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Binding of host factors to stabilized HIV-1 capsid tubes
Virology 523 (2018) 1–5
Contents lists available at ScienceDirect
Virology journal homepage: www.elsevier.com/locate/virology
Binding of host factors to stabilized HIV-1 capsid tubes ⁎
T
Anastasia Selyutina, Angel Bulnes-Ramos, Felipe Diaz-Griffero Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, NY 10461, USA
A R T I C LE I N FO
A B S T R A C T
Keywords: HIV-1 Capsid Binding Stabilized tubes TRIMCyp CPSF6 MxB Cyp A Human TRIM5α-R332P
The capsid-binding assay is an in vitro experiment used to determine whether cellular proteins interact with the HIV-1 core. In vitro assembled HIV-1 capsids recapitulate the surface of the HIV-1 core. The assay involves the incubation of in vitro assembled HIV-1 capsid-nucleocapsid (CA-NC) complexes with the protein in question. Subsequently, the mixture is spun through a sucrose cushion using an ultracentrifuge, and the pellet is analyzed for the presence of the protein in question. Although this binding assay is reliable, it is labor intensive and does not contain washing steps. Here we have developed a simpler and faster assay to measure whether a cellular protein is binding to capsid. More importantly, this novel capsid-binding assay contains washing steps. In this assay, we took advantage of the HIV-1 capsid mutant A14C/E45C protein, which is stabilized by disulfide bonds, and is resistant to washing steps. We validated the reliability and specificity of this novel assay by testing the capsid binding ability of TRIMCyp, CPSF6 and MxB with their corresponding controls. Overall, this novel assay provides a reliable and fast methodology to search for novel capsid binders.
1. Introduction The HIV-1 core is composed of ~1800 monomers of capsid assembled into a conical structure. Upon viral membrane fusion, the HIV1 core is delivered into the cytoplasm, where the uncoating process of the virus takes place. Uncoating is biochemically defined as the dissociation of monomeric capsids from the HIV-1 core over time during infection. Over the years, we have learned that host proteins directly interact with the HIV-1 core during the early steps of infection. Proteins that interact with the HIV-1 core can modulate its stability, which can have detrimental effects on infection. For example, TRIM5α and TRIMCyp destabilize the HIV-1 core during the early steps of infection, which inhibits productive infection (Diaz-Griffero et al., 2007; Stremlau et al., 2006). On the contrary, CPSF6 and Mx2 stabilize the HIV-1 core, which also inhibits infection (De Iaco et al., 2013; Fricke et al., 2013, 2014). These results suggested that the interaction of host proteins with the HIV-1 core could have detrimental effects on infectivity. The development of a capsid-binding assay for HIV-1 has been instrumental for the understanding of the nature and specificity of these interactions (Diaz-Griffero et al., 2006; Stremlau et al., 2006). Basically this assay allows testing a protein of choice for binding to in vitro assembled HIV-1 capsid-nucleocapsid (CA-NC) complexes, which recapitulate the surface of the HIV-1 core (Ganser et al., 1999). Briefly, the pure protein of choice or in cellular extracts is incubated with in vitro assembled HIV-1 CA-NC complexes for 1 h at 25 °C. The mixture is spun using a sucrose
⁎
cushion, which pellets the capsid complexes together with proteins that directly interact with the capsid. Although the assay is robust and uses specificity controls, it does not contain washing steps. Although the assay that uses CA-NC complexes did not have washing steps, it allowed the discovery of the interaction of proteins with the HIV-1 core such as TRIM5α, TRIMCyp, cleavage and polyadenylation specificity factor subunit 6 (CPSF6), Nup153, RanBP2/Nup358, cyclophilin A(Cyp A), MxB and others (Di Nunzio et al., 2012, 2013; Diaz-Griffero et al., 2006; Fribourgh et al., 2014; Fricke et al., 2014; Lee et al., 2010; Stremlau et al., 2006). The use of washing steps in a capsid-binding assay will provide several advantages: 1) remove the unbound protein of choice, 2) remove nonspecific binders, and 3) provides information of the relative strength of the interaction. Because of the aforementioned reasons, a capsid-binding assay that contains washing steps is very desirable. This assay will provide a solid platform to characterize the interaction of known capsid binders and to discover new proteins that bind capsid. 2. Results 2.1. Capsid-binding assay with washing steps To develop a capsid-binding assay with washing steps, we took advantage of the HIV-1 capsid mutations A14C and E45C. The HIV-1 capsid protein bearing changes A14C and E45C assembled into wild
Correspondence to: Albert Einstein College of Medicine, 1301 Morris Park – Price Center 501, New York, NY 10461, USA. E-mail address: Felipe.Diaz-Griff[email protected] (F. Diaz-Griffero).
https://doi.org/10.1016/j.virol.2018.07.019 Received 15 May 2018; Received in revised form 13 July 2018; Accepted 14 July 2018 0042-6822/ © 2018 Elsevier Inc. All rights reserved.
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Fig. 1. Diagram of the binding assay using stabilized HIV1 capsid tubes. (A) Assembly of stabilized HIV-1 capsid tubes. Recombinant capsid protein bearing the mutations A14C and E45C was purified from bacterial extracts, as previously described (Pornillos et al., 2010). The purified monomeric capsid protein is assembled into hexamers, which are subsequently stabilized by oxidation of adjacent cysteines 14 and 45 to a disulfide bond or disulfide bridge. Cysteines are depicted in red. Oxidized hexamers assemble into large stable tubes(stabilized HIV-1 capsid tubes). These tubes are readily stable in solution, and washing does not affect the integrity of the complexes. (B) Binding assay using stabilized HIV-1 capsid tubes. The tubes are incubated for 1 h with specific and nonspecific protein binders. Subsequently the tubes are washed in a buffer of choice to eliminate nonspecific binding preserving specific binding. The proteins that remain associated to the stabilized tubes are analyzed by Western blotting or mass spectrometry. (C) Cysteines 14 and 45 are illustrated forming disulfide bridges in the capsid hexameric structure 3h4e. The hexameric capsid monomers are depicted in red and orange while cysteines 14 and 45 forming disulfide bridges are shown in cyan. (D) Stabilized HIV-1 capsid tubes were negatively stained and analyzed by transmission electron microscopy. Representative fields are shown, and the scale bar corresponds to 50 nm.
