Volume 21, Issue 2, Pages 220-230 (February 2017) Heme Oxygenase 2 Binds Myristate to Regulate Retrovirus Assembly and TLR4 Signaling  Yiping Zhu, Shukun Luo, Yosef Sabo, Cheng Wang, Liang Tong, Stephen P. Goff  Cell Host & Microbe  Volume 21, Issue 2, Pages 220-230 (February 2017) DOI: 10.1016/j.chom.2017.01.002 Copyright © 2017 Elsevier Inc. Terms and Conditions

Cell Host & Microbe 2017 21, 220-230DOI: (10.1016/j.chom.2017.01.002) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 1 HO-2 Is a Myristate-Binding Protein (A) Proteins from cells expressing Myc-HO-2 and the indicated forms of MA-flag were immunoprecipitated using antiflag antibody, and Myc-HO-2 and MA-flag were detected by western blot. (B) Endogenous HO-2 interacts with wild-type HIV-1 MA (WT), but not G2A mutant. Proteins of cells expressing indicated MA-flags were immunoprecipitated with antiflag antibody. Endogenous HO-2 was detected by HO-2-specific antibody. (C) HO-2 interacts with various myristoylated proteins. Proteins of cells expressing empty vector (EV), flag-tagged versions of indicated variants of HIV-1 MA or MLV MA, or v-Src and Myc-HO-2 were immunoprecipitated using antiflag antibody. Myc-HO-2 and myristoylated proteins were detected by western blot. (D) Myristic acid competes with HIV-1 MA for binding to HO-2. Proteins of cells expressing Myc-tagged HO-2 and MA-flag as indicated were incubated with indicated concentrations of myristic acid and immunoprecipitated using antiflag antibody. Myc-HO-2, MA-flag, and control RRS proteins were detected by western blot. Also see Figure S1. Cell Host & Microbe 2017 21, 220-230DOI: (10.1016/j.chom.2017.01.002) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 2 Crystal Structures of HO-2 in Complex with Myristate and Laurate (A) Schematic drawing of the structure of HO-2 in complex with myristate. HO-2 is shown as ribbons (light cyan) and myristate as spheres (black for carbon atoms). (B) Molecular surface of myristate binding site of HO-2, colored by electrostatic potential. (C) Structural image showing detailed interactions between myristate (black) and HO-2 (light cyan). (D) Omit Fo–Fc electron density at 1.9 Å resolution for myristate in (A), contoured at 2.5 σ. The density for the carboxylate group becomes visible at 2 σ. (E) Structural image showing detailed interactions between laurate and HO-2. (F) Omit Fo–Fc electron density at 2.1 Å resolution for laurate in (E), contoured at 2.5σ. (G) Residues in HO-2 essential for myristate-binding activity. Proteins from cells expressing MA-flag and WT or indicated mutant Myc-HO-2 were immunoprecipitated with antiflag antibody. Myc-HO-2 and MA-flag were detected by immunoblot. Also see Figure S2. Cell Host & Microbe 2017 21, 220-230DOI: (10.1016/j.chom.2017.01.002) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 3 HO-2 Inhibits Retrovirus Production (A) HO-2 does not affect infection by HIV-1. The 293A cells were transfected twice with control nontargeting siRNA (NT) or an siRNA pool targeting HO-2 (siRNA HO-2) and then infected with VSV-G-pseudotyped NL4-3luc virus at 1:10 or 1:100 dilution. Luciferase activities were measured 48 hr after infection. The luciferase activity from cells transfected with control siRNA (NT) and infected with 1:10 diluted virus was set as 100. The data are expressed as means ± SD from three independent experiments. (B) Knockdown of HO-2 enhances the production of infectious HIV-1 virus. The 293A cells were transfected twice with the control nontargeting siRNA (NT) or an siRNA pool targeting HO-2 (siRNA HO-2) and then transfected with pNL4.3luc and pVSVG to package virus. Then, 48 hr after plasmid transfection, equal amounts of the supernatant from transfected cells were used to infect 293A cells. Luciferase activities were measured 48 hr after infection. The luciferase activity from cells infected with virus packaged from control siRNA (NT)-transfected cells was set as 1. The data are means ± SD from three independent experiments. (C) Knockdown of HO-2, but not HO-1, increases release of HIV-1 virus-like particles (VLPs). Cells were transfected with control nontargeting siRNA (NT) or siRNA pools targeting HO-2 or HO-1 and then with viral DNA genomes. Gag protein in the cells (cell lysate) and CA in released virion particles were detected by HIV-1-specific antibody. (D) Myristate-binding activity of HO-2 is required to inhibit VLP release. Cells expressing WT HO-2, or myristate nonbinding mutant F53A HO-2, were treated to knock down endogenous HO-2 and transfected to produce virus, and VLPs were collected 48 hr later. Gag expression in the cells (cell lysate) and CA in the VLPs were detected by HIV-1-specific antibody. (E) Knockdown of HO-2 increases the release of HIV-1 Gag from Jurkat T cells. JTag-SCR, JTag-HO-2i253, and JTag-HO-2i257 cells were transfected with pNL4.3luc. Then, 48 hr after transfection, VLPs were pelleted from supernatant of cells. Gag expression in the cells (cell lysate) and CA in the VLPs were detected by HIV-1 p24 antibody. (F) HO-2 knockdown increases MLV release. Cells were transfected with control nontargeting siRNA (NT) or siRNA pools targeting HO-2, and then with MLV DNA. Gag protein in