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A genetic approach to inactivating chemokine receptors using a modified viral protein

Abstract

We have developed a genetic system, called degrakine, that specifically and stably inactivates chemokine receptors (CKR) by redirecting the ability of the HIV-1 protein, Vpu, to degrade CD4 in the endoplasmic reticulum (ER) via the host proteasome machinery. To harness Vpu's proteolytic targeting capability to degrade new receptors, we fused a chemokine with the C terminal region of Vpu. The fusion protein, or degrakine, accumulates in the ER, trapping and functionally inactivating its target CKR. We have demonstrated that degrakines based on SDF-1 (CXCL12), MDC (CCL22) and RANTES (CCL5) specifically inactivate their respective receptor functions. Using a retroviral vector expressing the SDF-1 degrakine, we have established that CXCR4 is required for the homing of hematopoietic stem/progenitor cells (HSPC) to the bone marrow immediately after transplantation. Thus the degrakine provides an effective genetic tool to dissect receptor functions in a number of biological systems in vitro and in vivo.

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Figure 1: Design and function of the degrakine system.
Figure 2: The SDF-1 degrakine localizes to the ER and is stably expressed.
Figure 3: CXCR4 expression is stably and specifically downregulated by the SDF-1 degrakine.
Figure 4: The interaction between VpuC and β-TrCP is important for efficient CXCR4 downregulation.
Figure 5: Degrakines specifically inhibit their target CKR function.
Figure 6: The SDF-1 degrakine inhibits CXCR4-mediated HSPC migration in vitro and homing to the bone marrow in vivo.

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Acknowledgements

We thank S. Chen, K. Strebel, H. Golding and M. Zaitseva for kindly providing the SDF-1 intrakine construct, the Vpu2/6 mutant, anti-CXCR4-4N and CXCR4 western protocol, respectively; J. Smith, Y. Kim, M. Townsend and A. Elms for technical assistance; E. Meissner and W. Helms for critical reading of the manuscript; R. Bagnell for assistance with confocal microscopy; and L. Arnold of the UNC Flow Cytometry Facility for assistance with FACS. Anti-Vpu and anti-CXCR4 (12G5) were obtained through the NIH AIDS Reagent program. The project was partially supported by grants from the US National Institutes of Health (nos. HL72240 and AI53804 to L.S.).

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Correspondence to Lishan Su.

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Coffield, V., Jiang, Q. & Su, L. A genetic approach to inactivating chemokine receptors using a modified viral protein. Nat Biotechnol 21, 1321–1327 (2003). https://doi.org/10.1038/nbt889

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