CD4 deletion mutants evaluated for human immunodeficiency virus type 1 infectivity in a highly efficient system of expression and detection based on LTR-dependent reporter gene activation.
The human CD4 glycoprotein is thought to be involved at several stages of the infection process with the human immunodeficiency virus type 1. To pursue this line of investigation with CD4 deletion mutants, we combined a system of high transient cell-surface expression of the target molecule with an assay of HIV-1 infectivity based on induction of LTR-linked luciferase activity. The approach was also designed to distinguish between defects in gp120 binding and postbinding events. Optimal assay conditions were established with wild-type CD4 and the previously characterized CD4 mutant, d367-371. New deletions of CD4 domains D3 and D4 were then designed from a rat model of the D3D4 atomic coordinates with the concern of maintaining overall structural integrity. While all CD4 mutants were found to be defective towards HIV, it was demonstrated that the mutations affected different stages of the entry process. These data indicate that the system is well suited for studying the intricacy of molecular interactions involving HIV envelope glycoproteins and its receptors.
Moir
S
S
Centre de Recherche du CHUL, Québec, Canada.
Boissinot
M
M
Tremblay
M
M
Poulin
L
L
D000818:Animals; D015704:CD4 Antigens / Q000235:genetics* / Q000378:metabolism*; D016466:CHO Cells; D002460:Cell Line; D006224:Cricetinae; D005434:Flow Cytometry; D015967:Gene Expression Regulation, Viral; D017930:Genes, Reporter; D015699:HIV Envelope Protein gp120 / Q000032:analysis; D016325:HIV Long Terminal Repeat; D015497:HIV-1 / Q000235:genetics / Q000378:metabolism* / Q000472:pathogenicity; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D016133:Polymerase Chain Reaction; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D017384:Sequence Deletion; D015533:Transcriptional Activation
D015704:CD4 Antigens; D015699:HIV Envelope Protein gp120