HIV-1 gp41 fusogenic function triggers autophagy in uninfected cells

Cell-expressed HIV-1 envelope glycoproteins (gp120 and gp41, called Env) induce autophagy in uninfected CD4 T cells, leading to their apoptosis, a mechanism most likely contributing to immunodeficiency. The presence of CD4 and CXCR4 on target cells is required for this process, but Env-induced autophagy is independent of CD4 signaling. Here we demonstrate that CXCR4-mediated signaling pathways are not directly involved in autophagy and cell death triggering. Indeed, cells stably expressing mutated forms of CXCR4, unable to transduce different Gi-dependent and -independent signals, still undergo autophagy and cell death after coculture with effector cells expressing Env. After gp120 binding to CD4 and CXCR4, the N terminus fusion peptide (FP) of gp41 is inserted into the target membrane, and gp41 adopts a trimeric extended pre-hairpin intermediate conformation, target of HIV fusion inhibitors such as T20 and C34, before formation of a stable six-helix bundle structure and cell-to-cell fusion. Interestingly, Env-mediated autophagy is triggered in both single cells (hemifusion) and syncytia (complete fusion), and prevented by T20 and C34. The gp41 fusion activity is responsible for Env-mediated autophagy since the Val2Glu mutation in the gp41 FP totally blocks this process. On the contrary, deletion of the C-terminal part of gp41 enhances Env-induced autophagy. These results underline the major role of gp41 in inducing autophagy in the uninfected cells and indicate that the entire process leading to HIV entry into target cells through binding of Env to its receptors, CD4 and CXCR4, is responsible for autophagy and death in the uninfected, bystander cells.     

HIV-1 gp41 fusogenic function triggers autophagy in uninfected cells

Cell-expressed HIV-1 envelope glycoproteins (gp120 and gp41, called Env) induce autophagy in uninfected CD4 T cells, leading to their apoptosis, a mechanism most likely contributing to immunodeficiency. The presence of CD4 and CXCR4 on target cells is required for this process, but Env-induced autophagy is independent of CD4 signaling. Here, we demonstrate that CXCR4-mediated signaling pathways are not directly involved in autophagy and cell death triggering. Indeed, cells stably expressing mutated forms of CXCR4, unable to transduce different Gi-dependent and -independent signals, still undergo autophagy and cell death after coculture with effector cells expressing Env. After gp120 binding to CD4 and CXCR4, the N terminus fusion peptide (FP) of gp41 is inserted into the target membrane, and gp41 adopts a trimeric extended pre-hairpin intermediate conformation, target of HIV fusion inhibitors such as T20 and C34, before formation of a stable six-helix bundle structure and cell-to-cell fusion. Interestingly, Env-mediated autophagy is triggered in both single cells (hemifusion) and syncytia (complete fusion), and prevented by T20 and C34. The gp41 fusion activity is responsible for Env-mediated autophagy since the Val2Glu mutation in the gp41 FP totally blocks this process. On the contrary, deletion of the C-terminal part of gp41 enhances Env-induced autophagy. These results underline the major role of gp41 in inducing autophagy in the uninfected cells and indicate that the entire process leading to HIV entry into target cells through binding of Env to its receptors, CD4 and CXCR4, is responsible for autophagy and death in the uninfected, bystander cells.     

Apoptosis of bystander T cells induced by human immunodeficiency virus type 1 with increased envelope/receptor affinity and coreceptor binding site exposure