shown in Fig. 1B, capsid tubes are used as a substrate to determine the binding of proteins of choice (orange octagons). Washing steps remove the unbound protein and nonspecific binding (blue stars) (Fig. 1B). On Fig. 1C, we illustrate the location of residues C14 and C45 in the hexameric capsid structure 3h4e (Pornillos et al., 2009). C14 of each monomer is forming a disulfide bridge with C45 located in the adjacent monomer. Formation of these 6 disulfide bonds stabilizes the hexameric capsid protein, which assembles into tubular structures when analyzed
type tubes (Pornillos et al., 2010). The introduced cysteines allows the capsid to cross-link efficiently into hexamers under oxidative conditions (Fig. 1A) (Pornillos et al., 2010). Cross-linked capsid hexamers assembled into stabilized HIV-1 capsid tubes in solution (Fig. 1A), which could be used as substrate for binding of a protein of choice. Remarkably, these capsid tubes are more stable in solution when compared with CA-NC tubes. The steadiness of the stabilized HIV-1 capsid tubes in solution allowed washing steps with minimal loss of capsid. As 2
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this binding is sensitive to the small molecule cyclosporine A (CsA) (Diaz-Griffero et al., 2006; Stremlau et al., 2006). To this end, we incubated human cell extracts containing TRIMCyp-FLAG (INPUT) with stabilized HIV-1 capsid tubes for 1 h at 25 °C. Stabilized tubes were pelleted in eppendorf tubes at 21,000 x g for 2 min. Stabilized tubes were washed 2,4,6,8 or 10 times using capsid binding buffer (CBB) or phosphate-buffered saline (PBS). After every wash, stabilized HIV-1 capsid tubes were pelleted using an eppendorf centrifuge at 21,000 x g for 2 min. After the final washing step, stabilized tubes were resuspended in Laemmli loading buffer (BOUND) (Fig. 2). In order to optimize the binding conditions, washing steps were performed at 25 °C or 4 °C. Subsequently, INPUT and BOUND fractions were analyzed for the presence of TRIMCyp-FLAG by Western blotting using anti-FLAG antibodies. The BOUND fraction was also analyzed for the presence of p24 using anti-p24 antibodies. As a control, we performed bindings in the presence of the small molecule CsA, which prevents the binding of TRIMCyp to capsid. Washing the stabilized tubes with CBB at 25 °C (Fig. 2A) minimally lose TRIMCyp-FLAG binding when compared to the use of CBB at 4 °C (Fig. 2B). These experiments suggest that the use of CBB at 25 °C is the best option to preserve the binding of TRIMCyp. In agreement, the use of CsA prevented the binding of TRIMCyp-FLAG to capsid (Fig. 2A and B). One caveat of the assay is that extensive washes can start depleting the amount of stabilized tubes from the mixture. At the same time, we tested washing with PBS at 25 °C and 4 °C. Washing the stabilized tubes with PBS at 25 °C or 4 °C better preserved the stabilized tubes in the mixture, as shown by the p24 levels in the bound fraction (Fig. 2C and D). Similarly, the use of CsA prevented the binding of TRIMCyp to capsid. These results demonstrated that the binding of TRIMCyp to the assembled capsid is sensitive to CsA, as previously shown (Diaz-Griffero et al., 2006; Stremlau et al., 2006). These extensive binding studies validate this novel capsid-binding assay. 2.3. CPSF6 binding to stabilized HIV-1 capsid tubes using washing steps To further validate our assay, we tested the ability of CPSF6 to bind to stabilized HIV-1 capsid tubes. CPSF6 binds to the HIV-1 core, and this binding is sensitive to the small molecule PF74 (Fricke et al., 2013; Lee et al., 2010). To this end, we tested the ability of CPSF6-FLAG to bind stabilized tubes of capsid. As shown in Fig. 3, CPSF6-FLAG binds to the stabilized HIV-1 capsid tubes. In agreement with previous reports, this binding was sensitive to the use of increasing concentrations of PF74 (Fricke et al., 2013; Lee et al., 2010). These results account for the specificity of the binding assay and further validate our assay.
Fig. 2. TRIMCyp binding to stabilized HIV-1 capsid tubes. To test the ability of TRIMCyp to bind stabilized HIV-1 capsid tubes, we expressed TRIMCyp-FLAG in human HEK293 cells (INPUT). Human cell extracts containing TRIMCypFLAG were incubated with stabilized HIV-1 capsid tubes for 1 h at 25 °C. Subsequently, tubes were washed at 25 °C using 1 ml of capsid binding buffer (CBB) (A) or phosphate-buffered saline (PBS) (C) the indicated times. Stabilized HIV-1 capsid tubes were washed by inverting the eppendorf tube three times (this represents one washing step). Stabilized HIV-1 capsid tubes were pelleted at 21,000 × g, and resuspended in 1x Laemmli (BOUND). INPUT and BOUND fractions were analyzed by Western blotting using anti-FLAG and anti-p24 antibodies. As a control, we performed binding assays in the presence of the drug CsA, which prevents the binding of TRIMCyp to capsid. The binding of TRIMCyp to stabilized HIV-1 capsid tubes was also tested using washing steps at 4 °C using 1 ml of CBB (B) or PBS (D) the indicated times. Experiments were performed at least three times, and a representative experiment is shown.
Fig. 3. CPSF6 binding to stabilized HIV-1 capsid tubes. To test the ability of NES-CPSF6-FLAG to bind stabilized HIV-1 capsid tubes, we expressed NESCPSF6-FLAG in human HEK293 cells (INPUT). Human cell extracts containing NES-CPSF6-FLAG were incubated with stabilized HIV-1 capsid tubes for 1 h at 25 °C. Subsequently, tubes were washed at 25 °C using 1 ml of capsid binding buffer (CBB) the indicated times. Stabilized HIV-1 capsid tubes were pelleted at 21,000 x g, and resuspended in 1x Laemmli buffer (BOUND). INPUT and BOUND fractions were analyzed by Western blotting using anti-FLAG and antip24 antibodies. As a control, we performed binding assays in the presence of the drug PF74, which prevents the binding of CPSF6 to capsid. Experiments were performed at least three times, and a representative experiment is shown.
by negative staining under the electron microscope (Fig. 1D, left image). Electron microscope analysis allowed us to visualize the hexameric capsid units in the stabilized tubes (Fig. 1D, right image). 2.2. TRIMCyp binding to stabilized HIV-1 capsid tubes To validate this novel assay, we tested the ability of TRIMCyp to bind to HIV-1 capsid. TRIMCyp potently binds to the HIV-1 core, and 3
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TRIMCyp, which is known to interact with stabilized HIV-1 capsid tubes. 2.4.1. Endogenously expressed Cyp A binds to stabilized HIV-1 capsid tubes, but not stabilized HIV-1 P90A capsid tubes Here we sought to test the ability of endogenously expressed Cyp A from HeLa cells to bind stabilized HIV-1 capsid tubes. As expected, Cyp A binds to stabilized HIV-1 capsid tubes, but not to stabilized HIV-1 P90A capsid tubes (Fig. 4B). As a control, we performed similar bindings in the presence of CsA, which prevents the binding of Cyp A to capsid. These results showed that this assay could also be used to test the binding of proteins to tubes that were assembled using mutant capsids. 2.5. Comparison between capsid binding assays To compare the capsid binding assay that uses in vitro assembled HIV-1 CA-NC complexes to the novel binding assay described here, we performed these assays side-by-side. To this end, we simultaneously tested the binding of human TRIM5α (TRIM5αhu) bearing the mutation R332P to in vitro assembled HIV-1 CA-NC complexes and stabilized HIV-1 capsid tubes. Contrary to the wild type TRIM5αhu protein, TRIM5αhu-R332P binds to capsid and restrict HIV-1 (Li et al., 2006). As shown in Fig. 4C, our results showed that the new assay is more sensitive when compared to the assay that uses in vitro assembled HIV-1 CA-NC complexes. 3. Discussion This work validates a novel capsid-binding assay, which has several advantages over the capsid-binding assay that uses CA-NC complexes: 1) it contains washing steps, 2) assembly of the tubes does not require the use of oligo DNA, which increases nonspecific binding, 3) contrary to CA-NC tubes, stabilized HIV-1 capsid tubes do not contain nucleocapsid (NC), which increases nonspecific binding, 4) contrary to CA-NC tubes that last only a few days, stabilized HIV-1 capsid tubes preserve their binding ability for weeks, 5) salt is not required to stabilize the capsid tubes, which might interfere with binding, 6) the new assay does not require an ultracentrifugation step, which limits the binding to 6 samples per run in an ultracentrifuge, and can be performed on the bench, 7) the assay is shorter when compared to the assay that uses CANC complexes, and 8) this assay is more sensitive when compared the assay that uses CA-NC complexes. Overall this is a simpler and faster assay that contains washing steps. This assay will be instrumental for the search of novel HIV-1 capsid binders. In addition, it provides the investigator with the flexibility to perform the binding and washes in different buffer conditions.
Fig. 4. Binding of different factors to stabilized HIV-1 capsid tubes. (A) MxB binding to stabilized HIV-1 capsid tubes. To test the ability of MxB to bind stabilized HIV-1 capsid tubes, we expressed MxB-FLAG in human HEK293 cells (INPUT). Human cell extracts containing MxB-FLAG were incubated with stabilized HIV-1 capsid tubes for 1 h at 25 °C. Subsequently, tubes were washed at 25 °C using 1 ml of CBB the indicated times. Stabilized HIV-1 capsid tubes were pelleted at 21,000 x g, and resuspended in 1x Laemmli buffer (BOUND). INPUT and BOUND fractions were analyzed by Western blotting using anti-FLAG and anti-p24 antibodies. As a negative control, we tested the capsid binding ability of MxA, which is known not to bind. As a positive control, we tested the capsid binding ability of TRIMCyp. (B) Similarly, we tested the ability of endogenously expressed Cyp A in HeLa cells to bind wild type and P90A stabilized HIV-1 capsid tubes. As a control, we performed similar bindings in the presence of CsA, which prevents the binding of Cyp A to capsid. (C) Comparison of the capsid-binding assay that uses in vitro assembled HIV-1 CA-NC complexes with the assay that uses stabilized HIV-1 capsid tubes. The ability of TRIM5αhuR332P to bind in vitro assembled HIV-1 CA-NC complexes and stabilized HIV-1 capsid tubes were measured. Experiments were performed at least three times, and a representative experiment is shown.
4. Methods 4.1. Protein expression and purification pET-11a vectors were used to express the HIV-1 capsid protein. Point mutations, A14C and E45C were introduced using the QuikChange II site-directed mutagenesis kit (Stratagene) according to the manufacturer's instructions. All proteins were expressed in Escherichia coli one-shoot BL21star™ (DE3) cells (Invitrogen). Briefly, LB medium was inoculated with overnight cultures, which were grown at 30 °C until mid log-phase (A600, 0.6–0.8). Protein expression was induced with 1 mM isopropyl-β-d-thiogalactopyranoside (IPTG) overnight at 18 °C. Cells were harvested by centrifugation at 5000 × g for 10 min at 4 °C, and pellets were stored at − 80 °C until purification. Purification of capsid was carried out as follows. Pellets from 2 L of bacteria were lysed by sonication (Qsonica microtip: 4420; A=45; 2 min; 2 s on; 2 s off for 12 cycles), in 40 ml of lysis buffer (50 mM Tris pH=8, 50 mM NaCl, 100 mM β-mercaptoethanol and Complete EDTA-
2.4. MxB binding to stabilized HIV-1 capsid tubes using washing steps We have previously reported that MxB, contrary to MxA, binds to the HIV-1 core and to in vitro assembled HIV-1 CA-NC complexes (DiazGriffero et al., 2006; Fribourgh et al., 2014; Fricke et al., 2014; Stremlau et al., 2006). To test weather these results are reproducible using our novel assay, we tested the ability of MxB and MxA to bind to stabilized HIV-1 capsid tubes. As shown in Fig. 4A, MxB binds to stabilized HIV-1 capsid tubes. In agreement with previous reports, MxA did not interact with capsid. As a positive control, we included 4
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Acknowledgements
free protease inhibitor tablets). Cell debris were removed by centrifugation at 40,000 g for 20 min at 4 °C. Proteins from the supernatant were precipitated by incubation with 1/3 of volume of saturated ammonium sulfate containing 100 mM β-mercaptoethanol for 20 min at 4 °C and centrifugation at 8000 g for 20 min at 4 °C. Precipitated proteins were resuspended in 30 ml of buffer A (25 mM MES pH6.5, 100 mM β-mercaptoethanol) and sonicated 2–3 times (Qsonica microtip: 4420; A=45; 2 min; 1 s on; 2 s off). Sample was dialyzed 3 times in buffer A (2 h, overnight, 2 h). The sample was sonicated and diluted in 500 ml of buffer A and was chromatographed sequentially on a 5 ml HiTrap™ Q HP column and on a 5 ml HiTrap™ SP FF column (GE Healthcare), both pre-equilibrated with buffer A. The capsid protein was eluted from HiTrap™ SP FF column using a linear gradient from 0 to 2 M of NaCl. Absorbance at 280 nm was checked to take the eluted fraction that had higher protein levels. Pooled fractions were dialyzed 3 times (2 h, overnight, 2 h) in storage buffer (25 mM MES, 2 M NaCl, 20 mM β-mercaptoethanol). Sample was concentrated using centricons to a concentration of 20 mg/ml and stored at − 80 °C.
