Spontaneous activation of the lytic cycle in cells infected with a recombinant Kaposi's sarcoma-associated virus.
The genetic analysis of human herpesvirus 8 (HHV8), also termed Kaposi's sarcoma-associated virus, has been hampered by severe difficulties in producing infectious viral particles and modifying the viral genome. In this article, we report the successful cloning of the HHV8 complete genome onto a prokaryotic F-plasmid replicon which allows the propagation of the recombinant viral DNA in Escherichia coli. The insertion of the F-plasmid into the HHV8 genome interrupts the ORF56 gene, whose expression product-by homology with the Epstein-Barr virus BSLF1 gene--is supposed to be necessary for lytic DNA replication. After introduction of the recombinant HHV8 DNA into 293 cells, early viral antigens are expressed, suggesting that spontaneous lytic replication is initiated. However, completion of the lytic program is prevented by the absence of the ORF56 protein, and a quasi-latent state is established. Upon reintroduction of the ORF56 viral gene, the block is overcome and infectious HHV8 virions are produced. As the recombinant HHV8 genome can be easily modified in E. coli, this experimental system opens the way to an extensive genetic analysis of other HHV8 functions.
Department of Gene Vectors, GSF-National Research Center for Environment and Health, D-81377 Munich, Germany. H.Delacluse@bham.ac.uk
H J
HJ
Delecluse
M
M
Kost
R
R
Feederle
L
L
Wilson
W
W
Hammerschmidt
D002460:Cell Line; D004926:Escherichia coli / Q000235:genetics; D005144:F Factor; D016679:Genome, Viral; D019288:Herpesvirus 8, Human / Q000235:genetics* / Q000502:physiology*; D006801:Humans; D016366:Open Reading Frames / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D011995:Recombination, Genetic; D012093:Replicon; D014162:Transfection; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism; D014771:Virion / Q000378:metabolism
C110348:ORF59 protein, Human herpesvirus 8; D014764:Viral Proteins