[MoOrA1005] Palmitoylation-dependent CD4 distribution in rafts: consequence on HIV entry and -raft-associated kinases

14th International AIDS Conference

Barcelona, Spain — July 7-12, 2002

[TITLE:] Palmitoylation-dependent CD4 distribution in rafts: consequence on HIV entry and -raft-associated kinases

[AUTHOR(S):] Y. Percherancier, T. Planchenault, F. Arenzana-Seisdedos, J.L. Virelizier, F. Bachelerie¹, I. Semac, D. Hoessli²

Int Conf AIDS. 2002 Jul 7-12;14:Abstract No. MoOrA1005

BACKGROUND: Recent advances have lead to the identification of rafts as platforms for numerous cellular functions including entry or budding of viruses and bacteria. HIV entry into cells involves formation of a complex between the viral Envelope and receptors CD4, CCR5 or CXCR4. We have investigated whether palmitoylation, a known raft targeting signal, can affect the lateral distribution of CD4. The impact of viral attachment, membrane fusion, and CD4-associated p56Lck kinase activity on the lateral distribution of CD4 has been studied.

METHODS: CD4-palmitoylation (palm-) and -Lck interaction (Lck-) sites were modified through mutation of C-terminal Cys residues. Detergent TX-100-insoluble (rafts) or -soluble fractions were isolated. The distribution of CD4, CCR5 or Lck was compared in a CD4 negative T-cell line stably expressing CCR5 and WT or mutated CD4 molecules after retroviral transduction. Kinase assays were performed on TX-100-insoluble fractions and characterization of phosphorylated proteins was accomplished following immunoprecipitation.

RESULTS: Both palm- and Lck- CD4 mutants were delocalized from rafts as compared to the WT CD4 molecule. Localization of CCR5 was not detected in rafts in the presence or absence of HIV-virions. Regardless of CD4 palmitoylation, Lck was found to be partially distributed to rafts (30-50%). The presence of CD4 in rafts however, caused a 30-50% decrease in total kinase activity in raft membranes containing similar amounts of Lck and Fyn. Amplitude of R5-HIV-fusion and -replication were not, significantly affected by the modified localization of palm- and Lck- CD4 mutants.

CONCLUSIONS: Our data suggest that the presence of CD4 and CCR5 in raft is dispensable for HIV fusion and replication in this T cell line. It is unlikely that the minute amounts of CD4 mutants in rafts account for full HIV entry. On the other hand, our results suggest a new regulatory effect of CD4 towards the raft-associated kinase pools.

Presenting author: Yann Percherancier

1Institut Pasteur, Institut pasteur, 28 rue du Dr Roux, 75015 Paris, France.

2Centre médical universitaire, Geneva, Switzerland.

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