to developing CLASH datasets (cross-linking, ligation and sequencing of hybrid). This technique can directly map miRNA-mRNA interactions within the Argonaute protein (AGO). Initially, a NIH 3T3 stable cell line expressing AGO2 with a double affinity tag at the N terminus was generated. Analysis of the stable cell line revealed no significant alteration of miR-27 regulation or change in permissiveness to MCMV compared to wild type cells, making this amenable to further studies. Using the stable cell line, the CLASH protocol was carried out and preliminary data collected. In summary, this thesis identifies a direct target of miR-27, Mapkapk3, that is an important gene in MCMV replication that requires further investigation. Mapkapk3 is a substrate of p38 in the p38 MAP kinase pathway which is a signal transduction mediating numerous biological processes in response to cellular stresses including CMV infection. Furthermore, miR-27 overexpression was found to stimulate the G1/S transition of the cell cycle, and miR-27 inhibition had the opposite effect. Previous evidence has shown that MCMV and HCMV arrest the cell cycle in the G1 phase and inhibit host DNA synthesis to create an optimal condition for viral gene expression and DNA replication. Given that MCMV arrests host cells in the G1 phase, it is possible that degradation of miR-27 by MCMV contributes to this effect. Since miR-27 regulates both Mapkapk3 and the cell cycle, it seems likely that a number of targets and pathways underlie the antiviral properties of this miRNA.