the cells (cell lysate) and in released VLPs were detected by specific antibody against MLV Gag. (G) Knockdown of HO-2 has no effect on the release of RSV Gag from 293A cells. The 293A cells were transfected twice with the control nontargeting siRNA (NT) or the siRNA pool targeting HO-2 (siRNA HO-2) and then transfected with pCMV-RSVGag. Then, 48 hr after transfection, VLPs were pelleted from supernatant of cells. Gag expression in the cells (cell lysate) and CA in the VLPs were detected by specific antibodies against RSV Gag. Also see Figure S3. Cell Host & Microbe 2017 21, 220-230DOI: (10.1016/j.chom.2017.01.002) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 4 SnPP IX Inhibits HO-2’s Myristate-Binding Activity (A) Molecular surface of HO-2 catalytic active site and myristate binding site colored by electrostatic potential (heme in salmon and myristate in black). (B) Heme analog SnPP IX inhibits HO-2’s binding to HIV-1 MA. Proteins from cells expressing MA-flag and treated with indicated concentrations of SnPP IX were immunoprecipitated using antiflag antibody. Endogenous HO-2, RRS, and MA-flag were detected by immunoblot. (C) SnPP IX treatment increases VLP production. Virus-producing 293A cells were treated with SnPP IX at indicated concentrations for 48 hr, and VLPs were pelleted from culture supernatant. Gag in cell lysate and in the VLP was detected by HIV-1-specific antibody. Also see Figure S4. Cell Host & Microbe 2017 21, 220-230DOI: (10.1016/j.chom.2017.01.002) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 5 HO-2 Inhibits the Membrane Association of HIV-1 Gag (A) Knockdown of HO-2 enhances membrane association of HIV-1 Gag. The 293A cells were transfected twice with control nontargeting siRNA (NT) or the siRNA pool targeting HO-2 (siRNA HO-2) and then transfected with pNL4.3luc. Then, 48 hr after transfection, membrane floatation assay was used to determine the distribution of Gag in the cytosol and membrane fraction. ATPA1 is a membrane fraction marker, while GAPDH is a cytosol fraction marker. (B) HO-2’s myristate-binding activity inhibits HIV-1 Gag membrane association. Cells expressing WT HO-2, or myristate nonbinding mutant F53A HO-2, were treated to knock down endogenous HO-2 and transfected with pNL4.3luc. Lysates of cells were fractionated, and the subcellular distribution of Gag was determined by immunoblot using HIV-1-specific antibody. ATPA1 is a membrane fraction marker. Also see Figure S5. Cell Host & Microbe 2017 21, 220-230DOI: (10.1016/j.chom.2017.01.002) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 6 HO-2 Inhibits the TRAM-Dependent LPS-TLR4 Pathway via Its Myristate-Binding Activity (A) Interaction between HO-2 and TRAM requires TRAM N-myristoylation and the HO-2 myristate binding site. Cells expressing flag-tagged WT or mutant G2A TRAM were transfected with Myc-tagged versions of WT, myristate nonbinding mutant F53A, or catalytically inactive mutant H45A of HO-2. Proteins in lysates were immunoprecipitated using antiflag antibody and probed by immunoblot. (B) Knockout of HO-2 enhances TRAM signaling. Control or HO-2 KO cells were transfected with a RANTES-luciferase reporter and indicated amounts of TRAM expression DNA. Upper shows luciferase activities 24 hr after transfection relative to control cells without TRAM. Lower shows immunoblot of HO-2. (C) HO-2’s myristate-binding activity, but not its heme oxygenase activity, is required for inhibition of TRAM signaling. HO-2 KO cells were engineered to stably express WT, myristate-nonbinding mutant (F53A), or catalytically inactive mutant (H45A) of HO-2 and then transfected with a luciferase reporter of RANTES gene expression and indicated amounts of TRAM expression DNA. Upper shows luciferase activities 24 hr after transfection relative to control cells without TRAM. Lower shows immunoblot of HO-2. (D) Knockdown of HO-2 enhances the expression of RANTES induced by LPS. THP-1-MD2-CD14 cells stably expressing the indicated shRNAs were stimulated with the indicated concentration of LPS for 24 hr. Levels of RANTES in the supernatant were measured by ELISA. (E) LPS induces the expression of HO-2. THP-1-MD2-CD14 cells were treated with 10 ng/mL LPS for indicated time. Levels of HO-2 mRNA were measured by qRT-PCR normalized to that of GAPDH and presented relative to LPS untreated cells. Data in (A)–(D) are representative of at least three independent experiments (mean and SD). Data in (E) are mean and SD of triplicate determinations. Also see Figure S6. Cell Host & Microbe 2017 21, 220-230DOI: (10.1016/j.chom.2017.01.002) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 7 Working Model for Functions of HO-2 (A) The binding of HO-2 to the N-terminal myristate of HIV-1 Gag traps the myristate moiety and prevents it from inserting into the membrane in its proper conformation, thus inhibiting HIV-1 virion production. (B) HO-2 binds to the N-terminal myristate moiety and downregulates the function of TRAM. HO-2 is induced by LPS-TLR4 signaling and acts as a negative feedback regulator of the LPS-TLR4 pathway. Cell Host & Microbe 2017 21, 220-230DOI: (10.1016/j.chom.2017.01.002) Copyright © 2017 Elsevier Inc. Terms and Conditions