Comparison of trans-dominant inhibitory mutant human immunodeficiency virus type 1 genes expressed by retroviral vectors in human T lymphocytes.
trans-Dominant inhibitory mutant versions of the human immunodeficiency virus type 1 (HIV-1) regulatory genes tat and rev have previously been described. We have constructed a series of retroviral vectors to transduce these genes and compare their inhibitory activities. The inhibitory activities were measured with transient transfection assays by using a reporter which expresses an HIV-1 gag-Escherichia coli lacZ fusion protein with strict dependence on coexpression of both tat and rev. Additionally, the vectors were packaged as amphotropic virions and used to stably transduce human CEM T lymphocytes. The transduced CEM cells were challenged with HIV-1, and the effects of the mutant HIV-1 genes were determined by measuring the levels of HIV-1 p24gag produced. A tat gene substituted at amino acid 41 (tatk41a) retained partial trans-activating activity and lacked inhibitory activity. A tat gene with a premature stop codon at amino acid 54 (tat54ter) showed moderate trans-dominant inhibition of the reporter plasmid but failed to significantly inhibit HIV-1 replication. The M10 rev mutant, with a 2-amino-acid substitution, showed strong trans-dominant inhibitory activity both in the reporter plasmid and in the HIV-1 infection assay. The greatest inhibition of HIV-1 growth was seen when M10 was expressed under the transcriptional control of a human cytomegalovirus promoter; slightly less inhibition was achieved when expression of M10 was controlled by the Moloney murine leukemia virus long terminal repeat, and minimal inhibition was seen when the HIV-1 long terminal repeat controlled the M10 gene. These results demonstrate the potential utility of retroviral vectors expressing trans-dominant inhibitory mutant HIV-1 genes for gene therapy approaches to AIDS.
Division of Research Immunology/Bone Marrow Transplantation, Childrens Hospital Los Angeles, California.
I
I
Bahner
C
C
Zhou
X J
XJ
Yu
Q L
QL
Hao
J C
JC
Guatelli
D B
DB
Kohn
D000163:Acquired Immunodeficiency Syndrome / Q000235:genetics*; D000595:Amino Acid Sequence; D001483:Base Sequence; D003587:Cytomegalovirus / Q000235:genetics; D005799:Genes, Dominant / Q000235:genetics*; D005814:Genes, Viral / Q000235:genetics*; D015750:Genes, gag / Q000235:genetics; D015757:Genes, rev / Q000235:genetics; D015756:Genes, tat / Q000235:genetics; D005822:Genetic Vectors; D015497:HIV-1 / Q000235:genetics*; D006801:Humans; D007763:Lac Operon; D008969:Molecular Sequence Data; D008979:Moloney murine leukemia virus / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics; D011993:Recombinant Fusion Proteins; D012091:Repetitive Sequences, Nucleic Acid / Q000235:genetics; D013601:T-Lymphocytes; D015533:Transcriptional Activation; D014161:Transduction, Genetic; D014170:Transformation, Genetic; D014779:Virus Replication
D011993:Recombinant Fusion Proteins