Inhibition of human immunodeficiency virus-1 production resulting from transduction with a retrovirus containing an HIV-regulated diphtheria toxin A chain gene.
Expression of a gene encoding the diphtheria toxin A (DT-A) chain, under the control of human immunodeficiency virus-1 (HIV-1) proteins Tat and Rev, has previously been shown to confer on cells an impaired ability to produce HIV. That work was done in HeLa cell lines that had stably integrated the regulated DT-A gene in a plasmid context. To increase the efficiency with which the HIV-regulated DT-A gene could be introduced into cells, we studied a recombinant, amphotropic murine leukemia virus containing the HIV-regulated DT-A transcription unit. Here we demonstrate that such recombinant retroviruses can be packaged, for both wild-type DT-A and an attenuated version, tox 176. In transient transfection assays, the proviral constructs exhibited similar basal and trans-activated levels of DT-A expression to the parental plasmids. Transduced H9 cells expressed the integrated DT-A gene upon transfection with plasmids encoding Tat and Rev, as assayed by decreased expression of a cotransfected luciferase reporter gene. Furthermore, the transduced H9 cells were substantially impaired in their ability to produce HIV, as demonstrated by p24 assays of culture supernatants following either transfection with an HIV proviral clone or infection with HIV-IIIB. These data demonstrate that basal expression of the regulated DT-A gene has been reduced to a tolerable level, both in packaging cells and transduced H9 cells. The use of HIV-regulated retroviruses encoding the highly lethal DT-A product may eventually be applicable as a gene therapy approach for the acquired immunodeficiency syndrome (AIDS).
Harrison
G S
GS
Division of Medical Oncology, University of Colorado Health Sciences Center, Denver 80262.
Long
C J
CJ
Curiel
T J
TJ
Maxwell
F
F
Maxwell
I H
IH
D002460:Cell Line; D003001:Cloning, Molecular; D004167:Diphtheria Toxin / Q000008:administration & dosage / Q000235:genetics / Q000494:pharmacology*; D015967:Gene Expression Regulation, Viral; D015694:Gene Products, rev / Q000378:metabolism; D015696:Gene Products, tat / Q000378:metabolism; D015316:Genetic Therapy / Q000379:methods; D005822:Genetic Vectors; D016655:HIV Core Protein p24 / Q000378:metabolism; D015497:HIV-1 / Q000187:drug effects* / Q000235:genetics / Q000502:physiology; D006367:HeLa Cells; D006801:Humans; D009052:Leukemia Virus, Murine / Q000235:genetics*; D008156:Luciferases / Q000096:biosynthesis; D016133:Polymerase Chain Reaction; D015533:Transcriptional Activation; D014161:Transduction, Genetic; D014779:Virus Replication / Q000187:drug effects; D054321:rev Gene Products, Human Immunodeficiency Virus; D054322:tat Gene Products, Human Immunodeficiency Virus
D004167:Diphtheria Toxin; D015694:Gene Products, rev; D015696:Gene Products, tat; D016655:HIV Core Protein p24; D054321:rev Gene Products, Human Immunodeficiency Virus; D054322:tat Gene Products, Human Immunodeficiency Virus; D008156:Luciferases