University of Cambridge > > Cambridge Immunology > From Screens to Genes: Unraveling the Mystery of Poxvirus Genome Uncoating

From Screens to Genes: Unraveling the Mystery of Poxvirus Genome Uncoating

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Jason Mercer, hosted by Leo James

The main goal of all viruses is to deliver a replication-competent genome from one cell or organism to another. Thus, the uncoating of viral genomes from incoming capsids is a critical step in the lifecycle of all viruses. Genome delivery is an active process for which different mechanisms exist depending on the virus itself and the cellular location of release. Poxviruses, including the causative agent of smallpox and vaccinia virus (VACV) the smallpox vaccine, uncoat and replicate their genome in the cytoplasm. Interestingly, the poxviruses are the only mammalian viruses known to require both cellular and viral factors to mediate genome uncoating. For nearly 40 years the mechanisms and factors required for this process have remained a mystery. Since its discovery, RNA interference (RNAi) has been the method of choice for large-scale loss-of-function studies. Using parallel automated, high-throughput RNAi silencing screens we set out to identify host cell factors required for VACV infection and viral factors needed for genome uncoating. For cell factors: 7,000 genes were screened revealing a new mechanism for genome uncoating involving the ubiquitination of viral core proteins during virus assembly and subsequent released of the viral DNA from the core through the activity of the cell’s proteasomes. For viral factors: the first large-scale RNAi screen directed against a single virus was generated. Targeting of 80 conserved VACV genes led to the unambiguous identification of the VACV AAA + ATPase D5 as the poxvirus-uncoating factor. RNAi-mediated down-regulation of D5 in vivo resulted in reduced viral replication and spread, highlighting the potential of such libraries to provide simultaneous identification of antiviral targets and complementary therapeutics. The results accentuate the value of large-scale RNAi screens in providing directions for detailed cell biological investigation of complex pathways, investigation of large DNA virus gene function, and identification of effective antiviral RNAs.

This talk is part of the Cambridge Immunology series.

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