RNAi screening reveals proteasome- and Cullin3-dependent stages in vaccinia virus infection.
A two-step, automated, high-throughput RNAi silencing screen was used to identify host cell factors required during vaccinia virus infection. Validation and analysis of clustered hits revealed previously unknown processes during virus entry, including a mechanism for genome uncoating. Viral core proteins were found to be already ubiquitinated during virus assembly. After entering the cytosol of an uninfected cell, the viral DNA was released from the core through the activity of the cell's proteasomes. Next, a Cullin3-based ubiquitin ligase mediated a further round of ubiquitination and proteasome action. This was needed in order to initiate viral DNA replication. The results accentuate the value of large-scale RNAi screens in providing directions for detailed cell biological investigation of complex pathways. The list of cell functions required during poxvirus infection will, moreover, provide a resource for future virus-host cell interaction studies and for the discovery of antivirals.
Institute of Biochemistry, ETH Zürich, 8093 Zürich, Switzerland.
Jason
J
Mercer
Berend
B
Snijder
Raphael
R
Sacher
Christine
C
Burkard
Christopher Karl Ernst
CK
Bleck
Henning
H
Stahlberg
Lucas
L
Pelkmans
Ari
A
Helenius
D019295:Computational Biology; D044842:Cullin Proteins / Q000378:metabolism*; D004261:DNA Replication; D004279:DNA, Viral; D006367:HeLa Cells; D006801:Humans; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism*; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000378:metabolism; D054875:Ubiquitination; D014615:Vaccinia / Q000378:metabolism* / Q000473:pathology / Q000821:virology; D014616:Vaccinia virus / Q000235:genetics* / Q000378:metabolism
C503645:CUL3 protein, human; D044842:Cullin Proteins; D004279:DNA, Viral; D034741:RNA, Small Interfering; D046988:Proteasome Endopeptidase Complex