Submitted to: Journal of Virology
Publication Type: Peer reviewed journal
Publication Acceptance Date: 6/2/2006
Publication Date: 9/25/2006
Citation: Chang, A.C., Zsak, L., Feng, Y., Mosseri, R., Lu, Q., Kowalski, P., Zsak, A., Burrage, T.G., Neilan, J.G., Kutish, G.F., Lu, Z., Laegreid, W.W., Rock, D.L., Cohen, S.N. 2006. Phenotype-based identification of host genes required for replication of African swine fever virus. Journal of Virology. 80(17):8705-8717. Interpretive Summary: In this report the authors have described the use of an EST-based genome-wide inactivation procedure to functionally identify host cell genes implicated in ASFV replication. Among the genes identified are loci involved in the host cell immune response, signal transduction, mitochondrial stability, and functions related to actin cytoskeleton reorganization.The work have focused here on the effects of an EST from a gene that encodes a segment of BAT3, a member of the BAG1 protein family found in the MHCIII region of human chromosome, and which has been reported to modulate apoptosis and cell proliferation during mammalian developmentThe data indicate that dysfunction of BAT3 in mammalian cells results in the upregulation of apoptotic genes, and that such dysfunction in ASFV infected cells is associated with impairment of ASF viral replication. The gene silencing procedure described here has been proven useful in identifying genes and proteins whose perturbed function limits ASFV replication. The authors suggest that such CGEPs may be targets for pharmacological or immunological interventions that treat ASF. Agents that mimic the effects of the BAT3 dominant negative peptide may prove to be of particular value in this regard.
Technical Abstract: African Swine Fever Virus (ASFV) produces a fatal acute hemorrhagic fever in domesticated pigs that potentially is a worldwide economic threat. Using an expressed sequence tag (EST)-library-based antisense method of random gene inactivation and a phenotypic screen for limitation of ASFV replication in cultured human cells, we identified six host genes whose cellular functions are required by ASFV. These included three loci, BAT3 (HLA-B associated transcript 3), C1qTNF (C1q and tumor necrosis factor related protein 6), and TOM40 (translocase of outer mitochondrial membrane 40), for which antisense expression from a tetracycline (Tet)-regulated promoter resulted in reversible inhibition of ASFV production by >99%. The effects of antisense transcription of the BAT3 EST and also of expression in the sense orientation of this EST, which encodes amino acid residues 450 to 518 of the mature BAT3 protein, were investigated more extensively. Sense expression of the BAT3 peptide, which appears to reversibly interfere with BAT3 function by a dominant negative mechanism, resulted in decreased synthesis of viral DNA and proteins early after ASFV infection, altered transcription of apoptosis related genes as determined by cDNA microarray analysis, and increased cellular sensitivity to staurosporine-induced apoptosis. Antisense transcription of BAT3 reduced ASFV production without affecting abundance of the virus macromolecules we assayed. Our results, which demonstrate the utility of EST-based functional screens for the detection of host genes exploited by pathogenic viruses, reveal a novel collection of cellular genes previously not known to be required for ASFV infection.