Identification of targets of Human Cytomegalovirus microRNAs by cross linking and analysis of cDNA (CRAC).

The discovery of a class of small ribonucleic acid (RNA) molecules known as microRNAs (miRNAs) has led to extensive interest in their biological relevance and role. The first miRNA was discovered in Caenorhabditis elegans in 1993. Since then studies have shown that miRNAs represent a fundamental mechanism of gene expression regulation, regulating thousands of genes at the post-transcriptional level. Given that viruses are highly adept at exploiting cellular processes, it is perhaps unsurprising they have evolved miRNAs of their own. The majority of known viral miRNAs are expressed by herpes viruses underscoring their importance to this virus family. Identifying the targets of herpes virus miRNAs would aid in elucidating the role played during infection. In this study, we aim to identify and understand the targets of the human cytomegalovirus (HCMV) encoded miRNAs using a cutting edge biochemical technique, Cross-Linking and Analysis of cDNA (CRAC). HCMV is a member of the beta (β) herpes virus subfamily and is globally distributed causing clinically asymptotic infections in immune competent individuals. However, persistent and recurrent infections in AIDS and organ transplant patients, who have a compromised immune function causes a high degree of mortality and morbidity. Intra-uterine infected infants are also at high risk with infection causing congenital abnormalities and mental retardation. Scientists worldwide are trying to understand one special characteristic of HCMV and herpes viruses in general, which is latency i.e. the presence of an intact viral genome in the cell with a majority of the genes in a dormant or silent state. Viral encoded miRNAs regulate both the viral genome as well as the host’s and this has been postulated as a latency inducing mechanism. To date, there are 22 known HCMV encoded miRNAs. On-going research in our group using techniques such as bioinformatics, RISC immunoprecipitation (RISC-IP) and microarray analysis has identified both viral and cellular targets of HCMV miRNAs. Targets include a crucial viral transactivator thought to play an important role in latency as well as cellular targets involved in a variety of functions including cell cycle control, intrinsic defence and innate cellular defence. However, the majority of HCMV miRNAs have no known function. New approaches and technologies are required to elucidate the functions of these miRNAs. The initial goal of this project is to establish and optimise the CRAC technique. This will be performed in HEK293 cells transfected with a plasmid expressing a cluster of HCMV miRNAs termed miR-US25. The long-term goal of the project will be to use CRAC technology to identify miRNA targets in infected cells