Penetration of CD4 T cells by HIV-1. The CD4 receptor does not internalize with HIV, and CD4-related signal transduction events are not required for entry.
Receptor binding of HIV to the CD4 molecule is required for efficient infection of T cells, but the post-binding steps that result in penetration of HIV are not well understood. CD4 is induced to internalize upon T cell activation, and mAb to CD4 modify signal transduction and T cell activation as does HIV in some systems. It is not known whether HIV binding triggers CD4 endocytosis or whether signal transduction events are required for penetration. Selected inhibitors of signal transduction were evaluated for their effects on penetration using two assays that are dependent on penetration. After short term exposure to inhibitor and HIV, cells were analyzed for reverse-transcribed HIV DNA (DNA amplification assay), or productive infection is monitored (infectivity assay). Viral penetration was tested in the presence of H7 (protein kinase C inhibition), EGTA (extracellular Ca2+ chelation), cyclosporine A (inhibition of Ca2+/calmodulin-dependent activation), or pertussis toxin (inhibition of G protein function). All agents were used at concentrations that were inhibitory for their respective signal transduction pathways. None of the inhibitors affected viral penetration. We tracked the CD4 molecule with fluorescent probes that do not interfere with HIV binding in a system where CD4 T cells were saturated with HIV and the penetration event was relatively synchronized. Under conditions where detection of CD4 was more sensitive than the detection of HIV, HIV internalization was readily detected but CD4 internalization was not.
Orloff
G M
GM
Division of Immunologic, Oncologic and Hematologic Diseases, Centers for Disease Control, Atlanta, GA 30333.
Orloff
S L
SL
Kennedy
M S
MS
Maddon
P J
PJ
McDougal
J S
JS
D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D015704:CD4 Antigens / Q000276:immunology*; D015496:CD4-Positive T-Lymphocytes / Q000276:immunology*; D002118:Calcium / Q000502:physiology; D002121:Calcium Channel Blockers / Q000494:pharmacology; D002147:Calmodulin / Q000502:physiology; D002478:Cells, Cultured; D003524:Cyclosporins / Q000494:pharmacology; D003572:Cytochalasins / Q000494:pharmacology; D004533:Egtazic Acid / Q000494:pharmacology; D015497:HIV-1 / Q000276:immunology / Q000472:pathogenicity*; D006801:Humans; D066298:In Vitro Techniques; D007546:Isoquinolines / Q000494:pharmacology; D008213:Lymphocyte Activation; D009419:Nerve Tissue Proteins / Q000502:physiology; D037342:Pertussis Toxin; D010879:Piperazines / Q000494:pharmacology; D016133:Polymerase Chain Reaction; D011493:Protein Kinase C / Q000037:antagonists & inhibitors; D015398:Signal Transduction / Q000187:drug effects; D013449:Sulfonamides; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
D015704:CD4 Antigens; D002121:Calcium Channel Blockers; D002147:Calmodulin; D003524:Cyclosporins; D003572:Cytochalasins; C029152:G-substrate; D007546:Isoquinolines; D009419:Nerve Tissue Proteins; D010879:Piperazines; D013449:Sulfonamides; D010566:Virulence Factors, Bordetella; C007215:cytochalasin E; D004533:Egtazic Acid; D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; C043204:N-(2-guanidinoethyl)-5-isoquinolinesulfonamide; D037342:Pertussis Toxin; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium