Regulation and function of miR-199-3p in murine and human cytomegalovirus infections
Laqtom, Nouf Nasser Mohammad
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Human Cytomegalovirus (HCMV), the prototypic β-herpesvirus, is the most common cause of congenital infections as well as morbidity and mortality in immunocompromised patients. The anti-HCMV drugs currently available have a number of drawbacks (i.e. detrimental side-effects and/or the appearance of drug resistant strains), which limit their clinical usefulness. Therefore, a better understanding of host-virus interactions is important to develop new, safe and effective ways to treat HCMV. HCMV has evolved various strategies to make the host cell more conducive for the replication process, many of these involve modulation of host signalling pathways through proteins or non-coding RNAs. The focus of this thesis is on the regulation of one class of non-coding RNA, microRNAs (miRNA) by HCMV as well as murine CMV (MCMV). miRNAs are short ~22 nucleotide RNA sequences, which negatively regulate the stability and translational efficiency of specific target messenger RNAs (mRNAs). It has been previously shown that three host-encoded miRNAs, miR-199-3p, miR-199-5p and miR-214, are down-regulated in both MCMV and HCMV infected cells. Despite the biological and genomic differences between the two viruses, this down-regulation occurs in both infections, suggesting a possible conserved antiviral role of the miRNAs in mouse and human cells. Consistent with this, miR-199-3p and miR-214 manifest antiviral properties against MCMV and HCMV when over-expressed in vitro. This thesis investigates two hypotheses: 1) CMV down-regulates the expression of these host miRNAs through a mechanism involving viral factors, 2) The down-regulation of miR-199-3p leads to the up-regulation of its targets and this influences the cell in a way that favours some aspect of the viral life cycle. The first part of this project examined the regulation of miR-199-3p, miR-199-5p, and miR-214, which derive from a single primary transcript (pri-miRNA). The down-regulation of all three miRNAs was found to occur at the transcriptional level by 4 hours post infection. The promoter of the miR-199a/214 cluster was therefore cloned into a reporter vector in order to interrogate the factors regulating transcription of pri-miRNA in infection; this was carried out in the murine model based on availability of reagents. The reduction in the pri-miRNA was found to correlate with a decrease in the transcriptional activity of miR-199a/214 promoter in infected cells. Further analysis revealed the presence of a sequence between -421 to -273 relative to the transcription start site (TSS) that was critical for promoter activity. This sequence contains a putative serum response element (SRE), which includes two binding sites for the SRF dimer (serum response factor) and a binding site for a molecule of TCF (ternary complex factor), ELK-1. Initial knock-down studies suggest that these transcription factors are required for basal activity but it remains unknown whether they are involved in the differential expression of miR-199a/214 observed during infection. Another binding site for the transcription factor TWIST-1 was found outside this region, which is known to regulate the miR-199a/214 cluster in other cell types. Western blot analysis showed reduced expression of TWIST-1 in cells infected with HCMV and MCMV infections, by 24 and 48 hours, respectively, suggesting a role of TWIST-1 in regulating miR-199a/214 cluster during these infections. This regulation seems to be dependent on viral gene expression, as a replication deficient viral mutant fails to repress the promoter function and subsequent pri-miRNA production. Taken together, these results suggest an active viral mechanism for transcriptional repression of the miR-199a/214 promoter. To understand the antiviral function of miR-199-3p, the second part of this thesis examined whether miR-199-3p regulates host signalling pathways important for CMV replication and/or the life cycle. A microarray analysis was carried out with samples from cells transfected with miR- 199-3p mimic versus inhibitor. This revealed 198 genes significantly down-regulated by the miRNA. From the 198 genes, Ingenuity pathway analysis (IPA) software identified several host pathways with a potential role in HCMV infection including: PI3K/AKT signalling, the ERK-MAPK cascade, and prostaglandin production. This thesis examined the role of miR-199-3p in regulating the PI3K/AKT pathway in HCMV infection. It was found that miR-199-3p modulates the phosphorylation of the central regulator of PI3K/AKT signalling, AKT. Transfection of miR-199-3p before the infection impedes the complete phosphorylation of AKT, which is known to be required for the immediate early viral gene expression and replication. This provides an explanation for the antiviral function of miR-199-3p, through its ability to modulate AKT phosphorylation. An open question, however, is how the natural down-regulation of miR-199-3p from 24 to 72 hours post infection naturally affects AKT phosphorylation. Several predicted targets of miR-199-3p, such as PIK3CB, ITGA3, and ITGA6 were shown to be up-regulated at these late time points, correlating with the miR-199-3p down-regulation. The interaction of miR-199-3p with target sites in the 3′UTRs of PIK3CB and ITGA3 was validated by luciferase reporter assays and western blotting and qRT-PCR results indicated that protein and mRNA levels of ITGA6 were regulated by miR-199-3p mimic transfection. However, the knock-down of these three targets did not result in a significant decrease of the viral growth, and thus cannot alone explain the antiviral function of miR-199-3p. Overall, this study suggests that the transcriptional repression of miR- 199a/214 is likely a strategy employed by CMV to support its own growth through attenuating the biological effect of miR-199-3p within the host cell.