Lecithin:cholesterol acyltransferase activation by synthetic amphipathic peptides

The amphipathic helical theory of Segrest and colleagues (FEBS Lett.:38:247-253, 1974) proposes that the lipid-binding segments of serum apolipoproteins are in an alpha helical conformation. Furthermore the helices have a hydrophobic face and a hydrophilic face with a specific distribution of positively and negatively charged residues. The importance of the pattern of the charged residues in the lipid binding and lecithin:cholesterol acyltransferase (LCAT) activation by the segments is still debated. We designed a 30-residue peptide, GALA, which in the alpha helical conformation has a hydrophilic face composed of glutamic acid residues (Sabbarao et al.: Biochemistry 26:2964-2972, 1987). GALA behaves like the serum apolipoproteins in its interaction with dimyristoylphosphatidylcholine (DMPC) at neutral pH; the amino terminal tryptophan of GALA undergoes a blue shift in its fluorescence emission spectrum, and the circular dichroism (CD) spectrum indicates that GALA acquires alpha helical structure in the presence of DMPC. A DMPC-GALA:19/1 (molar ratio) complex can be isolated by gel-permeation chromatography. This complex has a discoidal structure with the approximate dimensions of 44-A edge thickness and a 170- to 350-A diameter. GALA activates LCAT with DMPC but not with unsaturated phospholipids as the substrate. The apparent partition coefficient of GALA into DMPC vesicles is 100-fold larger than into egg phosphatidylcholine vesicles. The interaction of GALA with unsaturated lipids at neutral pH is so weak that no detectable change in the spectroscopic properties of GALA or the structure of the liposomes can be detected under the conditions used here. The sequence of GALA differs from previously studied model Apo A1 peptides by the absence of positively charged residues on the hydrophilic face. This indicates that positive charges in Apo A1-like peptides are not required in order to form discoidal structures with saturated phospholipids or to activate LCAT with such lipid substrates.     

Biophysical studies on fragments of the α-factor receptor protein

The receptor for the α-factor mating pheromone of the yeast Saccharomyces cerevisiae consists of 431 amino acid residues and is a member of a family of membrane proteins predicted to have seven transmembrane helices. Fragments of the receptor corresponding to two of the transmembrane helices [residues 246–269 (M6) and 273–302 (M7)], two of the interhelical loops [residues 107–125 (E2) and 191–206 (E3)], and to a portion of the carboxyl terminus [residues 350–372 (CT)] were synthesized using solid-phase methodologies and purified to near homogeneity. CD was used to characterize the secondary structure of these peptides in trifluoroethanol (TFE), in TFE/water mixtures, in sodium dodecyl sulfate (SDS), and in the presence of dimyristoyl phosphatidylcholine (DMPC) liposomes. In TFE, M6 and M7 exhibited CD spectra consistent with highly helical peptides, whereas CT was partially helical. In contrast, E2 and E3 were either disordered or aggregated in this solvent. M6 did not partition well into DMPC vesicles whereas M7 remained helical. Both M6 and M7 assumed helical conformations in 25 mM SDS. The loop neptides and the carboxyl terminus peptide were either in a β-structure or disordered in the presence of lipid. These findings represent the first biophysical evidence for conformations assumed by specific segments of the STE2 receptor protein. © 1994 John Wiley & Sons, Inc.     

A CD4 domain important for HIV-mediated syncytium formation lies outside the virus binding site

HIV infection of chimpanzees results in a chronic viremia unaccompanied by the ultimately fatal immunodeficiency that marks HIV infection in man. We show here that expression of HIV envelope proteins allows syncytium formation between cells expressing human but not chimpanzee or macaque CD4. We find that the CD4 sequences regulating cell fusion lie outside the recognized virus binding site; in the simplest exchange, chimpanzee CD4 bearing human residue 87 supports syncytium formation, while human CD4 bearing chimpanzee residue 87 does not. Neither the equilibrium nor the forward rate constants for HIV-CD4 association are affected by substitution at position 87. Infection of human cells expressing chimpanzee CD4 is insensitive to lysosomotropic agents, suggesting that viral penetration under these circumstances does not require endocytosis. The benign course of HIV infection in chimpanzees may reflect the failure of the host to support direct cell to cell transmission of the virus.     

