Ceramide synthase 2 deletion decreases the infectivity of HIV-1
2021; Elsevier BV; Volume: 296; Linguagem: Inglês
10.1016/j.jbc.2021.100340
ISSN1083-351X
AutoresEric Barklis, Ayna Alfadhli, Jennifer Kyle, Lisa Bramer, Kent Bloodsworth, Robin Lid Barklis, Hans C. Leier, R. Max Petty, Iris D. Zelnik, Thomas Metz, Anthony H. Futerman, Fikadu Tafesse,
Tópico(s)Immune Cell Function and Interaction
ResumoThe lipid composition of HIV-1 virions is enriched in sphingomyelin (SM), but the roles that SM or other sphingolipids (SLs) might play in the HIV-1 replication pathway have not been elucidated. In human cells, SL levels are regulated by ceramide synthase (CerS) enzymes that produce ceramides, which can be converted to SMs, hexosylceramides, and other SLs. In many cell types, CerS2, which catalyzes the synthesis of very long chain ceramides, is the major CerS. We have examined how CerS2 deficiency affects the assembly and infectivity of HIV-1. As expected, we observed that very long chain ceramide, hexosylceramide, and SM were reduced in CerS2 knockout cells. CerS2 deficiency did not affect HIV-1 assembly or the incorporation of the HIV-1 envelope (Env) protein into virus particles, but it reduced the infectivites of viruses produced in the CerS2-deficient cells. The reduced viral infection levels were dependent on HIV-1 Env, since HIV-1 particles that were pseudotyped with the vesicular stomatitis virus glycoprotein did not exhibit reductions in infectivity. Moreover, cell–cell fusion assays demonstrated that the functional defect of HIV-1 Env in CerS2-deficient cells was independent of other viral proteins. Overall, our results indicate that the altered lipid composition of CerS2-deficient cells specifically inhibit the HIV-1 Env receptor binding and/or fusion processes. The lipid composition of HIV-1 virions is enriched in sphingomyelin (SM), but the roles that SM or other sphingolipids (SLs) might play in the HIV-1 replication pathway have not been elucidated. In human cells, SL levels are regulated by ceramide synthase (CerS) enzymes that produce ceramides, which can be converted to SMs, hexosylceramides, and other SLs. In many cell types, CerS2, which catalyzes the synthesis of very long chain ceramides, is the major CerS. We have examined how CerS2 deficiency affects the assembly and infectivity of HIV-1. As expected, we observed that very long chain ceramide, hexosylceramide, and SM were reduced in CerS2 knockout cells. CerS2 deficiency did not affect HIV-1 assembly or the incorporation of the HIV-1 envelope (Env) protein into virus particles, but it reduced the infectivites of viruses produced in the CerS2-deficient cells. The reduced viral infection levels were dependent on HIV-1 Env, since HIV-1 particles that were pseudotyped with the vesicular stomatitis virus glycoprotein did not exhibit reductions in infectivity. Moreover, cell–cell fusion assays demonstrated that the functional defect of HIV-1 Env in CerS2-deficient cells was independent of other viral proteins. Overall, our results indicate that the altered lipid composition of CerS2-deficient cells specifically inhibit the HIV-1 Env receptor binding and/or fusion processes. As an enveloped virus, the HIV-1 depends on host cell lipids to assemble replicating viruses (1Swanstrom R. Wills J.W. Synthesis, assembly and processing of viral proteins.in: Coffin J.M. Hughes S.H. Varmus H.E. Retroviruses. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1997: 263-334Google Scholar, 2Lorizate M. Kräusslich H.G. Role of lipids in virus replication.Cold Spring Harb. Perspect. Biol. 2011; 3a004820Crossref PubMed Scopus (151) Google Scholar). Among the lipids enriched in the envelopes of HIV-1 particles are phosphatidyl-4,5-bisphosphate (PIP2), phosphatidylinositol-3,4,5-trisphosphate, phosphatidylserine (PS), sphingomyelin (SM), and cholesterol (3Aloia R.C. Tian H. Jensen F.C. Lipid composition and fluidity of the human immunodeficiency virus envelope and host cell plasma membranes.Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 5181-5185Crossref PubMed Scopus (368) Google Scholar, 4Brügger B. Glass B. Haberkant P. Leibrecht I. Wieland F.T. Kräusslich H.G. 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Lüchtenborg C. Glass B. Traynor-Kaplan A. Schultz C. Brügger B. Kräusslich H.G. Quantification of phosphoinositides reveals strong enrichment of PIP2 in HIV-1 compared to producer cell membranes.Sci. Rep. 2019; 9: 17661Crossref PubMed Scopus (21) Google Scholar). Ample evidence has shown that binding of the matrix domains of HIV-1 precursor Gag (PrGag) proteins to PIP2 molecules directs virus assembly at the plasma membranes (PMs) of infected cells (9Ono A. Ablan S.D. Lockett S.J. Nagashima K. Freed E.O. Phosphatidylinositol (4,5) bisphosphate regulates HIV-1 Gag targeting to the plasma membrane.Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 14889-14894Crossref PubMed Scopus (385) Google Scholar, 10Saad J.S. Miller J. Tai J. Kim A. Ghanam R.H. Summers M.F. Structural basis for targeting HIV-1 Gag proteins to the plasma membrane for virus assembly.Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 11364-11369Crossref PubMed Scopus (423) Google Scholar). Investigations also have demonstrated that depletion of HIV-1 cholesterol levels reduces viral infectivity, and this effect can be mitigated in part by mutations in the cytoplasmic tail (CT) of the HIV-1 envelope (Env) protein (11Campbell S.M. Crowe S.M. Mak J. Virion-associated cholesterol is critical for the maintenance of HIV-1 structure and infectivity.AIDS. 2002; 16: 2253-2261Crossref PubMed Scopus (108) Google Scholar, 12Graham D.R. Chertova E. Hilburn J.M. Arthur L.O. Hildreth J.E. Cholesterol depletion of human immunodeficiency virus type 1 and simian immunodeficiency virus with beta-cyclodextrin inactivates and permeabilizes the virions: Evidence for virion-associated lipid rafts.J. Virol. 2003; 77: 8237-8248Crossref PubMed Scopus (162) Google Scholar, 13Waheed A.A. Ablan S.D. Mankowski M.K. Cummins J.E. Ptak R.G. Schaffner C.P. Freed E.O. Inhibition of HIV-1 replication by amphotericin B methyl ester: Selection for resistant variants.J. Biol. 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Chem. 2020; 295: 348-362Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar, 17Checkley M.A. Luttge B.G. Freed E.O. HIV-1 envelope glycoprotein biosynthesis, trafficking, and incorporation.J. Mol. Biol. 2011; 410: 582-608Crossref PubMed Scopus (282) Google Scholar). However, potential roles of other enriched viral lipids, including SM, have not been elucidated. As illustrated in Figure 1, cellular levels of SM and other sphingolipids (SLs) are closely associated with the metabolism of ceramide (Cer; (18Delgado A. Casas J. Llebaria A. Abad J.L. Fabrias G. Inhibitors of sphingolipid metabolism enzymes.Biochim. Biophys. Acta. 2006; 1758: 1957-1977Crossref PubMed Scopus (138) Google Scholar, 19Riley R.T. Merrill Jr., A.H. Ceramide synthase inhibition by fumonisins: A perfect storm of perturbed sphingolipid metabolism, signaling, and disease.J. Lipid Res. 2019; 60: 1183-1189Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar, 20Zitomer N.C. Mitchell T. Voss K.A. Bondy G.S. Pruett S.T. Garnier-Amblard E.C. Liebeskind L.S. Park H. Wang E. Sullards M.C. Merrill Jr., A.H. Riley R.T. Ceramide synthase inhibition by fumonisin B1 