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Differential control of CXCR4 and CD4 downregulation by HIV-1 Gag.

Valiathan RR, Resh MD - Virol. J. (2008)

Bottom Line: The effect of Gag was dependent on an ESCRT-I interacting motif within the C-terminal p6 region of Gag.In contrast, PMA-induced downregulation of the HIV-1 receptor CD4 was independent of ESCRT-I and Vps4; HIV-1 Gag had no effect on this process.These results establish that the HIV-1 receptor, CD4, and co-receptor, CXCR4 are differentially regulated by ESCRT proteins.

View Article: PubMed Central - HTML - PubMed

Affiliation: Cell Biology Program, Memorial Sloan-Kettering Cancer Center, New York, USA. rrv@umich.edu

ABSTRACT

Background: The ESCRT (endosomal sorting complex required for transport) machinery functions to sort cellular receptors into the lumen of the multivesicular body (MVB) prior to lysosomal degradation. ESCRT components can also be recruited by enveloped viruses to sites of viral assembly where they have been proposed to mediate viral egress. For example, HIV-1 budding is dependent on Gag-mediated recruitment of the cellular ESCRTs-I, -III, AIP1/Alix and Vps4 proteins. Viral recruitment of ESCRT proteins could therefore impact on host cell processes such as receptor downregulation.

Results: Here we show that downregulation of the HIV-1 co-receptor, CXCR4, by its ligand SDF-1, is ESCRT-I dependent. Expression of HIV-1 Gag attenuated downregulation of CXCR4, resulting in accumulation of undegraded receptors within intracellular compartments. The effect of Gag was dependent on an ESCRT-I interacting motif within the C-terminal p6 region of Gag. In contrast, PMA-induced downregulation of the HIV-1 receptor CD4 was independent of ESCRT-I and Vps4; HIV-1 Gag had no effect on this process.

Conclusion: These results establish that the HIV-1 receptor, CD4, and co-receptor, CXCR4 are differentially regulated by ESCRT proteins. HIV-1 Gag selectively modulates protein sorting at the MVB, interfering with ESCRT-I dependent but not ESCRT-I independent processes.

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SDF-1 induced CXCR4 downregulation is TSG101-dependent. (A) COS-1 cells co-transfected with HA-tagged CXCR4 and either GFP, TSG101-GFP, or siRNA directed against TSG101, as indicated, were incubated with an anti-HA antibody for 1 hour on ice. Cells were then either fixed and stained with a secondary antibody to detect cell surface CXCR4 (first row), or incubated in DMEM/10% FBS without SDF (second row) or with 100 nM SDF (third and fourth rows) for 3 hours at 37°C prior to fixation, permeabilization and secondary-antibody staining. The cells were then analysed for GFP fluorescence (green), or CXCR4 expression (red). Blue represents nuclear staining. Scale bars = 10 μm. (B) Immunoblot of COS-1 cells co-transfected with HA-tagged CXCR4 and siRNA directed against either TSG101 or LacZ. Top panel, TSG101 Western blot; bottom panel, actin Western blot.
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Figure 1: SDF-1 induced CXCR4 downregulation is TSG101-dependent. (A) COS-1 cells co-transfected with HA-tagged CXCR4 and either GFP, TSG101-GFP, or siRNA directed against TSG101, as indicated, were incubated with an anti-HA antibody for 1 hour on ice. Cells were then either fixed and stained with a secondary antibody to detect cell surface CXCR4 (first row), or incubated in DMEM/10% FBS without SDF (second row) or with 100 nM SDF (third and fourth rows) for 3 hours at 37°C prior to fixation, permeabilization and secondary-antibody staining. The cells were then analysed for GFP fluorescence (green), or CXCR4 expression (red). Blue represents nuclear staining. Scale bars = 10 μm. (B) Immunoblot of COS-1 cells co-transfected with HA-tagged CXCR4 and siRNA directed against either TSG101 or LacZ. Top panel, TSG101 Western blot; bottom panel, actin Western blot.

Mentions: Previous studies have established that SDF-1 induces internalization, endosomal trafficking and lysosomal degradation of CXCR4 and HA-tagged CXCR4 in a variety of cell types [19,20,30]. To study the role of TSG101 in CXCR4 downregulation, we used transfected COS-1 cells co-expressing GFP and HA-tagged CXCR4. HA-CXCR4 has previously been shown to be a valid marker for CXCR4 trafficking and degradation in COS-1 cells [19,20]. The plasma membrane population of HA-CXCR4 was first labeled using an anti-HA antibody. Cells were then incubated with or without SDF-1 for 3 hours. In the absence of SDF-1, a considerable amount of internalization of HA-CXCR4 was observed (Fig 1A). This observation confirms previous reports and likely reflects a combination of constitutive endocytosis [31] and antibody-induced, ligand-independent endocytosis of CXCR4 [17]. HA-CXCR4 that was internalized in the absence of SDF-1 appeared in punctate, endosomal structures and remained undegraded. In contrast, cells that were incubated in the presence of SDF-1 clearly exhibited a loss in receptor signal, confirming that SDF-1 induces degradation of HA-CXCR4.