We thank the NIH AIDS repository for important reagents such as the HIV-1 p24 monoclonal antibody (183-H12-5C). We would like to thank Thomas Fricke and Juan Manuel Carreno for technical assistance. The work was supported by an R01 grant from the NIH AI087390, to F.D.-G. References De Iaco, A., Santoni, F., Vannier, A., Guipponi, M., Antonarakis, S., Luban, J., 2013. TNPO3 protects HIV-1 replication from CPSF6-mediated capsid stabilization in the host cell cytoplasm. Retrovirology 10, 20. Di Nunzio, F., Danckaert, A., Fricke, T., Perez, P., Fernandez, J., Perret, E., Roux, P., Shorte, S., Charneau, P., Diaz-Griffero, F., Arhel, N.J., 2012. Human nucleoporins promote HIV-1 docking at the nuclear pore, nuclear import and integration. PLoS One 7, e46037. Di Nunzio, F., Fricke, T., Miccio, A., Valle-Casuso, J.C., Perez, P., Souque, P., Rizzi, E., Severgnini, M., Mavilio, F., Charneau, P., Diaz-Griffero, F., 2013. Nup153 and Nup98 bind the HIV-1 core and contribute to the early steps of HIV-1 replication. Virology 440, 8–18. Diaz-Griffero, F., Kar, A., Lee, M., Stremlau, M., Poeschla, E., Sodroski, J., 2007. Comparative requirements for the restriction of retrovirus infection by TRIM5alpha and TRIMCyp. Virology 369, 400–410. Diaz-Griffero, F., Vandegraaff, N., Li, Y., McGee-Estrada, K., Stremlau, M., Welikala, S., Si, Z., Engelman, A., Sodroski, J., 2006. Requirements for capsid-binding and an effector function in TRIMCyp-mediated restriction of HIV-1. Virology 351, 404–419. Fribourgh, J.L., Nguyen, H.C., Matreyek, K.A., Alvarez, F.J., Summers, B.J., Dewdney, T.G., Aiken, C., Zhang, P., Engelman, A., Xiong, Y., 2014. Structural insight into HIV1 restriction by MxB. Cell Host Microbe 16, 627–638. Fricke, T., Valle-Casuso, J.C., White, T.E., Brandariz-Nunez, A., Bosche, W.J., Reszka, N., Gorelick, R., Diaz-Griffero, F., 2013. The ability of TNPO3-depleted cells to inhibit HIV-1 infection requires CPSF6. Retrovirology 10, 46. Fricke, T., White, T.E., Schulte, B., de Souza Aranha Vieira, D.A., Dharan, A., Campbell, E.M., Brandariz-Nunez, A., Diaz-Griffero, F., 2014. MxB binds to the HIV-1 core and prevents the uncoating process of HIV-1. Retrovirology 11, 68. Ganser, B.K., Li, S., Klishko, V.Y., Finch, J.T., Sundquist, W.I., 1999. Assembly and analysis of conical models for the HIV-1 core. Science 283, 80–83. Lee, K., Ambrose, Z., Martin, T.D., Oztop, I., Mulky, A., Julias, J.G., Vandegraaff, N., Baumann, J.G., Wang, R., Yuen, W., Takemura, T., Shelton, K., Taniuchi, I., Li, Y., Sodroski, J., Littman, D.R., Coffin, J.M., Hughes, S.H., Unutmaz, D., Engelman, A., KewalRamani, V.N., 2010. Flexible use of nuclear import pathways by HIV-1. Cell Host Microbe 7, 221–233. Li, Y., Li, X., Stremlau, M., Lee, M., Sodroski, J., 2006. Removal of arginine 332 allows human TRIM5alpha to bind human immunodeficiency virus capsids and to restrict infection. J. Virol. 80, 6738–6744. Pornillos, O., Ganser-Pornillos, B.K., Banumathi, S., Hua, Y., Yeager, M., 2010. Disulfide bond stabilization of the hexameric capsomer of human immunodeficiency virus. J. Mol. Biol. 401, 985–995. Pornillos, O., Ganser-Pornillos, B.K., Kelly, B.N., Hua, Y., Whitby, F.G., Stout, C.D., Sundquist, W.I., Hill, C.P., Yeager, M., 2009. X-ray structures of the hexameric building block of the HIV capsid. Cell 137, 1282–1292. Stremlau, M., Perron, M., Lee, M., Li, Y., Song, B., Javanbakht, H., Diaz-Griffero, F., Anderson, D.J., Sundquist, W.I., Sodroski, J., 2006. Specific recognition and accelerated uncoating of retroviral capsids by the TRIM5alpha restriction factor. Proc. Natl. Acad. Sci. USA 103, 5514–5519.
4.2. Assembly of stabilized HIV-1 capsid tubes 1 ml of monomeric capsid (3 mg/ml or 1 mg/ml) was dialyzed in SnakeSkin dialysis tubing 10,000 MWCO (Thermo Scientific) against a buffer that is high in salt and contains a reducing agent (buffer 1: 50 mM Tris, pH 8, 1 M NaCl, 100 mM β-mercaptoethanol) at 4 °C for 8 h. Subsequently the protein was dialyzed against the same buffer without the reducing agent β-mercaptoethanol (buffer 2: 50 mM Tris, pH 8, 1 M NaCl) at 4 °C for 8 h. The absence of β-mercaptoethanol in the second dialysis allows formation of disulfide bonds between Cysteine 14 and 43 inter-capsid monomers in the hexamer. Finally the protein is dialyzed against buffer 3 (20 mM Tris, pH 8,0, 40 mM NaCl) at 4 °C for 8 h. Assembled complexes were kept at 4 °C up to 1 month. 4.3. Capsid binding assay protocol Human HEK293T cells were transfected for 24 h with a plasmid expressing the protein of interest. Cell media was completely removed and cells were lysed in 300 μL of capsid binding buffer (CBB: 10 mM Tris, pH 8,0, 1,5 mM MgCl2, 10 mM KCl) by scrapping off the plate. Cells were rotated at 4 °C for 15 min and then centrifuged to remove cellular debris (21,000 x g, 15 min, 4 °C). Cell lysates were incubated with stabilized HIV-1 capsid tubes for 1 h at 25 °C. Subsequently, stabilized HIV-1 capsid tubes were washed by pelleting the complexes by centrifugation at 21,000 × g for 2 min. Pellets were washed using by resuspension in CBB or PBS. Pellets were resuspended in Laemmli buffer 1X and analyzed by Western blotting using anti-p24 and the indicated antibodies.
5
Contents lists available at ScienceDirect
Virology journal homepage: www.elsevier.com/locate/virology
Binding of host factors to stabilized HIV-1 capsid tubes ⁎
T
Anastasia Selyutina, Angel Bulnes-Ramos, Felipe Diaz-Griffero Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, NY 10461, USA
A R T I C LE I N FO
A B S T R A C T
Keywords: HIV-1 Capsid Binding Stabilized tubes TRIMCyp CPSF6 MxB Cyp A Human TRIM5α-R332P
The capsid-binding assay is an in vitro experiment used to determine whether cellular proteins interact with the HIV-1 core. In vitro assembled HIV-1 capsids recapitulate the surface of the HIV-1 core. The assay involves the incubation of in vitro assembled HIV-1 capsid-nucleocapsid (CA-NC) complexes with the protein in question. Subsequently, the mixture is spun through a sucrose cushion using an ultracentrifuge, and the pellet is analyzed for the presence of the protein in question. Although this binding assay is reliable, it is labor intensive and does not contain washing steps. Here we have developed a simpler and faster assay to measure whether a cellular protein is binding to capsid. More importantly, this novel capsid-binding assay contains washing steps. In this assay, we took advantage of the HIV-1 capsid mutant A14C/E45C protein, which is stabilized by disulfide bonds, and is resistant to washing steps. We validated the reliability and specificity of this novel assay by testing the capsid binding ability of TRIMCyp, CPSF6 and MxB with their corresponding controls. Overall, this novel assay provides a reliable and fast methodology to search for novel capsid binders.
1. Introduction The HIV-1 core is composed of ~1800 monomers of capsid assembled into a conical structure. Upon viral membrane fusion, the HIV1 core is delivered into the cytoplasm, where the uncoating process of the virus takes place. Uncoating is biochemically defined as the dissociation of monomeric capsids from the HIV-1 core over time during infection. Over the years, we have learned that host proteins directly interact with the HIV-1 core during the early steps of infection. Proteins that interact with the HIV-1 core can modulate its stability, which can have detrimental effects on infection. For example, TRIM5α and TRIMCyp destabilize the HIV-1 core during the early steps of infection, which inhibits productive infection (Diaz-Griffero et al., 2007; Stremlau et al., 2006). On the contrary, CPSF6 and Mx2 stabilize the HIV-1 core, which also inhibits infection (De Iaco et al., 2013; Fricke et al., 2013, 2014). These results suggested that the interaction of host proteins with the HIV-1 core could have detrimental effects on infectivity. The development of a capsid-binding assay for HIV-1 has been instrumental for the understanding of the nature and specificity of these interactions (Diaz-Griffero et al., 2006; Stremlau et al., 2006). Basically this assay allows testing a protein of choice for binding to in vitro assembled HIV-1 capsid-nucleocapsid (CA-NC) complexes, which recapitulate the surface of the HIV-1 core (Ganser et al., 1999). Briefly, the pure protein of choice or in cellular extracts is incubated with in vitro assembled HIV-1 CA-NC complexes for 1 h at 25 °C. The mixture is spun using a sucrose
⁎
cushion, which pellets the capsid complexes together with proteins that directly interact with the capsid. Although the assay is robust and uses specificity controls, it does not contain washing steps. Although the assay that uses CA-NC complexes did not have washing steps, it allowed the discovery of the interaction of proteins with the HIV-1 core such as TRIM5α, TRIMCyp, cleavage and polyadenylation specificity factor subunit 6 (CPSF6), Nup153, RanBP2/Nup358, cyclophilin A(Cyp A), MxB and others (Di Nunzio et al., 2012, 2013; Diaz-Griffero et al., 2006; Fribourgh et al., 2014; Fricke et al., 2014; Lee et al., 2010; Stremlau et al., 2006). The use of washing steps in a capsid-binding assay will provide several advantages: 1) remove the unbound protein of choice, 2) remove nonspecific binders, and 3) provides information of the relative strength of the interaction. Because of the aforementioned reasons, a capsid-binding assay that contains washing steps is very desirable. This assay will provide a solid platform to characterize the interaction of known capsid binders and to discover new proteins that bind capsid. 2. Results 2.1. Capsid-binding assay with washing steps To develop a capsid-binding assay with washing steps, we took advantage of the HIV-1 capsid mutations A14C and E45C. The HIV-1 capsid protein bearing changes A14C and E45C assembled into wild
Correspondence to: Albert Einstein College of Medicine, 1301 Morris Park – Price Center 501, New York, NY 10461, USA. E-mail address: Felipe.Diaz-Griff[email protected] (F. Diaz-Griffero).
https://doi.org/10.1016/j.virol.2018.07.019 Received 15 May 2018; Received in revised form 13 July 2018; Accepted 14 July 2018 0042-6822/ © 2018 Elsevier Inc. All rights reserved.
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Fig. 1. Diagram of the binding assay using stabilized HIV1 capsid tubes. (A) Assembly of stabilized HIV-1 capsid tubes. Recombinant capsid protein bearing the mutations A14C and E45C was purified from bacterial extracts, as previously described (Pornillos et al., 2010). The purified monomeric capsid protein is assembled into hexamers, which are subsequently stabilized by oxidation of adjacent cysteines 14 and 45 to a disulfide bond or disulfide bridge. Cysteines are depicted in red. Oxidized hexamers assemble into large stable tubes(stabilized HIV-1 capsid tubes). These tubes are readily stable in solution, and washing does not affect the integrity of the complexes. (B) Binding assay using stabilized HIV-1 capsid tubes. The tubes are incubated for 1 h with specific and nonspecific protein binders. Subsequently the tubes are washed in a buffer of choice to eliminate nonspecific binding preserving specific binding. The proteins that remain associated to the stabilized tubes are analyzed by Western blotting or mass spectrometry. (C) Cysteines 14 and 45 are illustrated forming disulfide bridges in the capsid hexameric structure 3h4e. The hexameric capsid monomers are depicted in red and orange while cysteines 14 and 45 forming disulfide bridges are shown in cyan. (D) Stabilized HIV-1 capsid tubes were negatively stained and analyzed by transmission electron microscopy. Representative fields are shown, and the scale bar corresponds to 50 nm.
