Chemically induced CD4 mutants of a human T cell line. Evidence for dissociation between binding of HIV I envelope and susceptibility to HIV I infection and syncytia formation.
This study describes the derivation of a series of mutants from the human leukemic cell line CEM using the frame shift mutagen Ethyl-methanesulfonate followed by negative selection with multiple treatments of OKT4A + C, and sorting into CD4-, CD4-dull, and CD4-intermediate mutants. These mutants express reduced CD4 levels ranging from 0 to 60% of the parental line. The mutants were analyzed by staining with a battery of CD4-specific mAb, by assessing their ability to bind soluble gp120, and by their ability to form syncytia after infection with cell-free HIV I virus and a gp160-vaccinia vector. Two groups of particularly interesting mutants were identified: (1) CD4-dull mutants expressing only 5 to 10% of the wild type surface CD4 density, which nevertheless were infectable by HIV I and produced as many syncytia and reverse transcriptase activity as the parental line after infection with gp160-vaccinia or cell free HIV I. (2) CD4-intermediate mutants (30 to 60% of parental CD4 level), which express CD4-epitopes required for interaction with the HIV I envelope protein, yet are markedly deficient in their ability to form syncytia after gp160-vaccinia or HIV I infection. Two of these mutants did form syncytia after transient reconstitution with a wild type CD4 containing vaccinia vector. Inasmuch as they were found to bind soluble gp120 with the same avidity as other, functionally normal, CD4-intermediate mutants, these human T cell mutants may have a reduced susceptibility to HIV I infection due to the absence of a "fusogenic component" or to a structural alteration in a region of the CD4 molecule not required for binding of the HIV I envelope, but for the subsequent fusion and entry process.
Division of Virology, National Institute of Dental Research, National Institutes of Health, Bethesda, MD 20892.
K
K
Hillman
O
O
Shapira-Nahor
M F
MF
Gruber
J
J
Hooley
J
J
Manischewitz
R
R
Seeman
L
L
Vujcic
S J
SJ
Geyer
H
H
Golding
D015704:CD4 Antigens / Q000235:genetics* / Q000502:physiology; D015496:CD4-Positive T-Lymphocytes / Q000382:microbiology*; D002459:Cell Fusion; D002460:Cell Line; D005822:Genetic Vectors; D015699:HIV Envelope Protein gp120 / Q000378:metabolism*; D015497:HIV-1 / Q000378:metabolism*; D006801:Humans; D066298:In Vitro Techniques; D009154:Mutation; D012194:RNA-Directed DNA Polymerase / Q000032:analysis; D011994:Recombinant Proteins / Q000378:metabolism; D012995:Solubility; D014162:Transfection; D014616:Vaccinia virus
D015704:CD4 Antigens; D015699:HIV Envelope Protein gp120; D011994:Recombinant Proteins; D012194:RNA-Directed DNA Polymerase