Specific reactions between purified HIV-1 particles and CD4+ cell membrane fragments in a cell-free system of virus fusion or entry.
The initial step of human immunodeficiency virus type 1 (HIV-1) infection has been studied by Env-mediated fusion or entry assays with appropriate cells expressing CD4 or CXCR4/CCR5 receptors in cultures, where many factors underlying cellular activities likely regulate the fusion/entry efficiency. Here we attempted to develop a more simplified in vitro cell-free fusion/entry reaction that mimics HIV-1 infection in cultures. Membrane fragments of target cells and intact infectious HIV-1 particles were purified, mixed and incubated. The core p24 protein was released from the purified virions and detected by ELISA without detergents in the supernatant of the reaction mixtures. This release reaction proceeded temperature-dependently and in a dose-dependent manner between the virion and membrane fractions, and was specific for HIV-1 Env and CD4. Env-deleted or VSV-G-pseudotyped HIV-1 released little p24, if any. Pretreatment of the membrane fragments with anti-CD4 antibodies inhibited the p24 induction from both X4-tropic and R5-tropic HIV-1. Furthermore, X4 but not R5 HIV-1 reacted with the membrane prepared from intrinsically CXCR4-positive HeLa-CD4 cells, whereas both viruses reacted with that prepared from CCR5-transduced HeLa-CD4 cells, indicating that this cell-free reaction mimics coreceptor usage of HIV-1 infection. Therefore, a potent entry inhibitor of X4 HIV-1, SDF-1alpha, blocked the release from X4 but not R5 HIV-1. Inversely, a weak entry inhibitor of R5 HIV-1, MIP-1beta, partially affected only the release from R5 HIV-1. These results suggest that this cell-free reaction system provides a useful tool to study biochemical fusion/entry mechanisms of HIV-1 and its inhibitors.
Harada
Takayuki
T
Department of Pathology, National Institute of Infectious Diseases, Toyama 1-23-1, Shinjuku-ku, Tokyo 162-8640, Japan.
Tatsumi
Masashi
M
Takahashi
Hidehiro
H
Sata
Tetsutaro
T
Kurata
Takeshi
T
Kojima
Asato
A
D000998:Antiviral Agents / Q000494:pharmacology; D015704:CD4 Antigens / Q000302:isolation & purification / Q000378:metabolism; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism*; D002474:Cell-Free System; D054407:Chemokine CCL4; D054377:Chemokine CXCL12; D019743:Chemokines, CXC / Q000494:pharmacology; D015686:Gene Products, env / Q000235:genetics / Q000378:metabolism; D016655:HIV Core Protein p24 / Q000032:analysis / Q000276:immunology; D015497:HIV-1 / Q000302:isolation & purification / Q000378:metabolism*; D006801:Humans; D019402:Macrophage Inflammatory Proteins / Q000494:pharmacology; D019713:Receptors, CCR5 / Q000302:isolation & purification / Q000378:metabolism; D019718:Receptors, CXCR4 / Q000302:isolation & purification / Q000378:metabolism; D015728:Receptors, HIV / Q000302:isolation & purification / Q000378:metabolism*
D000998:Antiviral Agents; D015704:CD4 Antigens; C515255:CXCL12 protein, human; D054407:Chemokine CCL4; D054377:Chemokine CXCL12; D019743:Chemokines, CXC; D015686:Gene Products, env; D016655:HIV Core Protein p24; D019402:Macrophage Inflammatory Proteins; D019713:Receptors, CCR5; D019718:Receptors, CXCR4; D015728:Receptors, HIV