Plasma membrane receptors are a common feature found on cell surfaces across the body. These proteins, which are typically embedded directly in the cell membrane, can serve many functions, including cell-to-cell communication and identification to the immune system. Unfortunately, their exposure to the extracellular environment makes them a frequent target for infectious pathogens. By binding to receptors found on the surface of specific cell lines, viruses and other microorganisms can identify and target certain cells for infection and propagation.
Two of these membrane receptors in human immune cells, CD4 and CCR5, are targeted and bound by HIV-1, allowing the virus to properly identify and enter white blood cells. A small portion of the population, however, has a rare genetic mutation that results in CCR5’s absence at the cell surface, leading to HIV-1 infection resistance (1). Because of this, scientists have begun to develop anti-HIV therapies that target CCR5 expression. The most successful of these therapies binds directly to the CCR5 receptor, preventing HIV-1 binding and infection (2). This discovery has led researchers to investigate how the disruption of the transport of the CCR5 receptor to the cell membrane may provide an alternative method of HIV-1 treatment.
Before a membrane receptor is present in a cell surface, it must be synthesized and transported in a series of cell organelles that comprise its secretory pathway. This includes the Endoplasmic Reticulum (ER), where proteins are produced, the Golgi apparatus, which packages and ships proteins, and vesicles, which enclose the proteins and fuse to the cell membrane. By disrupting this pathway for CCR5 receptor delivery, scientists in a recent study were able to prevent its presence on the cell surface and therefore stop virus binding and HIV-1 infection.
To first gain an understanding of the secretory pathway of CCR5, the researchers, which represented the Curie Institute and the University of Paris, compared the receptor's membrane transport to that of tumor necrosis factor (TNF), another cell receptor. To accomplish this, they used an assay called RUSH which allows for the synchronized release of proteins in the secretory pathway after the addition of a molecule called biotin (3). Using this method, the researchers could time the transport of the proteins from one organelle to another. To visualize the movement of the proteins, they used a method called immunofluorescence, which labels the protein of interest with a dye that fluoresces under a light microscope. Together, these methods allowed them to observe and contrast the secretory pathways of the CCR5 and TNF protein receptors.
Using HeLa cells expressing CCR5 and TNF, the researchers induced transport with biotin and observed very different transport kinetics between the receptors. Visualization of the receptors in cells at 0, 15, and 20 minutes showed that CCR5 reached the plasma membrane more slowly and through different intermediate structures compared to TNF (Figure 1B,C). To confirm this kinetic transport difference, they used flow cytometry to measure the surface fluorescence intensity over time. This measurement found that CCR5, while transported more slowly than TNF, is more stable at the cell surface (Figure 1D). Graphically, this is seen by TNF intensity rising more rapidly than CCR5 but then almost immediately dissipating. These results indicate that CCR5 and TNF have different transport characteristics that are likely maintained by distinct molecular machineries that can be selectively targeted.
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| Figure 1. Transport Kinetics of CCR5 and TNF Receptors. (A) Immunofluorescence of HeLa cell expressing both CCR5 and TNF receptors (Top), CCR5 only (Middle), and TNF only (Bottom) at 0, 15, and 20 minutes after biotin addition. (C) Magnification (x2.8) of Golgi complex region. (D) Kinetics of arrival of CCR5 (magenta) or TNF (cyan) to the cell surface after release from the ER measured by fluorescence intensity. |
To search for molecules that may be candidates for inhibiting CCR5 secretion, HeLa cells expressing CCR5 and TNF were incubated with thousands of drugs from two chemical libraries: 1200 drugs from Prestwick Chemicals and 2824 drugs from the U.S. National Cancer Institute (NCI). By observing the transport of CCR5 and TNF by immunofluorescence, they determined which molecules inhibited transport for either or both receptor proteins. This allowed researchers to identify a small subset of fifteen molecules that specifically inhibited CCR5 transport.
Once a list of molecules that inhibited CCR5 was obtained, the researchers performed experiments on the specificity of the molecules, observing if any inhibited transport of the closely related CCR1 and CXCR4 receptors. If the molecules also inhibited these closely related receptors, they could be ruled out as potential CCR5-specific therapeutic candidates.
To observe the effect of the molecules on the transport of the closely related CCR1 and CXCR4 receptors relative to CCR5, they again used immunofluorescence to label the receptor proteins. This allowed surface fluorescence to be measured so they could determine whether receptor transport was inhibited in the cells. These surface expression results were graphed relative to two controls. The first control consisted of just a dimethyl surfoxide (DMSO) solution, which contained just the dissolved molecules and allowed transport to occur uninhibited. The second control did not induce transport with the addition of biotin, resulting in little to no surface fluorescence. This experiment yielded three potential molecules that strongly inhibited CCR5 (Figure 2B) while also minimally inhibiting CCR1 and CXCR4 (Figure 2C,D). In these graphs, surface expression closer to 0 indicated that the receptor transport was inhibited while surface expression closer to 100 indicated little no inhibition. These three molecules were termed molecules 13, 14, and 15 throughout the paper and across the figures.
