The host-cell restriction factor SERINC5 restricts HIV-1 infectivity without altering the lipid composition and organization of viral particles

Abstract

The host-cell restriction factor SERINC5 potently suppresses the infectivity of HIV, type 1 (HIV-1) particles, and is counteracted by the viral pathogenesis factor Nef. However, the molecular mechanism by which SERINC5 restricts HIV-1 particle infectivity is still unclear. Because SERINC proteins have been suggested to facilitate the incorporation of serine during the biosynthesis of membrane lipids and because lipid composition of HIV particles is a major determinant of the infectious potential of the particles, we tested whether SERINC5-mediated restriction of HIV particle infectivity involves alterations of membrane lipid composition. We produced and purified HIV-1 particles from SERINC5293T cells with very low endogenous SERINC5 levels under conditions in which ectopically expressed SERINC5 restricts HIV-1 infectivity and is antagonized by Nef and analyzed both virions and producer cells with quantitative lipid MS. SERINC5 restriction and Nef antagonism were not associated with significant alterations in steady-state lipid composition of producer cells and HIV particles. Sphingosine metabolism kinetics were also unaltered by SERINC5 expression. Moreover, the levels of phosphatidylserine on the surface of HIV-1 particles, which may trigger uptake into non-productive internalization pathways in target cells, did not change upon expression of SERINC5 or Nef. Finally, saturating the phosphatidylserine-binding sites on HIV target cells did not affect SERINC5 restriction or Nef antagonism. These results demonstrate that the restriction of HIV-1 particle infectivity by SERINC5 does not depend on alterations in lipid composition and organization of HIV-1 particles and suggest that channeling serine into lipid biosynthesis may not be a cardinal cellular function of SERINC5.

Keywords: Nef; SERINC5; host cell restriction factor; host-pathogen interaction; human immunodeficiency virus (HIV); infectious disease; lipid; lipid composition; viral protein.

Figure 1.

SERINC5 restriction to HIV-1 infectivity and antagonism by Nef. A, quantification of the RT activity of OptiPrep-purified virus particles. HIV-1 particles were produced in 293T cells in the absence or presence of SERINC5.intHA, respectively, and purified via velocity gradient centrifugation on an OptiPrep gradient. RT activity (microunits RT) was determined via SYBR Green I—based, product-enhanced reverse transcriptase analysis. Depicted are means of three independent experiment ± S.D., represented relative to HIV-1 WT + control (percent), which was arbitrarily set to 100%. ns, no statistical significance. B, relative infectivity of the HIV-1 particles analyzed in A. To determine relative virion infectivity, TZM-bl reporter cells were infected, and infection efficiency was assessed by measuring relative light units and normalized to virus release. Depicted are means of three independent experiment ± S.D., represented relative to HIV-1 WT + control (percent), which was arbitrarily set to 100% (n = 3). **, p < 0.01; ***, p < 0.001. C, Western blot analysis of HIV-1–producing cells and OptiPrep-purified virus particles. Shown is immunodetection of SERINC5.intHA, Nef, and HIV-1 capsid (p24). D, silver staining of an SDS-PAGE of OptiPrep-purified virus particles. HIV-1 capsid (p24) is readily detected in all samples.

Figure 2.

Quantitative lipid analysis of HIV-1–producing cells and purified HIV-1 particles. A and B, lipid composition of OptiPrep-purified HIV-1_NL43 WT or ΔNef particles produced in the absence or presence of SERINC5 (A) and of the corresponding 293T HIV-1–producing cells (B). Lipid classes are standardized to all lipids measured. Cer, ceramide; Chol, cholesterol; -O, ether or odd-numbered fatty acyl residue. Data are presented as mean ± S.D. of three independent experiments, except for ΔNef particles produced in the presence of SERINC5 (n = 2). Note that low-abundant lipid classes are presented in separate graphs on the right. C, relative contribution of SP, GP, and sterols (ST, cholesterol) to the lipid composition of HIV-1–producing cells and OptiPrep-purified HIV-1_NL43 WT or ΔNef particles produced in the absence or presence of SERINC5.

Figure 3.