Differential control of CXCR4 and CD4 downregulation by HIV-1 Gag.

Valiathan RR, Resh MD - Virol. J. (2008)

SDF-1 induced CXCR4 downregulation is TSG101-dependent. (A) COS-1 cells co-transfected with HA-tagged CXCR4 and either GFP, TSG101-GFP, or siRNA directed against TSG101, as indicated, were incubated with an anti-HA antibody for 1 hour on ice. Cells were then either fixed and stained with a secondary antibody to detect cell surface CXCR4 (first row), or incubated in DMEM/10% FBS without SDF (second row) or with 100 nM SDF (third and fourth rows) for 3 hours at 37°C prior to fixation, permeabilization and secondary-antibody staining. The cells were then analysed for GFP fluorescence (green), or CXCR4 expression (red). Blue represents nuclear staining. Scale bars = 10 μm. (B) Immunoblot of COS-1 cells co-transfected with HA-tagged CXCR4 and siRNA directed against either TSG101 or LacZ. Top panel, TSG101 Western blot; bottom panel, actin Western blot.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2262066&req=5

Figure 1: SDF-1 induced CXCR4 downregulation is TSG101-dependent. (A) COS-1 cells co-transfected with HA-tagged CXCR4 and either GFP, TSG101-GFP, or siRNA directed against TSG101, as indicated, were incubated with an anti-HA antibody for 1 hour on ice. Cells were then either fixed and stained with a secondary antibody to detect cell surface CXCR4 (first row), or incubated in DMEM/10% FBS without SDF (second row) or with 100 nM SDF (third and fourth rows) for 3 hours at 37°C prior to fixation, permeabilization and secondary-antibody staining. The cells were then analysed for GFP fluorescence (green), or CXCR4 expression (red). Blue represents nuclear staining. Scale bars = 10 μm. (B) Immunoblot of COS-1 cells co-transfected with HA-tagged CXCR4 and siRNA directed against either TSG101 or LacZ. Top panel, TSG101 Western blot; bottom panel, actin Western blot.
Mentions: Previous studies have established that SDF-1 induces internalization, endosomal trafficking and lysosomal degradation of CXCR4 and HA-tagged CXCR4 in a variety of cell types [19,20,30]. To study the role of TSG101 in CXCR4 downregulation, we used transfected COS-1 cells co-expressing GFP and HA-tagged CXCR4. HA-CXCR4 has previously been shown to be a valid marker for CXCR4 trafficking and degradation in COS-1 cells [19,20]. The plasma membrane population of HA-CXCR4 was first labeled using an anti-HA antibody. Cells were then incubated with or without SDF-1 for 3 hours. In the absence of SDF-1, a considerable amount of internalization of HA-CXCR4 was observed (Fig 1A). This observation confirms previous reports and likely reflects a combination of constitutive endocytosis [31] and antibody-induced, ligand-independent endocytosis of CXCR4 [17]. HA-CXCR4 that was internalized in the absence of SDF-1 appeared in punctate, endosomal structures and remained undegraded. In contrast, cells that were incubated in the presence of SDF-1 clearly exhibited a loss in receptor signal, confirming that SDF-1 induces degradation of HA-CXCR4.

Bottom Line: The effect of Gag was dependent on an ESCRT-I interacting motif within the C-terminal p6 region of Gag.In contrast, PMA-induced downregulation of the HIV-1 receptor CD4 was independent of ESCRT-I and Vps4; HIV-1 Gag had no effect on this process.These results establish that the HIV-1 receptor, CD4, and co-receptor, CXCR4 are differentially regulated by ESCRT proteins.

View Article: PubMed Central - HTML - PubMed

Affiliation: Cell Biology Program, Memorial Sloan-Kettering Cancer Center, New York, USA. rrv@umich.edu

ABSTRACT

Background: The ESCRT (endosomal sorting complex required for transport) machinery functions to sort cellular receptors into the lumen of the multivesicular body (MVB) prior to lysosomal degradation. ESCRT components can also be recruited by enveloped viruses to sites of viral assembly where they have been proposed to mediate viral egress. For example, HIV-1 budding is dependent on Gag-mediated recruitment of the cellular ESCRTs-I, -III, AIP1/Alix and Vps4 proteins. Viral recruitment of ESCRT proteins could therefore impact on host cell processes such as receptor downregulation.

Results: Here we show that downregulation of the HIV-1 co-receptor, CXCR4, by its ligand SDF-1, is ESCRT-I dependent. Expression of HIV-1 Gag attenuated downregulation of CXCR4, resulting in accumulation of undegraded receptors within intracellular compartments. The effect of Gag was dependent on an ESCRT-I interacting motif within the C-terminal p6 region of Gag. In contrast, PMA-induced downregulation of the HIV-1 receptor CD4 was independent of ESCRT-I and Vps4; HIV-1 Gag had no effect on this process.

Conclusion: These results establish that the HIV-1 receptor, CD4, and co-receptor, CXCR4 are differentially regulated by ESCRT proteins. HIV-1 Gag selectively modulates protein sorting at the MVB, interfering with ESCRT-I dependent but not ESCRT-I independent processes.

Show MeSH
Related in: MedlinePlus