shown in Fig. 1B, capsid tubes are used as a substrate to determine the binding of proteins of choice (orange octagons). Washing steps remove the unbound protein and nonspecific binding (blue stars) (Fig. 1B). On Fig. 1C, we illustrate the location of residues C14 and C45 in the hexameric capsid structure 3h4e (Pornillos et al., 2009). C14 of each monomer is forming a disulfide bridge with C45 located in the adjacent monomer. Formation of these 6 disulfide bonds stabilizes the hexameric capsid protein, which assembles into tubular structures when analyzed
type tubes (Pornillos et al., 2010). The introduced cysteines allows the capsid to cross-link efficiently into hexamers under oxidative conditions (Fig. 1A) (Pornillos et al., 2010). Cross-linked capsid hexamers assembled into stabilized HIV-1 capsid tubes in solution (Fig. 1A), which could be used as substrate for binding of a protein of choice. Remarkably, these capsid tubes are more stable in solution when compared with CA-NC tubes. The steadiness of the stabilized HIV-1 capsid tubes in solution allowed washing steps with minimal loss of capsid. As 2
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this binding is sensitive to the small molecule cyclosporine A (CsA) (Diaz-Griffero et al., 2006; Stremlau et al., 2006). To this end, we incubated human cell extracts containing TRIMCyp-FLAG (INPUT) with stabilized HIV-1 capsid tubes for 1 h at 25 °C. Stabilized tubes were pelleted in eppendorf tubes at 21,000 x g for 2 min. Stabilized tubes were washed 2,4,6,8 or 10 times using capsid binding buffer (CBB) or phosphate-buffered saline (PBS). After every wash, stabilized HIV-1 capsid tubes were pelleted using an eppendorf centrifuge at 21,000 x g for 2 min. After the final washing step, stabilized tubes were resuspended in Laemmli loading buffer (BOUND) (Fig. 2). In order to optimize the binding conditions, washing steps were performed at 25 °C or 4 °C. Subsequently, INPUT and BOUND fractions were analyzed for the presence of TRIMCyp-FLAG by Western blotting using anti-FLAG antibodies. The BOUND fraction was also analyzed for the presence of p24 using anti-p24 antibodies. As a control, we performed bindings in the presence of the small molecule CsA, which prevents the binding of TRIMCyp to capsid. Washing the stabilized tubes with CBB at 25 °C (Fig. 2A) minimally lose TRIMCyp-FLAG binding when compared to the use of CBB at 4 °C (Fig. 2B). These experiments suggest that the use of CBB at 25 °C is the best option to preserve the binding of TRIMCyp. In agreement, the use of CsA prevented the binding of TRIMCyp-FLAG to capsid (Fig. 2A and B). One caveat of the assay is that extensive washes can start depleting the amount of stabilized tubes from the mixture. At the same time, we tested washing with PBS at 25 °C and 4 °C. Washing the stabilized tubes with PBS at 25 °C or 4 °C better preserved the stabilized tubes in the mixture, as shown by the p24 levels in the bound fraction (Fig. 2C and D). Similarly, the use of CsA prevented the binding of TRIMCyp to capsid. These results demonstrated that the binding of TRIMCyp to the assembled capsid is sensitive to CsA, as previously shown (Diaz-Griffero et al., 2006; Stremlau et al., 2006). These extensive binding studies validate this novel capsid-binding assay. 2.3. CPSF6 binding to stabilized HIV-1 capsid tubes using washing steps To further validate our assay, we tested the ability of CPSF6 to bind to stabilized HIV-1 capsid tubes. CPSF6 binds to the HIV-1 core, and this binding is sensitive to the small molecule PF74 (Fricke et al., 2013; Lee et al., 2010). To this end, we tested the ability of CPSF6-FLAG to bind stabilized tubes of capsid. As shown in Fig. 3, CPSF6-FLAG binds to the stabilized HIV-1 capsid tubes. In agreement with previous reports, this binding was sensitive to the use of increasing concentrations of PF74 (Fricke et al., 2013; Lee et al., 2010). These results account for the specificity of the binding assay and further validate our assay.
Fig. 2. TRIMCyp binding to stabilized HIV-1 capsid tubes. To test the ability of TRIMCyp to bind stabilized HIV-1 capsid tubes, we expressed TRIMCyp-FLAG in human HEK293 cells (INPUT). Human cell extracts containing TRIMCypFLAG were incubated with stabilized HIV-1 capsid tubes for 1 h at 25 °C. Subsequently, tubes were washed at 25 °C using 1 ml of capsid binding buffer (CBB) (A) or phosphate-buffered saline (PBS) (C) the indicated times. Stabilized HIV-1 capsid tubes were washed by inverting the eppendorf tube three times (this represents one washing step). Stabilized HIV-1 capsid tubes were pelleted at 21,000 × g, and resuspended in 1x Laemmli (BOUND). INPUT and BOUND fractions were analyzed by Western blotting using anti-FLAG and anti-p24 antibodies. As a control, we performed binding assays in the presence of the drug CsA, which prevents the binding of TRIMCyp to capsid. The binding of TRIMCyp to stabilized HIV-1 capsid tubes was also tested using washing steps at 4 °C using 1 ml of CBB (B) or PBS (D) the indicated times. Experiments were performed at least three times, and a representative experiment is shown.
Fig. 3. CPSF6 binding to stabilized HIV-1 capsid tubes. To test the ability of NES-CPSF6-FLAG to bind stabilized HIV-1 capsid tubes, we expressed NESCPSF6-FLAG in human HEK293 cells (INPUT). Human cell extracts containing NES-CPSF6-FLAG were incubated with stabilized HIV-1 capsid tubes for 1 h at 25 °C. Subsequently, tubes were washed at 25 °C using 1 ml of capsid binding buffer (CBB) the indicated times. Stabilized HIV-1 capsid tubes were pelleted at 21,000 x g, and resuspended in 1x Laemmli buffer (BOUND). INPUT and BOUND fractions were analyzed by Western blotting using anti-FLAG and antip24 antibodies. As a control, we performed binding assays in the presence of the drug PF74, which prevents the binding of CPSF6 to capsid. Experiments were performed at least three times, and a representative experiment is shown.
