Abstract
The envelope of Human Immunodeficiency Virus type 1 (HIV-1) consists of a liquid-ordered membrane enriched in raft lipids and containing the viral glycoproteins. Previous studies demonstrated that changes in viral membrane lipid composition affecting membrane structure or curvature can impair infectivity. Here, we describe novel antiviral compounds that were identified by screening compound libraries based on raft lipid-like scaffolds. Three distinct molecular structures were chosen for mode-of-action studies, a sterol derivative (J391B), a sphingosine derivative (J582C) and a long aliphatic chain derivative (IBS70). All three target the viral membrane and inhibit virus infectivity at the stage of fusion without perturbing virus stability or affecting virion-associated envelope glycoproteins. Their effect did not depend on the expressed envelope glycoproteins or a specific entry route, being equally strong in HIV pseudotypes carrying VSV-G or MLV-Env glycoproteins. Labeling with laurdan, a reporter of membrane order, revealed different membrane structure alterations upon compound treatment of HIV-1, which correlated with loss of infectivity. J582C and IBS70 decreased membrane order in distinctive ways, whereas J391B increased membrane order. The compounds' effects on membrane order were reproduced in liposomes generated from extracted HIV lipids and thus independent both of virion proteins and of membrane leaflet asymmetry. Remarkably, increase of membrane order by J391B required phosphatidylserine, a lipid enriched in the HIV envelope. Counterintuitively, mixtures of two compounds with opposite effects on membrane order, J582C and J391B, did not neutralize each other but synergistically inhibited HIV infection. Thus, altering membrane order, which can occur by different mechanisms, constitutes a novel antiviral mode of action that may be of general relevance for enveloped viruses and difficult to overcome by resistance development.
Highlights
Human immunodeficiency virus type 1 (HIV-1) is an enveloped retrovirus, which infects CD4-positive human cells
MT-4 cells were infected with Human Immunodeficiency Virus type 1 (HIV-1) strain NL4-3 [24], and virus was harvested from cocultures of infected and uninfected cells before cytopathic effects were observed [25]. 293T cells were transfected with the proviral plasmid pNL4-3 [24] or with pCHIV [26] by calcium phosphate precipitation
For generation of pseudotyped particles, cells were co-transfected with pNL43 carrying a deletion of the envelope gene and a plasmid expressing either the G glycoprotein of Vesicular Stomatitis Virus (VSV) [27] or the envelope proteins of Friend ecotropic Murine Leukemia Virus (MLV) [28] at a molar ratio 1:2
Summary
Human immunodeficiency virus type 1 (HIV-1) is an enveloped retrovirus, which infects CD4-positive human cells. Studies of HIV-1 lipid composition indicated significant differences between the viral membrane and the host cell plasma membranes [4, 5]. This observation was later confirmed by more detailed analyses of the entire viral lipidome. Based on the concept of inverted cone-shaped lipids as fusion inhibitors [19], synthetic rigid amphipathic fusion inhibitors (RAFIs) have been designed as potential antivirals [20] These compounds insert into the viral membrane and promote positive curvature, increasing the energy barrier for fusion. Altering virion membrane structure by lipid-active compounds may be a promising approach for inhibiting HIV
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