Endocytic entry of HIV-1

Enveloped viruses enter target cells by membrane fusion or endocytosis. In the latter case, fusion of the viral envelope is induced by the acidic pH of the endocytic vesicle [1]. As with most other retroviruses, entry of the human immunodeficiency virus (HIV) is thought to be exclusively by pH-independent membrane fusion after interaction of its envelope with CD4 and a chemokine co-receptor on the target cell [2,3]. Expression of CD4 on the virus-producing cell impairs the release and infectivity of HIV-1(NL4-3) particles [4-6]. In sharp contrast, we found that the infectivity of another HIV isolate, HIV-1SF2, was enhanced by expression of CD4 on the producer cells, which correlated with significantly increased amounts of viral proteins in the vesicular fraction of target cells. Endocytic inhibitors decreased infectivity of HIV-1SF2 but enhanced that of HIV-1 NL4-3. Expression of CD4 in the producer cell did not remove gp41 from HIV-1SF2 virions. With these cells, the formation of syncytia could be induced by acidic medium. Thus, HIV-1SF2 can enter the cytoplasm by an endocytic route after activation of gp41 by the acidic pH of endocytic vesicles. Endocytic entry might expand the range of cells that HIV could infect and should be considered in antiviral strategies against AIDS.     

Failure of human immunodeficiency virus entry and infection in CD4-positive human brain and skin cells.

CD4 molecules on human cells function as a major receptor for human immunodeficiency virus (HIV); however, certain CD4-negative cell types may also be susceptible to infection. Therefore, we attempted to quantitate the relationship between HIV infection and CD4 expression on human cell lines before and after introduction of the CD4 gene by using a retrovirus vector. Prior to introduction of the CD4 expression vector, low levels of HIV infection were detected by a sensitive focal immunoassay on all three cell types studied. With several HIV strains in clones of human cervical carcinoma (HeLa) cells expressing different levels of CD4, HIV titer increased with increasing CD4 expression. In contrast, in squamous cell carcinoma cells (SCL1) and astroglial cells (U87MG), even high levels of CD4 expression failed to augment HIV infection. The CD4 protein expressed in these two cell lines had the expected molecular weight and was capable of binding HIV virions. However, in contrast to CD4-positive HeLa cells, CD4-positive U87MG and SCL1 cells were unable to form syncytia when cultured with cells expressing HIV envelope protein. Thus, the inability of HIV to infect these cells appeared to be due to lack of fusion between HIV virion envelope proteins and CD4-positive cell membranes. This block is infectivity was overcome when cells were infected with HIV which was pseudotyped with the envelope protein of amphotropic murine leukemia virus. Thus, in addition to CD4, other cell surface molecules appear to be required for successful HIV entry into and infection of these two human cell lines.     

Human immunodeficiency virus envelope glycoprotein/CD4-mediated fusion of nonprimate cells with human cells.

Human immunodeficiency virus (HIV) infects human cells by binding to surface CD4 molecules and directly fusing with the cell membrane. Although mouse cells expressing human CD4 bind HIV, they do not become infected, apparently because of a block in membrane fusion. To study this problem, we constructed a recombinant vaccinia virus that can infect and promote transient expression of full-length CD4 in mammalian cells. This virus, together with another vaccinia recombinant encoding biologically active HIV envelope glycoprotein gp160, allowed us to study CD4/gp160-mediated cell-cell fusion in a wide variety of human and nonhuman cells in the absence of other HIV proteins. By using syncytium formation assays in which a single cell type expressed both CD4 and gp160, we demonstrated membrane fusion in lymphoid and nonlymphoid human cells but not in any of the 23 tested nonhuman cell types, derived from African green monkey, baboon, rabbit, hamster, rat, or mouse. However, in mixing experiments with one cell type expressing CD4 and the other cell type expressing gp160, all of these nonhuman cells could form CD4/gp160-mediated syncytia when mixed with human cells; in 20 of 23 cases, membrane fusion occurred only if the CD4 molecule was expressed on the human cells whereas in the other three cases, CD4 could be expressed on either one of the fusing partners. Interestingly, in one mouse cell line, CD4-dependent syncytia formed without a human partner, but only if a C-terminally truncated form of the HIV envelope glycoprotein was employed. Our results indicate that nonhuman cells are intrinsically capable of undergoing CD4/gp160-mediated membrane fusion, but this fusion is usually prevented by the lack of helper or the presence of inhibitory factors in the nonhuman cell membranes.     