Apoptosis of uninfected bystander CD4(+) T cells contributes to T-cell depletion during human immunodeficiency virus type 1 (HIV-1) pathogenesis. The viral and host mechanisms that lead to bystander apoptosis are not well understood. To investigate properties of the viral envelope glycoproteins (Env proteins) that influence the ability of HIV-1 to induce bystander apoptosis, we used molecularly cloned viruses that differ only in specific amino acids in Env. The ability of these strains to induce bystander apoptosis was tested in herpesvirus saimiri-immortalized primary CD4(+) T cells (CD4/HVS), which resemble activated primary T cells. Changes in Env that increase affinity for CD4 or CCR5 or increase coreceptor binding site exposure enhanced the capacity of HIV-1 to induce bystander apoptosis following viral infection or exposure to nonreplicating virions. Apoptosis induced by HIV-1 virions was inhibited by CD4, CXCR4, and CCR5 antibodies or by the CXCR4 inhibitor AMD3100, but not the fusion inhibitor T20. HIV-1 virions with mutant Envs that bind CXCR4 but are defective for CD4 binding or membrane fusion induced apoptosis, whereas CXCR4 binding-defective mutants did not. These results demonstrate that HIV-1 virions induce apoptosis through a CXCR4- or CCR5-dependent pathway that does not require Env/CD4 signaling or membrane fusion and suggest that HIV-1 variants with increased envelope/receptor affinity or coreceptor binding site exposure may promote T-cell depletion in vivo by accelerating bystander cell death.     

Differential Role of Autophagy in CD4 T Cells and Macrophages during X4 and R5 HIV-1 Infection

Background

HIV-1 can infect and replicate in both CD4 T cells and macrophages. In these cell types, HIV-1 entry is mediated by the binding of envelope glycoproteins (gp120 and gp41, Env) to the receptor CD4 and a coreceptor, principally CCR5 or CXCR4, depending on the viral strain (R5 or X4, respectively). Uninfected CD4 T cells undergo X4 Env-mediated autophagy, leading to their apoptosis, a mechanism now recognized as central to immunodeficiency.

Methodology/Principal Findings

We demonstrate here that autophagy and cell death are also induced in the uninfected CD4 T cells by HIV-1 R5 Env, while autophagy is inhibited in productively X4 or R5-infected CD4 T cells. In contrast, uninfected macrophages, a preserved cell population during HIV-1 infection, do not undergo X4 or R5 Env-mediated autophagy. Autophagosomes, however, are present in macrophages exposed to infectious HIV-1 particles, independently of coreceptor use. Interestingly, we observed two populations of autophagic cells: one highly autophagic and the other weakly autophagic. Surprisingly, viruses could be detected in the weakly autophagic cells but not in the highly autophagic cells. In addition, we show that the triggering of autophagy in macrophages is necessary for viral replication but addition of Bafilomycin A1, which blocks the final stages of autophagy, strongly increases productive infection.

Conclusions/Significance

Taken together, our data suggest that autophagy plays a complex, but essential, role in HIV pathology by regulating both viral replication and the fate of the target cells.     

What is the role of autophagy in HIV-1 infection?

HIV-1 infection is characterized by a progressive CD4 T cell depletion. It is now accepted that apoptosis of uninfected bystander CD4 T lymphocytes plays a major role in AIDS development. Viral envelope glycoproteins (Env) are mainly involved in inducing this cell death process, but the mechanisms triggered by HIV-1 leading to immunodeficiency are still poorly understood. Recently, we have demonstrated that autophagy is a prerequisite for Env-mediated apoptosis in uninfected CD4 T cells, underlining its role in HIV-1 infection. However, occurrence of autophagy in HIV-1-infected cells has not yet been described. Several hypotheses are discussed, based on the comparison with data from other viral infections.     

What is the role of autophagy in HIV-1 infection?

HIV-1 infection is characterized by a progressive CD4 T cell depletion. It is now accepted that apoptosis of uninfected bystander CD4 T lymphocytes plays a major role in AIDS development. Viral envelope glycoproteins (Env) are mainly involved in inducing this cell death process, but the mechanisms triggered by HIV-1 leading to immunodeficiency are still poorly understood. Recently, we have demonstrated that autophagy is a prerequisite for Env-mediated apoptosis in uninfected CD4 T cells, underlining its role in HIV-1 infection. However, occurrence of autophagy in HIV-1-infected cells has not yet been described. Several hypotheses are discussed, based on the comparison with data from other viral infections.     

