Apart from the retroviral gag, pol and env the HIV genome contains the F (3' orf) gene which encodes a polypeptide of 206 amino acids which is myristylated at the N-terminal and whose function is unknown. We have expressed the F gene in Escherichia coli and from a recombinant vaccinia virus, VVTGfHIV. The F-protein produced in VVTGfHIV-infected mammalian cells is myristilated, and is phosphorylated by protein kinase C at a residue close to the N-terminus like pp60-src (ref. 5). Purified bacterial F-protein also shows the GTPase, autophosphorylation and GTP-binding activities reported for the ras gene product. Furthermore, we show that expression of F in a CD4+ cell line down-regulates the CD4(T4) antigen. These results suggest that F is important in the pathophysiology of AIDS (acquired immune deficiency syndrome).

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We previously documented persistent regulation of erythropoietin (Epo) secretion in mice after a single intramuscular (i.m.) injection of a recombinant adeno-associated virus (rAAV) vector harboring both the tetracycline-dependent transactivator (rtTA) and the Epo cDNA (D. Bohl, A. Salvetti, P. Moullier, and J. M. Heard, Blood 92:1512-1517, 1998). Using the same vector harboring the cynomolgus macaque Epo cDNA instead, the present study evaluated the ability of the tetracycline-regulatable (tetR) system to establish long-term transgene regulation in nonhuman primates. The vector was administered i.m., after which 5-day induction pulses were performed monthly for up to 13 months by using doxycycline (DOX), a tetracycline analog. We show that initial inductions were successful in all individuals and that there was a tight regulation and a rapid deinduction pattern upon DOX withdrawal. For one macaque, regulation of Epo secretion was maintained during the entire experimental period; for the five remaining macaques, secreted Epo became indistinguishable from endogenous Epo upon repeated DOX inductions. We investigated the mechanism involved and showed that, except in the animal in which secretion persisted, delayed humoral and cellular immune responses were directed against the rtTA transactivator protein associated with the reduction of vector DNA in transduced muscles. This study provides some evidence that, when the immune system is not mobilized against the rtTA transactivator, the tetR-regulatable system is able to support long-term transgene regulation in the context of an rAAV in nonhuman primates. In addition, our results suggest potential improvements for vector design.Recombinant adeno-associated virus (rAAV) vector-mediated gene transfer in skeletal muscle of mice (36), dogs (13), nonhuman primates (8, 37), and hemophilia patients (16) is well tolerated and is associated with long-term expression. As such, it becomes possible to evaluate strategies which allow long-term transgene regulation; such strategies are likely to be required for therapeutic applications and in some instances for safety reasons. A rather limited number of clinically translatable regulatory systems are available. They all have in common the use of chimeric transactivators, the activity of which is controlled by drugs including tetracycline (11), mifepristone (35), ecdysone (23), and rapamycin (25).The rapamycin-regulatable system uses rapamycin or its analog to bring together the functional units of bipartite chimeric transcription factor ZFHD1/FKBP-FRAP/p65 (25). Their corresponding cDNAs have been included in an rAAV vector and injected intramuscularly (i.m.) in macaques along with a second rAAV harboring the erythropoietin (Epo) cDNA under the control of a ZFHD1-dependent promoter. This resulted in long-term regulation of Epo secretion in mice and regulation for up to 3 months in one rhesus macaque out of three (37).The repressor of the Tn10 tetracycline resistance operon of Escherichia coli (tetR) recognizes its operator (tetO) ...

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