Herpesviral factors modulating the cellular miRNA processing machinery

Coordinator:    Prof. Dr. Dr. Jürgen Haas
Institution: Max von Pettenkofer Institu, Universität München
Herpesviruses such as the Kaposi’s sarcoma-associated herpesvirus (KSHV) have previously been shown to encode small RNA molecules termed microRNAs (miRNAs) which lead to a degradation or translational halt of messenger RNAs (mRNAs) and thereby regulate protein expression in eukaryotic cells. The objective of this project is to identify cellular targets which are regulated by KSHV-derived miRNAs and to analyse their role in the pathogenesis using a variety of different approaches. Since miRNAs can induce degradation of specifically targeted mRNAs, we will investigate the effect of KSHV infection on the amount of all cellular mRNAs by microarray analyses. In parallel, we will identify differentially expressed cellular proteins in miRNA expressing cells by a SILAC mass spectrometry approach. A third approach will be to purify miRNA-containing protein complexes (“RISC”, RNA-induced silencing complex) which recognise and degrade target mRNA molecules and identify the mRNAs associated with it by microarrays and real-time PCR. Cellular miRNAs are known to represent a defence mechanism against pathogens in plant and worm cells. Several viruses including hepatitis C virus (HCV), Human Immunodeficiency Virus 1 (HIV-1) and herpesviruses such as Epstein-Barr virus (EBV) encode proteins which interfere with the miRNA machinery, but it is not clear whether these proteins act as a viral counter-defence mechanism or whether the virus employs the miRNA machinery for its own replication or persistence. We will systematically screen several different herpesviruses for viral proteins interacting with cellular proteins involved in miRNA processing by high-throughput yeast-two-hybrid experiments. For this purpose, we will use a large collection of clones (>1.000) from a variety of pathogens including KSHV, Varicella Zoster Virus (VZV), EBV, Herpes simplex virus 1 (HSV-1) and mouse Cytomegalovirus (mCMV) and test them in a pairwise manner versus 40 cellular proteins of the miRNA-processing and -effector machinery.

Mature Micro-RNAs derive from hairpin-like structures, so-called pre-miRNAs. Kaposi’s Sarcoma Associated  Herpesvirus (KSHV, HHV-8) contains at least twelve such precursers, that are shown here.
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