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Fig. 1. HIV-1 phosphorylates endogenous ERM and redistributes F-actin. (A) Western blot analysis of rs-gp120-induced phosphorylation of C-terminal Thr residues of ERM proteins (P-ezrin and P-moesin). Results from untreated control CEM cells are shown in the blot below. Total ERM (ezrin and moesin) is shown for all experimental conditions. A representative experiment of three is shown. The graph shows quantification of Thr phosphorylation of ERMs from three independent experiments. (B) Time course of ERM Thr phosphorylation in PHA-activated PBL, during early infection with the X4-tropic HIV-1_NL4.3 viral strain (MOI, 5) compared with non-infected cells. Basal phosphorylation of endogenous ERM-Thr, total ERM and total -tubulin expression are shown for all experimental conditions. (C) Confocal microscopy shows polarized distribution (xy mid-sections) of activated endogenous ERM in CEM cells, 1 hour after HIV-1 infection (MOI, 1; HIV-1NL4.3). Untreated, non-infected cells. Quantification of HIV-1-induced ERM or ERM-P capping is indicated ± s.d. (D) Confocal analysis of ERM-P and endogenous ezrin and moesin localization and redistribution in non-infected or HIV-1-infected CEM cells (1 hour; MOI, 1). (E) Confocal analysis of F-actin and endogenous ERM localization and redistribution in non-infected or HIV-1-infected CEM cells (1 hour; MOI, 1). Quantification of HIV-1-induced ERM actin capping is indicated ± s.d. (F) Western blot analysis of ezrin and moesin phosphorylation induced by rs-gp120_IIIB (1 hour exposure) in CEM cells. Blots of total expression of ezrin, moesin and -tubulin are also shown. Rho-K (ROCK) and PKC inhibitors failed to prevent rs-gp120_IIIB-induced Thr phosphorylation of ezrin and moesin. A representative of three independent experiments is shown. (G) Blockade of rs-gp120_IIIB (5 µg/ml)-induced ERM Thr phosphorylation in CEM cells with neutralizing or non-neutralizing anti-CD4 mAbs (OKT4A or OKT4, respectively), or with the CXCR4 antagonist AMD3100. Blots of total ezrin and moesin proteins are shown. A representative example of three independent experiments is shown.

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