Dox-dependent SIVmac with tetracycline-inducible promoter in the U3 promoter region.
An attenuated live vaccine is a candidate in developing vaccines against human immunodeficiency virus type 1 (HIV-1). The study using macaques and simian immunodeficiency virus (SIVmac) showed an attenuated virus to be more effective than any other vaccine candidate. However, development of a safer vaccine is required for clinical application. In this study, we constructed pSIVmac Delta nef with tetracycline inducible promoter (pTet) and tried to control viral expression in a drug-dependent manner. Promoter/enhancer motifs in the U3 region of the long terminal repeats (LTRs) were serially deleted and replaced with pTet. In mutant LTRs, which lack NF-kappaB and Sp1 binding sites, TATA box motifs, and the 5' half of the U3 region, promoter activity was stringently controlled by doxycycline (Dox). Their activities were similar to or higher than that of wild-type LTR in the presence of Dox, based on the transient chloramphenicol acetyltransferase reporter assay. Three of these mutant LTRs were introduced into the pSIVmac239 Delta nef genome. Viral protein from these viruses was efficiently expressed in a Dox-dependent manner after transfection to a HeLa cell, which expresses reverse tetracycline transactivator (rtTA). The 2-LTR-form viral DNA of these viruses could be detected in M8166 cells that had been infected with supernatants from the transfected rtTA HeLa cell. These results suggest that pSIVmac Delta nef containing mutant LTRs can proceed through one viral replication cycle consisting of transcription, formation of viral particles, infection to cells, and reverse transcription. Although continuous replication of these Dox-dependent viruses requires a supply of rtTA as a constituent for the pTet-On viral genome, the successful replacement of the original promoter with a drug-dependent promoter suggests a new possibility for developing a safer attenuated live virus.
Xiao
Y
Y
Laboratory of Viral Pathogenesis, Research Center for AIDS, Institute for Virus Research, Kyoto University, Sakyo-ku, Kyoto, 606-8507, Japan.
Kuwata
T
T
Miura
T
T
Hayami
M
M
Shida
H
H
D004318:Doxycycline / Q000494:pharmacology*; D015967:Gene Expression Regulation, Viral / Q000187:drug effects; D006367:HeLa Cells; D006801:Humans; D011401:Promoter Regions, Genetic; D015302:Simian Immunodeficiency Virus / Q000235:genetics*; D013601:T-Lymphocytes / Q000821:virology*; D020079:Terminal Repeat Sequences; D013752:Tetracycline / Q000494:pharmacology*; D014158:Transcription, Genetic; D014765:Viral Vaccines / Q000138:chemical synthesis*
D014765:Viral Vaccines; D013752:Tetracycline; D004318:Doxycycline