Distinct mechanisms of CD4+ and CD8+ T-cell activation and bystander apoptosis induced by human immunodeficiency virus type 1 virions

Apoptosis of uninfected bystander T cells contributes to T-cell depletion during human immunodeficiency virus type 1 (HIV-1) infection. HIV-1 envelope/receptor interactions and immune activation have been implicated as contributors to bystander apoptosis. To better understand the relationship between T-cell activation and bystander apoptosis during HIV-1 pathogenesis, we investigated the effects of the highly cytopathic CXCR4-tropic HIV-1 variant ELI6 on primary CD4(+) and CD8(+) T cells. Infection of primary T-cell cultures with ELI6 induced CD4(+) T-cell depletion by direct cell lysis and bystander apoptosis. Exposure of primary CD4(+) and CD8(+) T cells to nonreplicating ELI6 virions induced bystander apoptosis through a Fas-independent mechanism. Bystander apoptosis of CD4(+) T cells required direct contact with virions and Env/CXCR4 binding. In contrast, the apoptosis of CD8(+) T cells was triggered by a soluble factor(s) secreted by CD4(+) T cells. HIV-1 virions activated CD4(+) and CD8(+) T cells to express CD25 and HLA-DR and preferentially induced apoptosis in CD25(+)HLA-DR(+) T cells in a CXCR4-dependent manner. Maximal levels of binding, activation, and apoptosis were induced by virions that incorporated MHC class II and B7-2 into the viral membrane. These results suggest that nonreplicating HIV-1 virions contribute to chronic immune activation and T-cell depletion during HIV-1 pathogenesis by activating CD4(+) and CD8(+) T cells, which then proceed to die via apoptosis. This mechanism may represent a viral immune evasion strategy to increase viral replication by activating target cells while killing immune effector cells that are not productively infected.     

Two discrete events, human T-cell leukemia virus type I Tax oncoprotein expression and a separate stress stimulus, are required for induction of apoptosis in T-cells

Apoptosis, or programmed cell death, is a key event in biologic homeostasis but is also involved in the pathogenesis of many human diseases including human immunodeficiency virus (HIV) infection. Although multiple mechanisms contribute to the gradual T cell decline that occurs in HIV-infected patients, programmed cell death of uninfected bystander T lymphocytes, including CD4+ and CD8+ T cells, is an important event leading to immunodeficiency. The HIV envelope glycoproteins (Env) play a crucial role in transducing this apoptotic signal after binding to its receptors, the CD4 molecule and a coreceptor, essentially CCR5 and CXCR4. Depending on Env presentation, the receptor involved and the complexity of target cell contact, apoptosis induction is related to death receptor and/or mitochondria-dependent pathways. This review summarizes current knowledge of Env-mediated cell death leading to T cell depletion and clinical complications and covers the sometimes conflicting studies that address the possible mechanisms of T cell death.     

HIV ENV glycoprotein-mediated bystander apoptosis depends on expression of the CCR5 co-receptor at the cell surface and ENV fusogenic activity

HIV-1 infections lead to a progressive depletion of CD4 cells culminating in AIDS. The coreceptor usage by HIV varies from CCR5 (R5) tropic early in infection to CXCR4 (X4) tropic in later infections. Although the coreceptor switch from R5 to X4 tropic HIV is well associated with progression to AIDS, the role of CCR5 in disease progression especially in patients infected exclusively with R5 isolates throughout the disease remains enigmatic. To better understand the role of CCR5 and R5 tropic HIV envelope in AIDS pathogenesis, we asked whether the levels of CCR5 and/or HIV Env-mediated fusion determine apoptosis of bystander cells. We generated CD4(+) T cell lines expressing varying levels of CCR5 on the cell surface to show that CCR5 expression levels correlate with bystander apoptosis induction. The mechanism of apoptosis involved caspase-3 activation and mitochondrial depolarization and was dependent on gp41 fusion activity as confirmed by fusion-restricted gp41 point mutants and use of the fusion inhibitor T20. Interestingly, lower levels of CCR5 were able to support virus replication in the absence of bystander apoptosis. Our findings suggest that R5 HIV-1-mediated bystander apoptosis is dependent on both CCR5 expression levels as well as fusogenic activity of the Env glycoprotein.     