causes accumulation of 1-deoxysphinganine: A novel category of bioactive 1-deoxysphingoid bases and 1-deoxydihydroceramides biosynthesized by mammalian cell lines and animals.J. Biol. Chem. 2009; 284: 4786-4795Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar, 21Levy M. Futerman A.H. Mammalian ceramide synthases.IUBMB Life. 2010; 62: 347-356Crossref PubMed Scopus (46) Google Scholar)). Notably, major pathways for the production of Cer from sphingosine and sphinganine (dihydrosphingosine) are governed by the activities of different ceramide synthase (CerS) enzymes (18Delgado A. Casas J. Llebaria A. Abad J.L. Fabrias G. Inhibitors of sphingolipid metabolism enzymes.Biochim. Biophys. Acta. 2006; 1758: 1957-1977Crossref PubMed Scopus (138) Google Scholar, 19Riley R.T. Merrill Jr., A.H. Ceramide synthase inhibition by fumonisins: A perfect storm of perturbed sphingolipid metabolism, signaling, and disease.J. Lipid Res. 2019; 60: 1183-1189Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar, 20Zitomer N.C. Mitchell T. Voss K.A. Bondy G.S. Pruett S.T. Garnier-Amblard E.C. Liebeskind L.S. Park H. Wang E. Sullards M.C. Merrill Jr., A.H. Riley R.T. Ceramide synthase inhibition by fumonisin B1 causes accumulation of 1-deoxysphinganine: A novel category of bioactive 1-deoxysphingoid bases and 1-deoxydihydroceramides biosynthesized by mammalian cell lines and animals.J. Biol. Chem. 2009; 284: 4786-4795Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar, 21Levy M. Futerman A.H. Mammalian ceramide synthases.IUBMB Life. 2010; 62: 347-356Crossref PubMed Scopus (46) Google Scholar). Mammals encode six CerS variants (CerS1–CerS6) that are differentially expressed in different tissues and have different chain length preferences (21Levy M. Futerman A.H. Mammalian ceramide synthases.IUBMB Life. 2010; 62: 347-356Crossref PubMed Scopus (46) Google Scholar, 22Wattenberg B.W. The long and the short of ceramides.J. Biol. Chem. 2018; 293: 9922-9923Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar, 23Tidhar R. Zelnik I.D. Volpert G. Ben-Dor S. Kelly S. Merrill Jr., A.H. Futerman A.H. Eleven residues determine the acyl chain specificity of ceramide synthases.J. Biol. Chem. 2018; 293: 9912-9921Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar, 24Feng S. Harayama T. Chang D. Hannich J.T. Winssinger N. Riezman H. Lysosome-targeted photoactivation reveals local sphingosine metabolism signatures.Chem. Sci. 2018; 10: 2253-2258Crossref PubMed Scopus (25) Google Scholar). In particular, CerS5 and CerS6 have short acyl chain specificities (C14 and C16), CerS1 and CerS4 have long chain specificities (C18 and C20), whereas CerS2 and CerS3 have very long chain specificities (C20–C30) (21Levy M. Futerman A.H. Mammalian ceramide synthases.IUBMB Life. 2010; 62: 347-356Crossref PubMed Scopus (46) Google Scholar). We have examined the effects of knocking out CerS2 on the assembly and infectivity of HIV-1. Lipid analyses showed that relative to WT cells, CerS2−/− cells had moderately reduced levels of long chain Cer species and greatly reduced levels of long chain SMs and hexosylceramide (HexCer) lipids. Importantly, while CerS2−/− cells supported the efficient assembly and release of viruses containing normal amounts of HIV-1 Env proteins, viruses so obtained were a third as infectious as those produced in WT cells. Similar results were obtained with HIV-1 virions carrying an Env protein cytoplasmic domain deletion (ΔCT; (17Checkley M.A. Luttge B.G. Freed E.O. HIV-1 envelope glycoprotein biosynthesis, trafficking, and incorporation.J. Mol. Biol. 2011; 410: 582-608Crossref PubMed Scopus (282) Google Scholar, 25Freed E.O. Martin M.A. Virion incorporation of envelope glycoproteins with long but not short cytoplasmic tails is blocked by specific, single amino acid substitutions in the human immunodeficiency virus type 1 matrix.J. Virol. 