PROTECTION OF HELA-T4+ CELLS AGAINST HUMAN-IMMUNODEFICIENCY-VIRUS (HIV) INFECTION AFTER STABLE TRANSFECTION WITH HIV LTR-2',5'-OLIGOADENYLATE SYNTHETASE HYBRID GENE

被引:30
作者
SCHRODER, HC
UGARKOVIC, D
MERZ, H
KUCHINO, Y
OKAMOTO, T
MULLER, WEG
机构
[1] UNIV MAINZ, INST PHYSIOL CHEM, ANGEW MOLEK BIOL ABT, DUESBERGWEG 6, W-6500 MAINZ, GERMANY
[2] RUDJER BOSKOVIC INST, CTR ORGAN CHEM & BIOCHEM, YU-41001 Zagreb, YUGOSLAVIA
[3] NATL CANC CTR, RES INST, DIV BIOPHYS, CHUO KU, TOKYO 104, JAPAN
[4] NATL CANC CTR, RES INST, DIV VIROL, CHUO KU, TOKYO 104, JAPAN
关键词
2'; 5'-oligoadenylate synthetase; HIV-1; Long terminal repeat; Reverse transcriptase; Tat protein;
D O I
10.1096/fasebj.4.13.1698680
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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.
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页码:3124 / 3130
页数:7
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