Final Report Summary - VIRUSSHCREEN (Discovery of novel aspects of host-pathogen interactions through the use of powerful genome-wide shRNA libraries.) RNA interference (RNAi) is a process of double-stranded RNA-dependent post-transcriptional gene silencing. It has become one of the most powerful and widely used strategies for genetic analysis based on the highly specific and efficient silencing of target genes. RNAi library screens have gained much attention from virologists resulting in the publication of several whole-genome short interfering RNA (siRNA) screens that identified numerous new host genes impacting RNA-virus replication. Although extremely powerful, one major shortcoming of siRNA screens was the inherent risk of off-target effects, leading to high false positive rates. This proposal was geared towards the development of ‘next-generation’ lentiviral shRNA library screens that solve many problems existing in RNAi screens and to exploit these libraries to identify host-factors critical for the infection cycle of human herpesviruses and for virus-induced ER-associated degradation (ERAD). Besides development of powerful whole genome shRNA library screens, these efforts lead to new insights into the biology of human herpesvirus infections and boosted research to explore the underlying cellular biology of these processes. We have constructed two genome-wide shRNA libraries and performed several genome-wide library screens with these reagents. We assessed what human genes are involved in the trafficking of the highly toxin Ricin within human cells. These studies yielded a wealth of information on proteins involved in this process and subsequent studies yielded valuable insights in trafficking of biomolecules in human cells in general. Additionally, we performed genome-wide assessments to determine what human genes impact herpesvirues-induced immune evasion through US11. This protein specifically interferes with antigen presentation, and using our genome-wide shRNA screens we discovered and studied key players in this process. One of these is a new E3 ubiquitin ligase that is essential for US11-mediated MHC class I downregulation. TMEM129 was also shown to be involved in the degradation of cellular targets outside the context of virus infection. Current studies are geared towards obtaining a better understanding of the host-pathogens interactions we have uncovered, and study of the function of these genes in normal physiology. Our screens and screening approaches are of wide interest to researchers and we have set up collaborations with multiple research labs that now perform genome-wide screens in their favorite model system. The results of these collaborations hold the promise to have a broad impact on basic science. In very recent years, the development of a new screenings platform making use of the CRISPR/Cas9 system was introduced. The swift developments in this field were possible by the earlier developments of genome-wide shRNA library reagents and screenings platforms. The work sponsored by the CIG enabled the fellow to secure additional funding from the Dutch Cancer Society and a permanent position as assistant professor at our Department.