1995; 69: 1984-1989Crossref PubMed Google Scholar, 26Staubus A.O. Alfadhli A. Barklis R.L. Barklis E. Replication of HIV-1 envelope protein cytoplasmic domain variants in permissive and restrictive cells.Virology. 2019; 538: 1-10Crossref PubMed Scopus (3) Google Scholar)), but infectivities of HIV-1 virions pseudotyped (27Johnson M.C. Mechanisms for Env glycoprotein acquisition by retroviruses.AIDS Res. Hum. Retroviruses. 2011; 27: 239-247Crossref PubMed Scopus (35) Google Scholar, 28Joglekar A.V. Sandoval S. Pseudotyped lentiviral vectors: One vector, many Guises.Hum. Gene Ther. Methods. 2017; 28: 291-301Crossref PubMed Scopus (41) Google Scholar) with the vesicular stomatitis virus (VSV) glycoprotein (G) were not so affected. Cell–cell fusion assay results mimicked virus infection results, indicating that the Env protein defect in CerS2−/− cells was independent of other virally encoded constituents. Overall, our results demonstrate that the HIV-1 Env binding and/or fusion functions are dependent on membrane SL compositions. To monitor the effects of CerS2 mutations on cellular lipid compositions, we performed lipidomic analyses on WT human embryonic kidney 293T (HEK293T) cells (29DuBridge R.B. Tang P. Hsia H.C. Leong P.M. Miller J.H. Calos M.P. Analysis of mutation in human cells by using an Epstein-Barr virus shuttle system.Mol. Cell. Biol. 1987; 7: 379-387Crossref PubMed Scopus (896) Google Scholar) and on HEK293T cells in which both CerS2 alleles were edited by CRISPR/Cas9 technology to possess premature stop codons after 63 residues (23Tidhar R. Zelnik I.D. Volpert G. Ben-Dor S. Kelly S. Merrill Jr., A.H. Futerman A.H. Eleven residues determine the acyl chain specificity of ceramide synthases.J. Biol. Chem. 2018; 293: 9912-9921Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). CerS2 is the predominant CerS expressed in the kidney (21Levy M. Futerman A.H. Mammalian ceramide synthases.IUBMB Life. 2010; 62: 347-356Crossref PubMed Scopus (46) Google Scholar, 30Laviad E.L. Albee L. Pankova-Kholmyansky I. Park H. Merrill Jr., A.H. Futerman A.H. Characterization of ceramide synthase 2: Tissue distribution, substrate specificity and inhibition by sphingosine 1-phosphate.J. Biol. Chem. 2008; 283: 5677-5684Abstract Full Text Full Text PDF PubMed Scopus (335) Google Scholar), and proteomic analyses of HEK293 cells have revealed that CerS2 is expressed at 10-fold higher levels than CerS5, whereas CerS1, CerS3, CerS4, and CerS6 were either not detected or detected at trace levels (31Geiger T. Wehner A. Schaab C. Cox J. Mann M. Comparative proteomic analysis of eleven common cell lines reveals ubiquitous but varying expression of most proteins.Mol. Cell. Proteomics. 2012; 11 (M111.014050)Abstract Full Text Full Text PDF Scopus (547) Google Scholar). Although some ceramides are present in the fetal bovine serum (32Brovkovych V. Aldrich A. Li N. Atilla-Gokcumen G.E. Frasor J. Removal of serum lipids and lipid-derived metabolites to investigate breast cancer cell biology.Proteomics. 