Viruses are simple, obligatory, intracellular parasites that depend on the host cell for most of the steps in the replication cycle. Not only do they rely on the cells biosynthetic machinery, they exploit cellular processes for signaling, membrane trafficking, intra-cellular transport, nuclear import and export, molecular sorting, transcriptional regulation, etc. To access the full spectrum of host cell functions in the infection of three different viruses in an unbiased and systematic fashion, we will identify the critical host cell proteins needed by monitoring infection in human tissue culture cells after silencing individual genes using genome-wide siRNA libraries and large sub-libraries based on the human genome. The three viruses are vaccinia virus (a poxvirus), human papilloma virus 16, and Uukuniemi virus (a bunyavirus). They are members of important but poorly analyzed pathogen families representing enveloped and nonenveloped viruses, RNA and DNA viruses, viruses that replicate in the nucleus and in the cytosol. The infection assays to be used are fully automated, and make use of high-content microscopic read-outs for infection and virus production. Identification of the viral infectomes , in this way, offers a valuable, new perspective into the complexities of the infection process, and opens wide new areas of basic and applied research. After validation of hits , extensive biochemical and cell biological analysis will be performed on a selected set of host proteins and pathways identified through the infectome analysis. We will determine which steps in the replication cycle are affected, which mechanisms are involved, and which cellular pathways play a role. For detailed analysis, we will focus on mechanisms in entry, uncoating, and early intracellular events. A large spectrum of techniques including live cell imaging and single particle tracking will be used to follow up the screening results with functional and mechanistic studies.