Association of chemokine-mediated block to HIV entry with coreceptor internalization.
Chemokines inhibit entry of HIV into CD4(+) T cells more effectively than into macrophages or transfected adherent cells. Here, we tested whether chemokine receptor internalization could account for cell type differences in the effectiveness of chemokines. Infection of CEM T cells expressing stably transduced wild-type CCR5 was much more readily inhibited by chemokine than were transduced HOS cells. This response correlated with the efficiency of CCR5 internalization. A mutated CCR5, termed M7-CCR5, in which the Ser/Thr phosphorylation sites in the cytoplasmic tail were changed to Ala, did not internalize in response to MIP-1alpha. M7-CCR5 was expressed at slightly higher levels than wild-type on stably transduced cell lines and was somewhat more potent as an HIV-1 coreceptor. The mutated receptor mobilized intracellular Ca(2+) in response to chemokine to a level 4-fold higher than did the wild type CCR5. Unexpectedly, the receptor was desensitized as efficiently as wild type, suggesting that desensitization does not require cytoplasmic tail phosphorylation. Entry of R5 HIV-1 reporter virus into cells stably expressing M7-CCR5 was largely resistant to blocking by MIP-1alpha. As much as 80% of entry inhibition was attributed to receptor internalization. Aminooxypentane (AOP)-MIP-1alpha was able to induce a low level of M7-CCR5 internalization in HOS and to weakly inhibit HIV-1 entry. Introduction of dominant negative dynamin into HOS cells reduced the ability of chemokine to inhibit infection. The inefficiency of internalization of chemokine receptors in some cell types could allow virus to replicate in vivo in the presence of endogenous chemokine. Last, M7-CCR5 is a useful tool for discriminating coreceptor internalization from binding site masking in the evaluation of small molecule inhibitors of HIV-1 entry.
Brandt
Stephanie M
SM
Salk Institute for Biological Studies, Infectious Disease Laboratory, La Jolla, California 92037, USA.
Mariani
Roberto
R
Holland
Anne U
AU
Hope
Thomas J
TJ
Landau
Nathaniel R
NR
D000595:Amino Acid Sequence; D001665:Binding Sites; D002460:Cell Line; D054405:Chemokine CCL3; D054407:Chemokine CCL4; D018925:Chemokines / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D015497:HIV-1 / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008024:Ligands; D008156:Luciferases / Q000378:metabolism; D019402:Macrophage Inflammatory Proteins / Q000378:metabolism; D008561:Membrane Fusion / Q000187:drug effects; D008856:Microscopy, Fluorescence; D008954:Models, Biological; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D010957:Plasmids / Q000378:metabolism; D019713:Receptors, CCR5 / Q000378:metabolism; D013997:Time Factors; D014162:Transfection; D014407:Tumor Cells, Cultured; D014779:Virus Replication / Q000187:drug effects
D054405:Chemokine CCL3; D054407:Chemokine CCL4; D018925:Chemokines; D008024:Ligands; D019402:Macrophage Inflammatory Proteins; D019713:Receptors, CCR5; D008156:Luciferases