2019; 19e1800370Crossref PubMed Scopus (14) Google Scholar), and Cer can be generated by hydrolysis of SM or glucosylceramide (Fig. 1), the CerS-mediated pathways are major routes of SL production (18Delgado A. Casas J. Llebaria A. Abad J.L. Fabrias G. Inhibitors of sphingolipid metabolism enzymes.Biochim. Biophys. Acta. 2006; 1758: 1957-1977Crossref PubMed Scopus (138) Google Scholar, 19Riley R.T. Merrill Jr., A.H. Ceramide synthase inhibition by fumonisins: A perfect storm of perturbed sphingolipid metabolism, signaling, and disease.J. Lipid Res. 2019; 60: 1183-1189Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar, 20Zitomer N.C. Mitchell T. Voss K.A. Bondy G.S. Pruett S.T. Garnier-Amblard E.C. Liebeskind L.S. Park H. Wang E. Sullards M.C. Merrill Jr., A.H. Riley R.T. Ceramide synthase inhibition by fumonisin B1 causes accumulation of 1-deoxysphinganine: A novel category of bioactive 1-deoxysphingoid bases and 1-deoxydihydroceramides biosynthesized by mammalian cell lines and animals.J. Biol. Chem. 2009; 284: 4786-4795Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar, 21Levy M. Futerman A.H. Mammalian ceramide synthases.IUBMB Life. 2010; 62: 347-356Crossref PubMed Scopus (46) Google Scholar). Because of this, we anticipated that CerS2−/− HEK293T cells might show reduced levels of Cer-derived SLs and, in particular, very long chain SLs. For our analyses, lipids extracted from WT and CerS2−/− HEK293T cells were analyzed by LC/MS. In all, we identified 366 lipid species for which comparisons could be made (Table S1). These included phosphatidylcholines, phosphatidylethanolamines, phosphatidylinositols, PSs, phosphatidylglycerols, diacylglycerols, triacylglycerols, cholesterol ester, cardiolipins, Cers, SMs, and HexCers. Our results are visualized as volcano plots (Fig. 2), in which X-axes indicate log2 fold change of mutant/WT, and Y-axes indicate −log10 adjusted significance (p) values. Of the lipid classes, levels of phosphatidylcholines, phosphatidylethanolamines, phosphatidylinositols, PSs, and cardiolipins were only marginally affected in the CerS2−/− cells (Fig. 2). In contrast, diacylglycerols and triacylglycerols were slightly increased, and cholesterol ester and phosphatidylglycerols were slightly decreased. Interestingly, levels of shorter chain Cer species (d18:1/16:0 and d18:1/18:0) were increased in CerS2−/− cells, whereas the longest chain species (d18:1/23:0, d18:1/24:0, and d18:1/24:1) were decreased: these results are consistent with the role of CerS2 in very long chain Cer synthesis (21Levy M. Futerman A.H. Mammalian ceramide synthases.IUBMB Life. 2010; 62: 347-356Crossref PubMed Scopus (46) Google Scholar, 22Wattenberg B.W. The long and the short of ceramides.J. Biol. Chem. 2018; 293: 9922-9923Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar, 23Tidhar R. Zelnik I.D. Volpert G. Ben-Dor S. Kelly S. Merrill Jr., A.H. Futerman A.H. Eleven residues determine the acyl chain specificity of ceramide synthases.J. Biol. Chem. 2018; 293: 9912-9921Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar) and suggest a larger role for CerS5 in Cer production in mutant versus WT HEK293T cells. However, the most significant SL reductions in CerS2−/− cells were very long chain HexCer (d18:1/22:0, d18:1/23:0, d18:1/24:0, and d18:1/24:1) and SM (d18:1/22:1, d18:1/24:0, d18:1/24:1 [or d18:2/24:0], d18:1/25:0, and d18:1/26:0) species. Given that SMs are enriched in HIV-1 virions relative to host cell membranes (4Brügger B. Glass B. Haberkant P. Leibrecht I. Wieland F.T. Kräusslich H.G. The HIV lipidome: A raft with an unusual composition.Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 2641-2646Crossref PubMed Scopus (536) Google Scholar, 6Lorizate M. Brügger B. Akiyama H. Glass B. Müller B. Anderluh G. Wieland F.T. Kräusslich H.G. Probing HIV-1 membrane liquid order by Laurdan staining reveals producer cell-dependent differences.J. Biol. Chem. 2009; 284: 22238-22247Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar, 7Lorizate M. Sachsenheimer T. Glass B. Habermann A. Gerl M.J. Kräusslich H.G. Brügger B. Comparative lipidomics analysis of HIV-1 particles and their producer cell membrane in different cell lines.Cell Microbiol. 