Reduced susceptibility to HIV-1 infection of ethyl-methanesulfonate-treated CEM subclones correlates with a blockade in their protein kinase C signaling pathway.
We have described the isolation of chemically induced CEM subclones that express CD4 receptors and bind soluble gp120, yet show a markedly reduced susceptibility to infection with HIV-1. Two subclones were found to have an abnormal response to the protein kinase C (PKC) activator PMA. PMA treatment induced CD3 and CD25 (IL-2R) receptors on the parental line and on other ethyl-methanesulfonate-derived subclones, but not on these two mutants. Direct assays of PKC activity were conducted. Total cellular PKC enzymatic activity was found to be normal in these subclones. PMA-induced CD4 down-modulation occurred normally. In addition, activation of c-raf kinase was normal. Since HIV-1 long terminal repeat contains two functional nuclear factor kB (NF-kB) regulatory elements, we studied the ability of PMA to induce NF-kB binding activity by different assays. Chloramphenicol acetyl transferase (CAT) assays using the HIV-1 (-139)long terminal repeat-CAT construct showed no PMA induction of CAT activity in these subclones (unlike the parental line and other subclones). Okadaic acid, an inhibitor of phosphatases 1 and 2A, did not overcome the defect in these subclones. Gel retardation assays, using a 32P-probe containing the HIV-1 NF-kB probe and nuclear extracts from PMA-treated cells, showed significantly reduced induction of nuclear NF-kB binding proteins in these two subclones compared with wild type CEM and a control subclone. Deoxycholate treatment of cytoplasmic extracts from these subclones released much reduced NF-kB binding proteins from their cytoplasmic pools. Thus, reduced levels of PKC-induced nuclear NF-kB activity in two T cell subclones did not affect their normal cell growth, but correlated with a pronounced reduction in their susceptibility to HIV-1 infection.
Hillman
K
K
Division of Virology, Food and Drug Administration, National Institutes of Health, Bethesda, MD 20892.
Qian
J
J
Siegel
J N
JN
Roderiquez
G
G
Blackburn
R
R
Manischewitz
J
J
Norcross
M
M
Golding
H
H
D001483:Base Sequence; D015496:CD4-Positive T-Lymphocytes / Q000382:microbiology*; D002451:Cell Compartmentation; D002478:Cells, Cultured; D002999:Clone Cells; D003593:Cytoplasm / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D004988:Ethers, Cyclic / Q000494:pharmacology; D005020:Ethyl Methanesulfonate / Q000494:pharmacology; D015870:Gene Expression; D015658:HIV Infections / Q000503:physiopathology*; D016325:HIV Long Terminal Repeat; D006801:Humans; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D009154:Mutation; D016328:NF-kappa B / Q000378:metabolism; D009687:Nuclear Proteins / Q000378:metabolism; D019319:Okadaic Acid; D011493:Protein Kinase C / Q000502:physiology*; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D019908:Proto-Oncogene Proteins c-raf; D015398:Signal Transduction; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D004268:DNA-Binding Proteins; D004988:Ethers, Cyclic; D016328:NF-kappa B; D009687:Nuclear Proteins; D011518:Proto-Oncogene Proteins; D019319:Okadaic Acid; D005020:Ethyl Methanesulfonate; D019908:Proto-Oncogene Proteins c-raf; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate