Background: Membrane trafficking cooperates with HIV-1 fusion with plasma membrane of target cells independently of virus tropism. TIRFM is a tool that can dynamically study CD4- dependent HIV-1 fusion, entry, viral assembly and release from the surface of permissive cells. While regulation of early HIV-1 infection by Arf6 activity seems to be related to fusion and entry steps of the viral cycle we agreed to characterize by TIRFM the function of Arf6-mediated membrane dynamics on HIV-1 entry and infection. Observations: We observed that TZM-bl cells over-expressing the Arf6-Q67L and Arf6-T44N constructs exhibited accumula- tion of PIP2-associated structures on plasma membrane. TIRFM studies revealed that wt-Arf6-, Arf6-Q67L- and Arf6-T44N-ECFP constructs did not co-localize with cell-surface CD4-DsRed, and HIV-1 binding to CD4 did not promote co-distribution of CD4 with Arf6 constructs. Finally, we performed TIRFM studies from the CD4-dependent HIV-1 uptake process by using fluorescent HIV-1-Gag-EGFP viral particles in permissive TZM-bl (CD4+/ CXCR4+/CCR5+) cells transiently expressing fluorescent CD4-DsRed together with one of the different Arf6-HA construct and the PH-ECFP probe. Our results indicated that alteration of Arf6-mediated PIP2-membrane dynamics by over-express- ing Arf6-Q67L-HA or Arf6-T44-HA mutant prevented produc- tive CD4-dependent HIV-1 uptake. Moreover, endogenous Arf6 knock-down did not affect the first CD4-DsRed/HIV-1-Gag-EGFP interaction, but prevented CD4-dependent viral uptake. Blocking plasma membrane dynamics by Arf6-Q67L and Arf6-T44N over-expression or by specific Arf6 silencing did not inhibit cell infection by HIV-1 vectors pseudotyped with the VSV-G protein. Conclusions: Arf6-GTP/GDP cooperates with HIV-1-cell receptors interactions, by maintaining PIP2-associated membrane dynamics to promote viral fusion and entry. Arf6- coordinated plasma membrane dynamics is required for an efficient HIV-1 membrane fusion, viral entry and infection of permissive CD4+T lymphocytes.

Productive CD4-dependent HIV-1 fusion, entry and infection dynamically studied by Total Internal Reflection Fluorescence Microscopy in living cells

ZIGLIO, Serena;
2012

Abstract

Background: Membrane trafficking cooperates with HIV-1 fusion with plasma membrane of target cells independently of virus tropism. TIRFM is a tool that can dynamically study CD4- dependent HIV-1 fusion, entry, viral assembly and release from the surface of permissive cells. While regulation of early HIV-1 infection by Arf6 activity seems to be related to fusion and entry steps of the viral cycle we agreed to characterize by TIRFM the function of Arf6-mediated membrane dynamics on HIV-1 entry and infection. Observations: We observed that TZM-bl cells over-expressing the Arf6-Q67L and Arf6-T44N constructs exhibited accumula- tion of PIP2-associated structures on plasma membrane. TIRFM studies revealed that wt-Arf6-, Arf6-Q67L- and Arf6-T44N-ECFP constructs did not co-localize with cell-surface CD4-DsRed, and HIV-1 binding to CD4 did not promote co-distribution of CD4 with Arf6 constructs. Finally, we performed TIRFM studies from the CD4-dependent HIV-1 uptake process by using fluorescent HIV-1-Gag-EGFP viral particles in permissive TZM-bl (CD4+/ CXCR4+/CCR5+) cells transiently expressing fluorescent CD4-DsRed together with one of the different Arf6-HA construct and the PH-ECFP probe. Our results indicated that alteration of Arf6-mediated PIP2-membrane dynamics by over-express- ing Arf6-Q67L-HA or Arf6-T44-HA mutant prevented produc- tive CD4-dependent HIV-1 uptake. Moreover, endogenous Arf6 knock-down did not affect the first CD4-DsRed/HIV-1-Gag-EGFP interaction, but prevented CD4-dependent viral uptake. Blocking plasma membrane dynamics by Arf6-Q67L and Arf6-T44N over-expression or by specific Arf6 silencing did not inhibit cell infection by HIV-1 vectors pseudotyped with the VSV-G protein. Conclusions: Arf6-GTP/GDP cooperates with HIV-1-cell receptors interactions, by maintaining PIP2-associated membrane dynamics to promote viral fusion and entry. Arf6- coordinated plasma membrane dynamics is required for an efficient HIV-1 membrane fusion, viral entry and infection of permissive CD4+T lymphocytes.
HIV; TIRFM; ARF6
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/11562/500361
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