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| Figure 2. Molecules 13, 14, and 15 inhibit CCR5 transport and do not inhibit CCR1 or CXCR4 transport. (A) Kinetics of synchronized transport of three chemokine receptors-CCR5 (black), CCR1 (red), and CXCR4 (green)-to the cell surface using the RUSH assay. Receptor transport was induced by the addition of biotin at time 0. Surface expression fluorescence intensity measured at end-point (2 hours) of the effects of the CCR5 inhibitory molecules on the trafficking of CCR5 (B), CCR1 (C), and CXCR4 (D) in HeLa cells. |
Once three candidate molecules were found to specifically inhibit CCR5, their potential therapeutic application against HIV-1 was finally tested. To accomplish this, the researchers first confirmed that the molecules inhibited CCR5 transport in human immune cells rather than just HeLa cells. After this was demonstrated, they then tested if HIV-1 infection decreased in these cells with the addition of the molecules.
Confirmation that the molecules inhibited CCR5 transport in immune cells was performed by again measuring surface expression of the receptor. However, the immune cells were isolated from various healthy blood donors. This experiment found a reduction in the surface expression of the receptor, confirming that CCR5 receptor transport was inhibited in living human immune cells.
To test if any of the molecules decreased HIV-1 infection, they used an assay called BlamM-Vpr (BV) which detects viral entry into cells by a change in protein fluorescence (4). Observing HIV-1 entry in immune cells with the addition of the three molecules, the researchers found all molecules to reduce cell infection by 45.7 to 78.0% (Figure 3C). To confirm that the molecules’ inhibitory effects were due to the absence of CCR5, a CCR5-independent HIV-1 virus termed VSVG-pseudotyped virus was used a control. As expected, this virus experienced no significant difference in virus entry into the immune cells, able to enter cells without the CCR5 receptor (Figure 3D). As an additional means of measuring HIV-1 infectivity, they quantified the total amount of viral protein produced by infected immune cells. HIV-1 viral production and secretion were both strongly reduced by 31.4 to 76.0% as a result of the addition of the molecules (Figure 3E).
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| Figure 3. Treatment with molecules 13, 14, and 15 decrease HIV-1 infection in human immune cells. Inhibition of entry of HIV-1ADA (C) or HIV-1VSVG (VSVG pseudotyped) (D) containing BlaM-Vpr (BV) with immune cells mediated by compounds. Fraction of viral protein produced by HIV-1ADA infected cells relative to control (E). Each black point represents one donor analyzed independently. |
The results of this study have significant therapeutic implications for patients infected with the HIV-1 virus. By disrupting the presence of the CCR5 receptor at the surface of immune cells, HIV-1 infection can be inhibited. Assessing the effect of thousands of molecules on CCR5 transport and further testing their effects on other receptor transport yielded three molecules that specifically disrupted the CCR5 receptor’s secretory pathway. Immune cells treated with these molecules showed a significant reduction in HIV-1 infection (Figure 3). These findings suggest that these molecules could serve as potential drug candidates for treating HIV-1 and therefore help prevent its development into AIDS.
This paper offers exciting new insight into using the secretory pathway of proteins as a means of treating medical problems on a cellular level. Because little is known about protein-specific regulation of transport in cells, there is huge potential for new medical treatments to arise with further research. Many of these treatments could follow the lead of this study and disrupt the secretion of receptors that viruses rely on for infection. However, the full potential of using the secretory pathway to prevent pathogenesis is yet to be discovered.
1. Liu, R., Paxton, W.A., Choe, S., Ceradini, D., Martin, S.R., Horuk, R., MacDonald, M.E., Stuhlmann, H., Koup, R.A., and Landau, N.R. (1996). Homozygous defect in HIV-1 coreceptor accounts for resistance of some multiply-exposed individuals to HIV-1 infection. Cell, 86:367–377, doi:10.1016/s0092-8674(00)80110-5
2. Tebas, P., Stein, D., Tang, W.W., Frank, I., Wang, S.Q., Lee, G., Spratt, S.K., Surosky, R.T., Giedlin, M.A., Nichol, G., Holmes, M.C., Gregory, P.D., Ando, D.G., Kalos, M., Collman, R.G., Binder-Scholl, G., Plesa, G., Hwang, W.T., Levine, B.L., and June, C.H. (2014). Gene editing of CCR5 in autologous CD4 T cells of persons infected with HIV. N. Engl. J. Med., 370:901–910, doi: 10.1056/NEJMoa1300662
3. Boncompain, G., Divoux, S., Gareil, N., de Forges, H., Lescure, A., Latreche, L., Mercanti, V., Jollivet, F., Raposo, G., and Perez, F. (2012). Synchronization ofsecretory protein traffic in populations of cells. Nat. Methods, 9:493–498, doi: 10.1038/nmeth.1928
4. Cavrois, M., De Noronha, C., Greene, W.C. (2002). A sensitive andspecific enzyme-based assay detecting HIV-1 virion fusion inprimary T lymphocytes. Nat. Biotechnol., 20:1151–1154, doi: 10.1038/nbt745
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