Lipid species compositions of HIV-1–producing cells and purified HIV-1 particles. Data are presented as mean ± S.D. of three independent experiments, except for ΔNef particles produced in the presence of SERINC5 (n = 2). A, molecular lipid species distributions of PC (left panel) and SM (right panel). PC species are shown as number of total carbon atoms:number of double bonds in fatty acyl residues. SM species are annotated with their number of carbon atoms, double bonds, and hydroxyl groups in the ceramide backbone, e.g. SM 34:2;1 contains in total in the sphingosine backbone and the fatty acyl moiety 34 carbon atoms, 2 double bonds, and 1 hydroxyl group. Species are standardized to 100% within each lipid class. B and C, distributions of chain lengths (B) and double bonds (C) within the categories GP (left panels) and SP (right panels). Species are standardized to 100% within each lipid category.

Figure 4.

Analysis of sphingosine metabolism in the absence or presence of SERINC5. A, metabolism of sphingosine (Sph). pacSph fed to cells can either enter the biosynthetic (orange) or degradative (purple) pathway. In SP biosynthesis, ceramide serves as a branching point for the synthesis of phosphosphingolipids such as SM and glycosphingolipids such as glucosylceramide (GlcCer). Breakdown of sphingosine yield shuttles the hydrocarbon backbone via palmitoyl-CoA into biosynthesis of GL and GP. B, pacSph contains a photoactivatable diazirine group and a terminal alkyne moiety. pacSph is metabolized like its endogenous counterpart, entering both the biosynthetic and degradation pathways, giving rise to functionalized cellular lipids. Following lysis at different times of labeling, cells are subjected to a click reaction to couple a fluorescent reporter to alkyne-containing lipids. Lipid extracts were analyzed by TLC and fluorescence imaging to monitor Sph metabolism. C, cells were labeled with pacSph for 15 min, 60 min, or 18 h, lysed, and subjected to a click reaction using an azide-activated fluorophore. Following TLC separation, lipids were visualized by fluorescence imaging. Lipid classes were distinguished based on co-migrating standards, except for neutral lipids. Fluorescence signals were quantified, and relative contributions of lipid classes were calculated. Cer, ceramide; DAG, diacylglycerol; GlcCer, glucosylceramide; TAG/CE, triacylglycerol/cholesteryl ester.

Figure 5.

PS surface levels on liposomes and HIV-1 particles and liposome competition for PS binding sites in the context of HIV-1 infection. A, percentages of PS-positive liposomes detected with PS-specific antibody. PC- and PS/PC-containing liposomes (100 μl of 1 mm liposome stock solution) were incubated with a PS-specific antibody (1:100, 1 h at 4 °C) followed by an anti-mouse APC-coupled secondary antibody (1:200, 1 h, 4 °C) and analyzed by flow cytometry. Unstained liposomes served as a control. B, percentages of PS-positive liposomes detected with Mfge8.eGFP. PC- and PS-containing liposomes (100 μl of 1 mm liposome stock solution) were incubated with recombinant Mfge8.eGFP (1:100) and analyzed via flow cytometry. Unstained liposomes served as a control. C, percentages of PS-positive HIV-1 particles stained with recombinant Mfge8.eGFP. HIV-1_NL43 WT or ΔNef particles (50 μl with approximately 5 × 10¹⁰ picounits RT/μl) produced in the absence (control) or presence of SERINC5 (SERINC5) were incubated with Mfge8.eGFP (1:100) and analyzed by flow cytometry. Unstained virus particles served as a control. D, quantification of PS-positive HIV-1 particles as analyzed in C. Percentages of PS-positive particles as determined by the Mfge8.eGFP signal are shown relative to HIV-1 WT particles + control (set to 100%) (n = 3). E, quantification of PS-positive HIV-1 particles analyzed with an anti-PS antibody. Percentages of PS-positive particles (average values from four independent experiments) are shown relative to HIV-1 WT particles + control (set to 100%). F, Western blot analysis of Huh7.5 cells infected with HCV (multiplicity of infection = 10) at 37 °C for 8 h in the absence (w/o) or presence of different concentrations (10 and 30 μm) of PC-, PE-, or PS-containing liposomes. Cells were lysed 48 h after infection and subjected to SDS-PAGE, and HCV NS5A was detected via immunostaining. TfR served as a loading control. Shown is one representative of two independent experiments. G, HIV-1 infection of TZM-bl cells in the absence or presence of different concentrations (10 and 30 μm) of PC-, PE-, or PS-containing liposomes. HIV-1 WT or ΔNef (2 × 10⁸ picounits RT/μl) produced in the absence (control) or presence of SERINC5 (SERINC5) was used for the infection. 48 h after infection, cells were lysed, and infection rates were determined via luciferase assay. Infection rates are presented relative to HIV-1 WT + control, which was arbitrarily set to 100%. Shown are means of two independent experiments, each performed in triplicate.