Phenotype-Based Identification of Host Genes Required for Replication of African Swine Fever Virus

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 tetracyclineregulated 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.

C1q/TNF-Related Proteins, A Family of Novel Adipokines, Induce Vascular Relaxation Through the Adiponectin Receptor-1/AMPK/eNOS/Nitric Oxide Signaling Pathway

“…25 CTRP6 is requisite for African swine fever virus replication in HeLa andHT144 cells. 26 Secreted from adipose tissue, CTRP9 reduces serum glucose in mice, and forms heterotrimers with adiponectin. 27 CTRPs 8 and 10 are human specific, and their biologic function has not been previously studied.…”

African Swine Fever Virus

“…Cellular genes important for ASFV replication have been identified using a novel phenotype-based screen in cultured cells (Chang et al 2006). A random HeLa cell library with clones having single genes inactivated through antisense expression of expressed sequence tags (EST) was used to screen for a phenotype in which ASFV replication was inhibited.…”

Calpain-dependent cytoskeletal rearrangement exploited for anthrax toxin endocytosis

“…We used regulated transcription from a lentivirusbased human EST library to perturb host gene expression globally and randomly (14,15,36,37) in murine RAW267.4 macrophages and isolated 96 macrophage colonies that survived exposure to the PA-LF complex under conditions that normally are lethal. Doxycycline, which represses a modified CMV pro-…”