Phenotype-based identification of host genes required for replication of African swine fever virus.
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-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.
Chang
Annie C Y
AC
Departments of Genetics, Stanford University School of Medicine, California, 94305, USA.
Zsak
Laszlo
L
Feng
Yanan
Y
Mosseri
Ronen
R
Lu
Quan
Q
Kowalski
Paul
P
Zsak
Aniko
A
Burrage
Thomas G
TG
Neilan
John G
JG
Kutish
Gerald F
GF
Lu
Zhiqiang
Z
Laegreid
Will
W
Rock
Daniel L
DL
Cohen
Stanley N
SN
D000358:African Swine Fever Virus / Q000472:pathogenicity* / Q000502:physiology; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002522:Chlorocebus aethiops; D018076:DNA, Complementary / Q000235:genetics; D020224:Expressed Sequence Tags; D015723:Gene Library; D020868:Gene Silencing; D006367:HeLa Cells; D006801:Humans; D018832:Molecular Chaperones; D008969:Molecular Sequence Data; D020411:Oligonucleotide Array Sequence Analysis; D010641:Phenotype; D011506:Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D014709:Vero Cells; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism; D014779:Virus Replication
C092009:BAG6 protein, human; D018076:DNA, Complementary; D018832:Molecular Chaperones; D011506:Proteins; D014764:Viral Proteins