Abstract
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.
Highlights
Some ceramides are present in the fetal bovine serum [32], and Cer can be generated by hydrolysis of SM or glucosylceramide (Fig. 1), the ceramide synthase (CerS)-mediated pathways are major routes of SL production [18,19,20,21]
Lipids extracted from WT and CerS2−/− human embryonic kidney 293T (HEK293T) cells were analyzed by LC/MS
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 [21,22,23] and suggest a larger role for CerS5 in Cer production in mutant versus WT HEK293T cells
Summary
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 [33] WT HIV-1 proviral plasmid and monitored particle production, Env levels in virus particles, and infection efficiencies. We pseudotyped Env-minus HIV-1 particles assembled by the HIV-Luc plasmid [40, 41] with VSV G and examined viruses produced in WT versus CerS2−/− HEK293T cells (see Experimental procedures section).
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