by negative staining under the electron microscope (Fig. 1D, left image). Electron microscope analysis allowed us to visualize the hexameric capsid units in the stabilized tubes (Fig. 1D, right image). 2.2. TRIMCyp binding to stabilized HIV-1 capsid tubes To validate this novel assay, we tested the ability of TRIMCyp to bind to HIV-1 capsid. TRIMCyp potently binds to the HIV-1 core, and 3
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TRIMCyp, which is known to interact with stabilized HIV-1 capsid tubes. 2.4.1. Endogenously expressed Cyp A binds to stabilized HIV-1 capsid tubes, but not stabilized HIV-1 P90A capsid tubes Here we sought to test the ability of endogenously expressed Cyp A from HeLa cells to bind stabilized HIV-1 capsid tubes. As expected, Cyp A binds to stabilized HIV-1 capsid tubes, but not to stabilized HIV-1 P90A capsid tubes (Fig. 4B). As a control, we performed similar bindings in the presence of CsA, which prevents the binding of Cyp A to capsid. These results showed that this assay could also be used to test the binding of proteins to tubes that were assembled using mutant capsids. 2.5. Comparison between capsid binding assays To compare the capsid binding assay that uses in vitro assembled HIV-1 CA-NC complexes to the novel binding assay described here, we performed these assays side-by-side. To this end, we simultaneously tested the binding of human TRIM5α (TRIM5αhu) bearing the mutation R332P to in vitro assembled HIV-1 CA-NC complexes and stabilized HIV-1 capsid tubes. Contrary to the wild type TRIM5αhu protein, TRIM5αhu-R332P binds to capsid and restrict HIV-1 (Li et al., 2006). As shown in Fig. 4C, our results showed that the new assay is more sensitive when compared to the assay that uses in vitro assembled HIV-1 CA-NC complexes. 3. Discussion This work validates a novel capsid-binding assay, which has several advantages over the capsid-binding assay that uses CA-NC complexes: 1) it contains washing steps, 2) assembly of the tubes does not require the use of oligo DNA, which increases nonspecific binding, 3) contrary to CA-NC tubes, stabilized HIV-1 capsid tubes do not contain nucleocapsid (NC), which increases nonspecific binding, 4) contrary to CA-NC tubes that last only a few days, stabilized HIV-1 capsid tubes preserve their binding ability for weeks, 5) salt is not required to stabilize the capsid tubes, which might interfere with binding, 6) the new assay does not require an ultracentrifugation step, which limits the binding to 6 samples per run in an ultracentrifuge, and can be performed on the bench, 7) the assay is shorter when compared to the assay that uses CANC complexes, and 8) this assay is more sensitive when compared the assay that uses CA-NC complexes. Overall this is a simpler and faster assay that contains washing steps. This assay will be instrumental for the search of novel HIV-1 capsid binders. In addition, it provides the investigator with the flexibility to perform the binding and washes in different buffer conditions.
Fig. 4. Binding of different factors to stabilized HIV-1 capsid tubes. (A) MxB binding to stabilized HIV-1 capsid tubes. To test the ability of MxB to bind stabilized HIV-1 capsid tubes, we expressed MxB-FLAG in human HEK293 cells (INPUT). Human cell extracts containing MxB-FLAG were incubated with stabilized HIV-1 capsid tubes for 1 h at 25 °C. Subsequently, tubes were washed at 25 °C using 1 ml of CBB the indicated times. Stabilized HIV-1 capsid tubes were pelleted at 21,000 x g, and resuspended in 1x Laemmli buffer (BOUND). INPUT and BOUND fractions were analyzed by Western blotting using anti-FLAG and anti-p24 antibodies. As a negative control, we tested the capsid binding ability of MxA, which is known not to bind. As a positive control, we tested the capsid binding ability of TRIMCyp. (B) Similarly, we tested the ability of endogenously expressed Cyp A in HeLa cells to bind wild type and P90A stabilized HIV-1 capsid tubes. As a control, we performed similar bindings in the presence of CsA, which prevents the binding of Cyp A to capsid. (C) Comparison of the capsid-binding assay that uses in vitro assembled HIV-1 CA-NC complexes with the assay that uses stabilized HIV-1 capsid tubes. The ability of TRIM5αhuR332P to bind in vitro assembled HIV-1 CA-NC complexes and stabilized HIV-1 capsid tubes were measured. Experiments were performed at least three times, and a representative experiment is shown.
4. Methods 4.1. Protein expression and purification pET-11a vectors were used to express the HIV-1 capsid protein. Point mutations, A14C and E45C were introduced using the QuikChange II site-directed mutagenesis kit (Stratagene) according to the manufacturer's instructions. All proteins were expressed in Escherichia coli one-shoot BL21star™ (DE3) cells (Invitrogen). Briefly, LB medium was inoculated with overnight cultures, which were grown at 30 °C until mid log-phase (A600, 0.6–0.8). Protein expression was induced with 1 mM isopropyl-β-d-thiogalactopyranoside (IPTG) overnight at 18 °C. Cells were harvested by centrifugation at 5000 × g for 10 min at 4 °C, and pellets were stored at − 80 °C until purification. Purification of capsid was carried out as follows. Pellets from 2 L of bacteria were lysed by sonication (Qsonica microtip: 4420; A=45; 2 min; 2 s on; 2 s off for 12 cycles), in 40 ml of lysis buffer (50 mM Tris pH=8, 50 mM NaCl, 100 mM β-mercaptoethanol and Complete EDTA-
2.4. MxB binding to stabilized HIV-1 capsid tubes using washing steps We have previously reported that MxB, contrary to MxA, binds to the HIV-1 core and to in vitro assembled HIV-1 CA-NC complexes (DiazGriffero et al., 2006; Fribourgh et al., 2014; Fricke et al., 2014; Stremlau et al., 2006). To test weather these results are reproducible using our novel assay, we tested the ability of MxB and MxA to bind to stabilized HIV-1 capsid tubes. As shown in Fig. 4A, MxB binds to stabilized HIV-1 capsid tubes. In agreement with previous reports, MxA did not interact with capsid. As a positive control, we included 4
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Acknowledgements
free protease inhibitor tablets). Cell debris were removed by centrifugation at 40,000 g for 20 min at 4 °C. Proteins from the supernatant were precipitated by incubation with 1/3 of volume of saturated ammonium sulfate containing 100 mM β-mercaptoethanol for 20 min at 4 °C and centrifugation at 8000 g for 20 min at 4 °C. Precipitated proteins were resuspended in 30 ml of buffer A (25 mM MES pH6.5, 100 mM β-mercaptoethanol) and sonicated 2–3 times (Qsonica microtip: 4420; A=45; 2 min; 1 s on; 2 s off). Sample was dialyzed 3 times in buffer A (2 h, overnight, 2 h). The sample was sonicated and diluted in 500 ml of buffer A and was chromatographed sequentially on a 5 ml HiTrap™ Q HP column and on a 5 ml HiTrap™ SP FF column (GE Healthcare), both pre-equilibrated with buffer A. The capsid protein was eluted from HiTrap™ SP FF column using a linear gradient from 0 to 2 M of NaCl. Absorbance at 280 nm was checked to take the eluted fraction that had higher protein levels. Pooled fractions were dialyzed 3 times (2 h, overnight, 2 h) in storage buffer (25 mM MES, 2 M NaCl, 20 mM β-mercaptoethanol). Sample was concentrated using centricons to a concentration of 20 mg/ml and stored at − 80 °C.
