Characterization of CMV miRNAs in vitro and in vivo

Coordinator:    Prof. Dr. Dr. Ulrich Koszinowski
Institution: Max von Pettenkofer-Institut, Universität München
Although usually asymptomatic in healthy individuals, human cytomegalovirus (HCMV) is the major cause of morbidity in immunocompromised patients and allogeneic bone-marrow or organ-transplant recipients. It is also the leading agent of birth defects among congenitally transmitted infections. During primary infection and reactivation, HCMV encounters an array of innate and adaptive immune responses. MicroRNAs (miRNAs) represent a novel entity of viral factors counteracting these defenses requiring no protein expression to exert their function. To date, a total of 11 miRNAs have been identified in HCMV. The infection of the mouse with the murine cytomegalovirus (MCMV) is the model of choice to study the biology of CMV infection and to explore the role of CMV genes and of host genes in vivo. We have identified 18 MCMV encoded miRNAs (Dölken et al., J Virol 2007). MCMV infection leads to a massive over-expression of small RNAs. Already after 24 h post infection of fibroblasts viral miRNAs constitute about 40% of all cellular miRNAs rising to >60% at 72 h post infection (see Figure 1). Therefore, significant effects on the cellular miRNA profile and function can be observed. We have created single miRNA knock-out mutants of the most abundant MCMV miRNAs. These viruses are currently being characterized both in vitro and in vivo to get a better understanding of the importance of viral miRNAs for productive infection, viral latency and reactivation. In addition, we have established biochemical assays (RISC pull-downs) to identify cellular and viral targets of these miRNAs. Primary goal of these experiments is to determine whether viral miRNAs may serve as readily accessible targets for new antiviral drugs (e.g. Antagomirs).

Figure 1

Accumulation of MCMV miRNAs during lytic infection of murine fibroblasts.
The three most abundant miRNAs (miR-m01-4, miR-m21-1 and miR-M23-2) are depicted.
Abbreviations:  h p.i. = hours post infection

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