siRNA screen of early poxvirus genes identifies the AAA+ ATPase D5 as the virus genome-uncoating factor.
Poxvirus genome uncoating is a two-step process. First, cytoplasmic viral cores are activated and early viral genes are expressed. Next, cores are disassembled and the genomes released. This second step depends on an early viral factor(s) that has eluded identification for over 40 years. We used a large-scale, high-throughput RNAi screen directed against vaccinia virus (VACV) to identify the VACV AAA+ ATPase D5 as the poxvirus uncoating factor. We show that the ATPase activity of D5 is required for uncoating. Superresolution microscopy suggests that D5 acts directly at viral cores for genome release. Thus, the putative helicase D5 is a multifunctional protein required for genome uncoating and replication. Additionally, in vivo delivery of anti-D5 siRNAs reduced virus production in a mouse model of VACV infection. These results demonstrate the use of virus-targeting RNAi libraries to investigate viral gene function and suggest therapeutic avenues.
Institute of Biochemistry, ETH Zurich, 8093 Zurich, Switzerland.
Samuel
S
Kilcher
Institute of Biochemistry, ETH Zurich, 8093 Zurich, Switzerland.
Florian Ingo
FI
Schmidt
Institute of Molecular Health Sciences, ETH Zurich, 8093 Zurich, Switzerland.
Christoph
C
Schneider
Institute of Molecular Health Sciences, ETH Zurich, 8093 Zurich, Switzerland.
Manfred
M
Kopf
Institute of Biochemistry, ETH Zurich, 8093 Zurich, Switzerland.
Ari
A
Helenius
MRC Laboratory for Molecular Cell Biology, University College London, London WC1E 6BT, UK. Electronic address: jason.mercer@ucl.ac.uk.
Jason
J
Mercer
D000251:Adenosine Triphosphatases / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism; D000818:Animals; D004265:DNA Helicases / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism; D016679:Genome, Viral; D006367:HeLa Cells; D057166:High-Throughput Screening Assays; D006801:Humans; D008099:Liver / Q000821:virology; D008168:Lung / Q000821:virology; D051379:Mice; D008810:Mice, Inbred C57BL; D034741:RNA, Small Interfering / Q000008:administration & dosage / Q000235:genetics / Q000378:metabolism; D013154:Spleen / Q000821:virology; D014615:Vaccinia / Q000821:virology; D014616:Vaccinia virus / Q000502:physiology*; D014758:Viral Core Proteins / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism; D014779:Virus Replication; D057646:Virus Uncoating / Q000235:genetics*
D034741:RNA, Small Interfering; D014758:Viral Core Proteins; D000251:Adenosine Triphosphatases; D004265:DNA Helicases