TY - JOUR
T1 - Spontaneous activation of the lytic cycle in cells infected with a recombinant Kaposi's sarcoma-associated virus
AU - Delecluse, Henri-Jacques
AU - Kost, M
AU - Feederle, Regina
AU - Wilson, Lisa
AU - Hammerschmidt, W
PY - 2001/3/15
Y1 - 2001/3/15
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=0035127538&partnerID=8YFLogxK
U2 - 10.1128/JVI.75.6.2921-2928.2001
DO - 10.1128/JVI.75.6.2921-2928.2001
M3 - Article
C2 - 11222717
SN - 1098-5514
VL - 75
SP - 2921
EP - 2928
JO - Journal of virology
JF - Journal of virology
ER -