We thank the NIH AIDS repository for important reagents such as the HIV-1 p24 monoclonal antibody (183-H12-5C). We would like to thank Thomas Fricke and Juan Manuel Carreno for technical assistance. The work was supported by an R01 grant from the NIH AI087390, to F.D.-G. References De Iaco, A., Santoni, F., Vannier, A., Guipponi, M., Antonarakis, S., Luban, J., 2013. TNPO3 protects HIV-1 replication from CPSF6-mediated capsid stabilization in the host cell cytoplasm. Retrovirology 10, 20. Di Nunzio, F., Danckaert, A., Fricke, T., Perez, P., Fernandez, J., Perret, E., Roux, P., Shorte, S., Charneau, P., Diaz-Griffero, F., Arhel, N.J., 2012. Human nucleoporins promote HIV-1 docking at the nuclear pore, nuclear import and integration. PLoS One 7, e46037. Di Nunzio, F., Fricke, T., Miccio, A., Valle-Casuso, J.C., Perez, P., Souque, P., Rizzi, E., Severgnini, M., Mavilio, F., Charneau, P., Diaz-Griffero, F., 2013. Nup153 and Nup98 bind the HIV-1 core and contribute to the early steps of HIV-1 replication. Virology 440, 8–18. Diaz-Griffero, F., Kar, A., Lee, M., Stremlau, M., Poeschla, E., Sodroski, J., 2007. Comparative requirements for the restriction of retrovirus infection by TRIM5alpha and TRIMCyp. Virology 369, 400–410. Diaz-Griffero, F., Vandegraaff, N., Li, Y., McGee-Estrada, K., Stremlau, M., Welikala, S., Si, Z., Engelman, A., Sodroski, J., 2006. Requirements for capsid-binding and an effector function in TRIMCyp-mediated restriction of HIV-1. Virology 351, 404–419. Fribourgh, J.L., Nguyen, H.C., Matreyek, K.A., Alvarez, F.J., Summers, B.J., Dewdney, T.G., Aiken, C., Zhang, P., Engelman, A., Xiong, Y., 2014. Structural insight into HIV1 restriction by MxB. Cell Host Microbe 16, 627–638. Fricke, T., Valle-Casuso, J.C., White, T.E., Brandariz-Nunez, A., Bosche, W.J., Reszka, N., Gorelick, R., Diaz-Griffero, F., 2013. The ability of TNPO3-depleted cells to inhibit HIV-1 infection requires CPSF6. Retrovirology 10, 46. Fricke, T., White, T.E., Schulte, B., de Souza Aranha Vieira, D.A., Dharan, A., Campbell, E.M., Brandariz-Nunez, A., Diaz-Griffero, F., 2014. MxB binds to the HIV-1 core and prevents the uncoating process of HIV-1. Retrovirology 11, 68. Ganser, B.K., Li, S., Klishko, V.Y., Finch, J.T., Sundquist, W.I., 1999. Assembly and analysis of conical models for the HIV-1 core. Science 283, 80–83. Lee, K., Ambrose, Z., Martin, T.D., Oztop, I., Mulky, A., Julias, J.G., Vandegraaff, N., Baumann, J.G., Wang, R., Yuen, W., Takemura, T., Shelton, K., Taniuchi, I., Li, Y., Sodroski, J., Littman, D.R., Coffin, J.M., Hughes, S.H., Unutmaz, D., Engelman, A., KewalRamani, V.N., 2010. Flexible use of nuclear import pathways by HIV-1. Cell Host Microbe 7, 221–233. Li, Y., Li, X., Stremlau, M., Lee, M., Sodroski, J., 2006. Removal of arginine 332 allows human TRIM5alpha to bind human immunodeficiency virus capsids and to restrict infection. J. Virol. 80, 6738–6744. Pornillos, O., Ganser-Pornillos, B.K., Banumathi, S., Hua, Y., Yeager, M., 2010. Disulfide bond stabilization of the hexameric capsomer of human immunodeficiency virus. J. Mol. Biol. 401, 985–995. Pornillos, O., Ganser-Pornillos, B.K., Kelly, B.N., Hua, Y., Whitby, F.G., Stout, C.D., Sundquist, W.I., Hill, C.P., Yeager, M., 2009. X-ray structures of the hexameric building block of the HIV capsid. Cell 137, 1282–1292. Stremlau, M., Perron, M., Lee, M., Li, Y., Song, B., Javanbakht, H., Diaz-Griffero, F., Anderson, D.J., Sundquist, W.I., Sodroski, J., 2006. Specific recognition and accelerated uncoating of retroviral capsids by the TRIM5alpha restriction factor. Proc. Natl. Acad. Sci. USA 103, 5514–5519.
4.2. Assembly of stabilized HIV-1 capsid tubes 1 ml of monomeric capsid (3 mg/ml or 1 mg/ml) was dialyzed in SnakeSkin dialysis tubing 10,000 MWCO (Thermo Scientific) against a buffer that is high in salt and contains a reducing agent (buffer 1: 50 mM Tris, pH 8, 1 M NaCl, 100 mM β-mercaptoethanol) at 4 °C for 8 h. Subsequently the protein was dialyzed against the same buffer without the reducing agent β-mercaptoethanol (buffer 2: 50 mM Tris, pH 8, 1 M NaCl) at 4 °C for 8 h. The absence of β-mercaptoethanol in the second dialysis allows formation of disulfide bonds between Cysteine 14 and 43 inter-capsid monomers in the hexamer. Finally the protein is dialyzed against buffer 3 (20 mM Tris, pH 8,0, 40 mM NaCl) at 4 °C for 8 h. Assembled complexes were kept at 4 °C up to 1 month. 4.3. Capsid binding assay protocol Human HEK293T cells were transfected for 24 h with a plasmid expressing the protein of interest. Cell media was completely removed and cells were lysed in 300 μL of capsid binding buffer (CBB: 10 mM Tris, pH 8,0, 1,5 mM MgCl2, 10 mM KCl) by scrapping off the plate. Cells were rotated at 4 °C for 15 min and then centrifuged to remove cellular debris (21,000 x g, 15 min, 4 °C). Cell lysates were incubated with stabilized HIV-1 capsid tubes for 1 h at 25 °C. Subsequently, stabilized HIV-1 capsid tubes were washed by pelleting the complexes by centrifugation at 21,000 × g for 2 min. Pellets were washed using by resuspension in CBB or PBS. Pellets were resuspended in Laemmli buffer 1X and analyzed by Western blotting using anti-p24 and the indicated antibodies.
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