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Protection of HeLa‐T4 + cells against human immunodeficiency virus (HIV) infection after stable transfection with HIV LTR‐2‘,5‘‐oligoadenylate synthetase hybrid gene 1
Author(s) -
Schröder Heinz C.,
Ugarkovic Durdica,
Merz Helmut,
Kuchino Yoshiyuki,
Okamoto Takashi,
Müller Werner E. G.
Publication year - 1990
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.4.13.1698680
Subject(s) - transfection , hela , microbiology and biotechnology , biology , virus , plasmid , virology , complementary dna , cell culture , reverse transcriptase , viral replication , gene , polymerase chain reaction , biochemistry , genetics
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 non‐transfected 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.— S chröder , H. C.; U garkovic , D.; M erz , H.; K uchino , Y.; O kamoto , T.; M üller , W. E. G. Protection of HeLa‐T4 + cells against human immunodeficiency virus (HIV) infection after stable transfection with HIV LTR‐2',5'‐oligoadenylate synthetase hybrid gene. FASEB J. 4: 3124‐3130; 1990.