Protection of HeLa-T4+ cells against human immunodeficiency virus (HIV) infection after stable transfection with HIV LTR-2',5'-oligoadenylate synthetase hybrid gene.
An expression vector (pU3R-III/2-5AS) of human 2',5'-oligoadenylate (2-5A) synthetase was constructed in which a cDNA encoding an active form of the enzyme was located 3' to a 3'-long terminal repeat (LTR) of human immunodeficiency virus type 1 (HIV-1). The LTR-directed expression of this hybrid DNA could be activated in trans by the HIV tat gene product. This vector was used for transfection of HeLa-T4+ cells, which are permissive to HIV infection, as well as of normal HeLa cells. HIV replication after infection of the CD4-receptor-bearing HeLa-T4+ cells with HIV-1 was found to be strongly reduced when drug-selected cells cotransfected with pU3R-III/2-5AS and a hygromycin B resistance gene containing plasmid were used. In nontransfected cultures or after transfection with the selectable marker plasmid only, about 60% p17- and p24-positive cells were found 5 days after infection. However, after stable transfection with pU3R-III/2-5AS the number of positive cells was decreased to about 2%. The reverse transcriptase (RT) activity, as a measure for virus production, was markedly decreased in the culture fluids of pU3R-III/2-5AS transfected cells compared with the mock-transfected controls. In parallel experiments it was established that Tat-mediated trans-activation of HIV-1 LTR-directed 2-5A synthetase expression resulted in a great increase in both 2-5A synthetase mRNA level and activity as well as in cellular 2-5A content. Similar results were found in HeLa-T4+ cells and in HeLa cells (without CD4 receptor) cotransfected with pU3R-III/2-5AS and a tat gene containing plasmid or after introduction of purified Tat protein into the pU3R-III/2-5AS transfected cells by using a modified scrape loading procedure. These results indicate that HIV-trans-activated 2-5A synthetase can selectively inhibit HIV replication in vitro, and might be a promising gene therapeutical approach.
Schröder
H C
HC
Institut für Physiologische Chemie, Universität, Mainz, West Germany.
Ugarkovic
D
D
Merz
H
H
Kuchino
Y
Y
Okamoto
T
T
Müller
W E
WE
D015088:2',5'-Oligoadenylate Synthetase / Q000235:genetics* / Q000378:metabolism; D015704:CD4 Antigens / Q000032:analysis* / Q000235:genetics; D002472:Cell Transformation, Viral; D015756:Genes, tat; D016325:HIV Long Terminal Repeat; D015497:HIV-1 / Q000201:enzymology / Q000235:genetics*; D006367:HeLa Cells / Q000201:enzymology / Q000276:immunology; D006801:Humans; D010957:Plasmids; D012194:RNA-Directed DNA Polymerase / Q000378:metabolism; D015183:Restriction Mapping; D014162:Transfection
D015704:CD4 Antigens; D012194:RNA-Directed DNA Polymerase; D015088:2',5'-Oligoadenylate Synthetase