In vivo evolution of human immunodeficiency virus type 1 toward increased pathogenicity through CXCR4-mediated killing of uninfected CD4 T cells

The destruction of the immune system by progressive loss of CD4 T cells is the hallmark of AIDS. CCR5-dependent (R5) human immunodeficiency virus type 1 (HIV-1) isolates predominate in the early, asymptomatic stages of HIV-1 infection, while CXCR4-dependent (X4) isolates typically emerge at later stages, frequently coinciding with a rapid decline in CD4 T cells. Lymphocyte killing in vivo primarily occurs through apoptosis, but the importance of apoptosis of HIV-1-infected cells relative to apoptosis of uninfected bystander cells is controversial. Here we show that in human lymphoid tissues ex vivo, apoptosis of uninfected bystander CD4 T cells is a major mechanism of lymphocyte depletion caused by X4 HIV-1 strains but is only a minor mechanism of depletion by R5 strains. Further, X4 HIV-1-induced bystander apoptosis requires the interaction of the viral envelope glycoprotein gp120 with the CXCR4 coreceptor on CD4 T cells. These results emphasize the contribution of bystander apoptosis to HIV-1 cytotoxicity and suggest that in association with a coreceptor switch in HIV disease, T-cell killing evolves from an infection-restricted stage to generalized toxicity that involves a high degree of bystander apoptosis.     

Site-specific mutations in HIV-1 gp41 reveal a correlation between HIV-1-mediated bystander apoptosis and fusion/hemifusion

The loss of CD4(+) T cells in HIV-1 infections is hypothesized to be caused by apoptosis of bystander cells mediated by cell surface-expressed HIV-1 Env glycoprotein. However, the mechanism by which Env mediates this process remains controversial. Specifically, the role of HIV-1 gp120 binding to CD4 and CXCR4 versus the fusion process mediated by gp41 remains unresolved. Env-induced apoptosis in bystander cells has been shown to be gp41-dependent and correlates with the redistribution of membrane lipids between Env-expressing cells and target cells (hemifusion). Using a rational mutagenesis approach aimed at targeting Env function via the gp41 subunit, we examined the role of HIV gp41 in bystander apoptosis. A mutation in the fusion domain of gp41 (V513E) resulted in a fusion-defective Env that failed to induce apoptosis. A mutation in the gp41 N-terminal helix (G547D) reduced cell fusion capacity and apoptosis; conversely, an Env mutant with a deletion of the gp41 cytoplasmic tail (Ct Del) enhanced both cell-to-cell fusion and apoptosis. Most significantly, an Env mutant containing a substitution in the loop region of gp41 (D589L) mediated transfer of lipids (hemifusion) to bystander cells but was defective in cell-to-cell and to a lesser degree virus-to-cell fusion. This mutant was still able to induce apoptosis in bystander cells. Hence, we have provided the first direct evidence that gp41-mediated hemifusion is both required and sufficient for induction of apoptosis in bystander cells. These results may help to explain the mechanism of HIV-1 Env-induced T cell depletion.     