2013; 15: 292-304Crossref PubMed Scopus (117) Google Scholar, 8Mücksch F. Citir M. Lüchtenborg C. Glass B. Traynor-Kaplan A. Schultz C. Brügger B. Kräusslich H.G. Quantification of phosphoinositides reveals strong enrichment of PIP2 in HIV-1 compared to producer cell membranes.Sci. Rep. 2019; 9: 17661Crossref PubMed Scopus (21) Google Scholar), we investigated how the lipid changes observed in CerS2−/− cells affected HIV-1 replication. To examine how a deficiency in CerS2 might affect HIV-1 assembly, release, and replication, we transfected WT and CerS2−/− HEK293T cells with the full-length NL4-3 (33Adachi A. Gendelman H.E. Koenig S. Folks T. Willey R. Rabson A. Martin M.A. Production of acquired immunodeficiency syndrome-associated retrovirus in human and nonhuman cells transfected with an infectious molecular clone.J. Virol. 1986; 59: 284-291Crossref PubMed Google Scholar) WT HIV-1 proviral plasmid and monitored particle production, Env levels in virus particles, and infection efficiencies. As shown in Figure 3A, cell lysate samples immunoblotted with an antibody against the Gag capsid (CA) protein showed similar staining patterns of PrGag, CA, and processing intermediates. Virus samples immunoblotted with the anti-CA antibody yielded the expected enrichment of CA over PrGag, with roughly equivalent levels of each species detected in samples produced from WT versus CerS2−/− cells (Fig. 3B). Virus samples immunoblotted with an antibody to the transmembrane (gp41) portion of Env also appeared similar (Fig. 3C), with appreciably higher levels of gp41 than the full-length unprocessed Env protein (gp160). Quantitation of immunoblots from 10 independent pairwise transfections allowed us to compare WT and CerS2−/− HEK293T virus release levels (defined as viral versus cellular Gag levels), Gag-normalized Env levels in virions (Env/Gag), and ratios of gp41 to gp160 (41/160) in virions (Fig. 4A). As shown, no significant differences in these parameters were observed, indicating that CerS2 deficiency had little effect on HIV-1 assembly efficiencies, Env incorporation levels, or Env processing. To assay virus infectivities, viruses produced from WT and mutant cells were used to infect TZM-bl cells, which respond to HIV-1 infection, integration, and expression via the transactivation of their Tat-inducible β-galactosidase (β-gal) genes (34Platt E.J. Wehrly K. Kuhmann S.E. Chesebro B. Kabat D. Effects of CCR5 and CD4 cell surface concentrations on infections by macrophagetropic isolates of human immunodeficiency virus type 1.J. Virol. 1998; 72: 2855-2864Crossref PubMed Google Scholar, 35Wei X. 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Virion-associated cholesterol is critical for the maintenance of HIV-1 structure and infectivity.AIDS. 2002; 16: 2253-2261Crossref PubMed Scopus (108) Google Scholar, 12Graham D.R. Chertova E. Hilburn J.M. Arthur L.O. Hildreth J.E. Cholesterol depletion of human immunodeficiency virus type 1 and simian immunodeficiency virus with beta-cyclodextrin inactivates and permeabilizes the virions: Evidence for virion-associated lipid rafts.J. Virol. 2003; 77: 8237-8248Crossref PubMed Scopus (162) Google Scholar, 13Waheed A.A. Ablan S.D. Mankowski M.K. Cummins J.E. Ptak R.G. Schaffner C.P. Freed E.O. Inhibition of HIV-1 replication by amphotericin B methyl ester: Selection for resistant variants.J. Biol. Chem. 2006; 281: 28699-28711Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar, 14Waheed A.A. Ablan S.D. Roser J.D. Sowder R.C. Schaffner C.P. Chertova E. Freed E.O. HIV-1 escape from the entry-inhibiting effects of a cholesterol-binding compound via cleavage of gp41 by the viral protease.Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 8467-8471Crossref PubMed Scopus (39) Google Scholar, 15Waheed A.A. Ablan S.D. Sowder R.C. Roser J.D. Schaffner C.P. Chertova E. Freed E.O. Effect of mutations in the human immunodeficiency virus type 1 protease on cleavage of the gp41 cytoplasmic tail.J. Virol. 2010; 84: 3121-3126Crossref PubMed Scopus (13) Google Scholar, 16Salimi H. Johnson J. Flores M.G. Zhang M.S. O'Malley Y. Houtman J.C. Schlievert P.M. Haim H. The lipid membrane of HIV-1 stabilizes the viral envelope glycoproteins and modulates their sensitivity to antibody neutralization.J. Biol. Chem. 2020; 295: 348-362Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar, 17Checkley M.A. Luttge B.G. Freed E.O. HIV-1 envelope glycoprotein biosynthesis, trafficking, and incorporation.J. Mol. Biol. 2011; 410: 582-608Crossref PubMed Scopus (282) Google Scholar). Notably, HIV-1 resistance to cholesterol depletion has been mapped to the Env CT (13Waheed A.A. Ablan S.D. Mankowski M.K. Cummins J.E. Ptak R.G. Schaffner C.P. Freed E.O. Inhibition of HIV-1 replication by amphotericin B methyl ester: Selection for resistant variants.J. Biol. Chem. 2006; 281: 28699-28711Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar, 14Waheed A.A. Ablan S.D. Roser J.D. Sowder R.C. Schaffner C.P. Chertova E. Freed E.O. HIV-1 escape from the entry-inhibiting effects of a cholesterol-binding compound via cleavage of gp41 by the viral protease.Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 8467-8471Crossref PubMed Scopus (39) Google Scholar, 15Waheed A.A. Ablan S.D. Sowder R.C. Roser J.D. Schaffner C.P. Chertova E. Freed E.O. Effect of mutations in the human immunodeficiency virus type 1 protease on cleavage of the gp41 cytoplasmic tail.J. Virol. 2010; 84: 3121-3126Crossref PubMed Scopus (13) Google Scholar). Because of this, we also subjected an NL4-3 HIV-1 variant that carries a CT deletion (ΔCT) to our analyses. Although our first experiment suggested that ΔCT viruses might be slightly less sensitive than WT HIV-1 to CerS2 depletion, subsequent repeats showed that ΔCT viruses produced in CerS2−/− cells were as impaired for infectivity as WT viruses (Fig. 4B). We also tested two additional HIV-1 Env CT mutants: one of these was an Env P714L mutant (also called P203L (14Waheed A.A. Ablan S.D. Roser J.D. Sowder R.C. Schaffner C.P. Chertova E. Freed E.O. HIV-1 escape from the entry-inhibiting effects of a cholesterol-binding compound via cleavage of gp41 by the viral protease.Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 8467-8471Crossref PubMed Scopus (39) Google Scholar, 15Waheed A.A. Ablan S.D. Sowder R.C. Roser J.D. Schaffner C.P. Chertova E. Freed E.O. Effect of mutations in the human immunodeficiency virus type 1 protease on cleavage of the gp41 cytoplasmic tail.J. Virol. 2010; 84: 3121-3126Crossref PubMed Scopus (13) Google Scholar)) that was selected for resistance to cholesterol depletion; and the other was the Env 716Ins-R∗ mutant, which is cleaved by the HIV-1 protease (PR) in a similar fashion to P714L (26Staubus A.O. Alfadhli A. Barklis R.L. Barklis E. Replication of HIV-1 envelope protein cytoplasmic domain variants in permissive and restrictive cells.Virology. 2019; 538: 1-10Crossref PubMed Scopus (3) Google Scholar). These variants also showed significant infectivity reductions for viruses produced in mutant versus WT HEK293T cells (CerS2−/− P714L inf/Gag = 6% WT cells; CerS2−/− 716Ins-R∗ = 30 ± 11% WT cells). Overall, these results indicated that infectivity defects were independent of the Env CT. 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