Cytolysis by CCR5-using human immunodeficiency virus type 1 envelope glycoproteins is dependent on membrane fusion and can be inhibited by high levels of CD4 expression

T-tropic (X4) and dualtropic (R5X4) human immunodeficiency virus type 1 (HIV-1) envelope glycoproteins kill primary and immortalized CD4(+) CXCR4(+) T cells by mechanisms involving membrane fusion. However, because much of HIV-1 infection in vivo is mediated by M-tropic (R5) viruses whose envelope glycoproteins use CCR5 as a coreceptor, we tested a panel of R5 and R5X4 envelope glycoproteins for their ability to lyse CCR5(+) target cells. As is the case for CXCR4(+) target cells, HIV-1 envelope glycoproteins expressed by single-round HIV-1 vectors killed transduced CD4(+) CCR5(+) cells in a membrane fusion-dependent manner. Furthermore, a CD4-independent R5 HIV-1 envelope glycoprotein was able to kill CD4-negative target cells expressing CCR5, demonstrating that CD4 is not intrinsically required for the induction of death. Interestingly, high levels of CD4 expression protected cells from lysis and syncytium formation mediated by the HIV-1 envelope glycoproteins. Immunoprecipitation experiments showed that high levels of CD4 coexpression inhibited proteolytic processing of the HIV-1 envelope glycoprotein precursor gp160. This inhibition could be overcome by decreasing the CD4 binding ability of gp120. Studies were also undertaken to investigate the ability of virion-bound HIV-1 envelope glycoproteins to kill primary CD4(+) T cells. However, neither X4 nor R5X4 envelope glycoproteins on noninfectious virions caused death in primary CD4(+) T cells. These results demonstrate that the interaction of CCR5 with R5 HIV-1 envelope glycoproteins capable of inducing membrane fusion leads to cell lysis; overexpression of CD4 can inhibit cell killing by limiting envelope glycoprotein processing.     

Genetic and functional analysis of R5X4 human immunodeficiency virus type 1 envelope glycoproteins derived from two individuals homozygous for the CCR5delta32 allele

We characterized human immunodeficiency virus type 1 (HIV-1) envelope glycoproteins (Env) isolated from two HIV-1-infected CCR5delta32 homozygotes. Envs from both subjects used CCR5 and CXCR4 for entry into transfected cells. Most R5X4 Envs were lymphocyte-tropic and used CXCR4 exclusively for entry into peripheral blood mononuclear cells (PBMC), but a subset was dually lymphocyte- and macrophage-tropic and used either CCR5 or CXCR4 for entry into PBMC and monocyte-derived macrophages. The persistence of CCR5-using HIV-1 in two CCR5delta32 homozygotes suggests the conserved CCR5 binding domain of Env is highly stable and provides new mechanistic insights important for HIV-1 transmission and persistence.     

Flavonoid baicalin inhibits HIV-1 infection at the level of viral entry

Baicalin (BA) is a flavonoid compound purified from medicinal plant Scutellaria baicalensis Georgi and has been shown to possess anti-inflammatory and anti-HIV-1 activities. In an effort to elucidate the mechanism of the anti-inflammatory effect of BA, we recently found that this flavonoid compound was able to form complexes with selected chemokines and attenuated their capacity to bind and activate receptors on the cell surface. These observations prompted us to investigate whether BA could inhibit HIV-1 infection by interfering with viral entry, a process known to involve interaction between HIV-1 envelope proteins and the cellular CD4 and chemokine receptors. We found that BA at the noncytotoxic concentrations, inhibited both T cell tropic (X4) and monocyte tropic (R5) HIV-1 Env protein mediated fusion with cells expressing CD4/CXCR4 or CD4/CCR5. Furthermore, presence of BA at the initial stage of HIV-1 viral adsorption blocked the replication of HIV-1 early strong stop DNA in cells. Since BA did not inhibit binding of HIV-1 gp120 to CD4, we propose that BA may interfere with the interaction of HIV-1 Env with chemokine coreceptors and block HIV-1 entry of target cells. Therefore, BA can be used as a basis for developing novel anti-HIV-1 agents.     

Autophagy: An overlooked mechanism of HIV-1 pathogenesis and NeuroAIDS?

Human immunodeficiency virus type 1 (HIV-1) establishes a persistent infection characterized by progressive depletion of CD4⁺ lymphocytes and immunosuppression. Although extensive research has examined the importance of apoptosis as a cause of cell death associated with HIV-1 infection, the role of autophagy has been largely ignored. Our laboratory has examined the autophagic process in HIV-1-infected cells. Following infection of human peripheral blood CD4+ T-cells or U937 cells with HIV-1 for 48 hours, the autophagy proteins Beclin 1 and LC3-II were found to be markedly decreased. Beclin 1 mRNA expression and autophagosomes were also reduced in HIV-1 infected cells. Thus, our data indicate that HIV-1 infection inhibits autophagy in infected cells in contrast to the previously described induction of autophagy by gp120 in uninfected bystander cells. It is likely that HIV-1 has evolved this mechanism as part of an elaborate attempt to evade the immune system while promoting its own replication. We believe that autophagy is an overlooked mechanism in HIV-1 pathogenesis and plays a particularly important role in the early cognitive impairment and dementia often associated with advanced AIDS. A model is presented that describes the potential role of autophagy in NeuroAIDS.

Addendum to: Zhou D, Spector SA. Human immunodeficiency virus type-1 infection inhibits autophagy. Aids 2008;22:695-9.     

Autophagy: an overlooked mechanism of HIV-1 pathogenesis and neuroAIDS?

Human immunodeficiency virus type 1 (HIV-1) establishes a persistent infection characterized by progressive depletion of CD4(+) lymphocytes and immunosuppression. Although extensive research has examined the importance of apoptosis as a cause of cell death associated with HIV-1 infection, the role of autophagy has been largely ignored. Our laboratory has examined the autophagic process in HIV-1-infected cells. Following infection of human peripheral blood CD4(+) T-cells or U937 cells with HIV-1 for 48 hours, the autophagy proteins Beclin 1 and LC3-II were found to be markedly decreased. Beclin 1 mRNA expression and autophagosomes were also reduced in HIV-1 infected cells. Thus, our data indicate that HIV-1 infection inhibits autophagy in infected cells in contrast to the previously described induction of autophagy by gp120 in uninfected bystander cells. It is likely that HIV-1 has evolved this mechanism as part of an elaborate attempt to evade the immune system while promoting its own replication. We believe that autophagy is an overlooked mechanism in HIV-1 pathogenesis and plays a particularly important role in the early cognitive impairment and dementia often associated with advanced AIDS. A model is presented that describes the potential role of autophagy in NeuroAIDS.     

CCR5- and CXCR4-tropic HIV-1 are equally cytopathic for their T-cell targets in human lymphoid tissue

A rapid decline in T-cell counts and the progression to AIDS is often associated with a switch from CCR5-tropic (R5) HIV-1 to CXCR4-tropic (X4) HIV-1 or R5/X4 HIV-1 variants. Experimental infection with R5 HIV-1 causes less T-cell depletion than infection with X4 or R5/X4 variants in T-cell cultures, in ex vivo infected human lymphoid tissue and in SCID/hu mice, despite similar replication levels. Experimental genetic changes in those sequences in gp120 that transform R5 HIV-1 variants into otherwise isogenic X4 viruses make them highly cytopathic. Thus, it is now believed that R5 variants are less cytopathic for T cells than are X4 variants. However, it is not known why CCR5-mediated HIV-1 infection does not lead to a massive CD4+ T-cell depletion, as occurs in CXCR4-mediated HIV-1 infection. Here we demonstrate that R5 HIV-1 isolates are indeed highly cytopathic, but only for CCR5+/CD4+ T cells. Because these cells constitute only a small fraction of CD4+ T cells, their depletion does not substantially change the total CD4+ T-cell count. These results may explain why the clinical stage of HIV disease correlates with viral tropism.     

Wild-type-like viral replication potential of human immunodeficiency virus type 1 envelope mutants lacking palmitoylation signals

Palmitoylation of the cytoplasmic domain of the human immunodeficiency type virus type 1 (HIV-1) envelope (Env) transmembrane protein, gp41, has been implicated in Env targeting to detergent-resistant lipid rafts, Env incorporation into the virus, and viral infectivity. In contrast, we provide evidence here to show that HIV-1 infectivity, Env targeting to lipid rafts, and Env incorporation into the virus are independent of cytoplasmic tail palmitoylation. The T-cell (T)-tropic HXB2-based virus, which utilizes CXCR4 as the entry coreceptor, carrying a Cys-to-Ser mutation at residue 764 or 837 or at both replicated with wild-type (WT) virus replication kinetics in CD4+ T cells. The properties of Env expression, precursor processing, cell surface expression, and Env incorporation of these three mutant viruses were normal compared to those of the WT virus. These three mutant Env proteins all effectively mediated one-cycle virus infection. When the Cys residues were replaced by Ala residues, all single and double mutants still retained the phenotypes of infectivity, Env incorporation, and lipid raft localization of the WT Env. When Cys-to-Ala substitutions were introduced into the macrophage (M)-tropic ConB virus, which utilizes CCR5 as the coreceptor, these mutations did not affect the replication potential, Env phenotypes, lipid raft targeting, or Env assembly into the virus of the WT Env. These T- and M-tropic mutants also productively replicated in human primary CD4+ T cells. Moreover, mutations at both Cys residues significantly reduced the level of palmitoylation of the Env. Our results together support the notion that palmitoylation of the cytoplasmic tail of the HIV-1 Env is not essential for the HIV-1 virus life cycle.     

Vaccinia virus replication is not affected by APOBEC3 family members

Background

HIV-1 entry into cells is a multifaceted process involving target cell CD4 and the chemokine receptors, CXCR4 or CCR5. The lipid composition of the host cell plays a significant role in the HIV fusion process as it orchestrates the appropriate disposition of CD4 and co-receptors required for HIV-1 envelope glycoprotein (Env)-mediated fusion. The cell membrane is primarily composed of sphingolipids and cholesterol. The effects of lipid modulation on CD4 disposition in the membrane and their role in HIV-1 entry have extensively been studied. To focus on the role of lipid composition on chemokine receptor function, we have by-passed the CD4 requirement for HIV-1 Env-mediated fusion by using a CD4-independent strain of HIV-1 Env.

Results

Cell fusion mediated by a CD4-independent strain of HIV-1 Env was monitored by observing dye transfer between Env-expressing cells and NIH3T3 cells bearing CXCR4 or CCR5 in the presence or absence of CD4. Chemokine receptor signaling was assessed by monitoring changes in intracellular [Ca²⁺] mobilization induced by CCR5 or CXCR4 ligand. To modulate target membrane cholesterol or sphingolipids we used Methyl-β-cyclodextrin (MβCD) or 1-phenyl-2-hexadecanoylamino-3-morpholino-1-propanol (PPMP), respectively. Treatment of the target cells with these agents did not change the levels of CD4 or CXCR4, but reduced levels of CCR5 on the cell surface. Chemokine receptor signalling was inhibited by cholesterol removal but not by treatment with PPMP. HIV-1 Env mediated fusion was inhibited by >50% by cholesterol removal. Overall, PPMP treatment appeared to slow down the rates of CD4-independent HIV-1 Env-mediated Fusion. However, in the case of CXCR4-dependent fusion, the differences between untreated and PPMP-treated cells did not appear to be significant.

Conclusion

Although modulation of cholesterol and sphingolipids has similar effects on CD4 -dependent HIV-1 Env-mediated fusion, sphingolipid modulation had little effect on CD4-independent HIV-1 Env-mediated fusion. Chemokine receptor function remained intact following treatment of cells with PPMP. Therefore such treatment may be considered a more suitable agent to inhibit CD4 dependent HIV-1 infection.