HIV infection does not require endocytosis of its receptor, CD4

The T cell surface molecule CD4 interacts with class II MHC molecules on the surface of target cells as well as with the envelope glycoprotein of human immunodeficiency virus (HIV). Internalization of CD4 molecules is observed after exposure of CD4+ T cells to either phorbol esters or appropriate antigen-bearing target cells. To determine whether HIV entry proceeds via receptor-mediated endocytosis or direct viral fusion with the cell membrane, we have constructed two mutants in the cytoplasmic domain of the CD4 protein that severely impair the ability of CD4 molecules to undergo endocytosis. Quantitative infectivity studies reveal that HeLa cell lines expressing wild-type or mutant CD4 molecules are equally susceptible to HIV infection. In addition, HIV binding does not lead to CD4 endocytosis. These studies indicate that although the CD4 molecule can be internalized, HIV entry proceeds via direct fusion of the viral envelope with the cell membrane.     

pH-independent HIV entry into CD4-positive T cells via virus envelope fusion to the plasma membrane

CD4 functions as the cell-surface receptor for human immunodeficiency virus (HIV); however, the mechanism of virus entry into susceptible cells is unknown. To explore this question we used a human T lymphoblastic cell line (VB) expressing high levels of surface CD4. Neutralization of endosomal compartments (pH greater than 6.4) with lysosomotropic agents did not effectively inhibit HIV nucleocapsid entry into the cytoplasm, and virus treated at low pH (5.5) failed to induce rapid cell-to-cell fusion in uninfected cells. Electron microscopy of VB cells acutely exposed to HIV at neutral pH revealed direct fusion of the virus envelope with the plasma membrane within minutes at 4 degrees C. No endocytosed virions were visualized upon rewarming the HIV-exposed cells to 37 degrees C for as long as 60 min. These results indicate that HIV penetrates CD4-positive T cells via pH-independent membrane fusion.     

Inactivation of retroviruses with preservation of structural integrity by targeting the hydrophobic domain of the viral envelope

We describe a new approach for the preparation of inactivated retroviruses for vaccine application. The lipid domain of the viral envelope was selectively targeted to inactivate proteins and lipids therein and block fusion of the virus with the target cell membrane. In this way, complete elimination of the infectivity of human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) could be achieved with preservation of antigenic determinants on the surface of the viral envelope. Inactivation was accomplished by modification of proteins and lipids in the viral envelope using the hydrophobic photoinduced alkylating probe 1,5 iodonaphthylazide (INA). Treatment of HIV and SIV isolates with INA plus light completely blocked fusion of the viral envelope and abolished infectivity. The inactivated virus remained structurally unchanged, with no detectable loss of viral proteins. Modifications to envelope and nucleocapsid proteins were detected by changes in their elution pattern on reverse-phase high-performance liquid chromatography. These modifications had no effect on primary and secondary structure epitopes as determined by monoclonal antibodies. Likewise, the inactivated HIV reacted as well as the live virus with the conformation-sensitive and broadly neutralizing anti-HIV type 1 monoclonal antibodies 2G12, b12, and 4E10. Targeting the lipid domain of biological membranes with hydrophobic alkylating compounds could be used as a general approach for inactivation of enveloped viruses and other pathogenic microorganisms for vaccine application.     

Complementation of murine cells for human immunodeficiency virus envelope/CD4-mediated fusion in human/murine heterokaryons.

Murine cell lines expressing human CD4 are resistant to the fusogenic effect of the human immunodeficiency virus (HIV) envelope. Consequently, they cannot be infected by HIV or form syncytia with HIV envelope-expressing cells. Murine cells could either lack human-specific cofactors necessary for the CD4/envelope-mediated membrane fusion or express inhibitors of this process. To address this question, we have tested the ability of heterokaryons made from CD4-expressing murine cells and human cells to undergo HIV envelope-mediated fusion. We have devised a rapid and specific assay based on the induction of lacZ expression, in which membrane fusion events with HIV-infected cells can be detected by a simple histochemical technique. CD4-positive murine/human heterokaryons, but not murine/simian heterokaryons, were found able to fuse with HIV envelope-expressing cells. In these experiments, the fusion resistant phenotype of murine-CD4 cells could be complemented by human cellular factors.     

Solid-state nuclear magnetic resonance studies of HIV and influenza fusion peptide orientations in membrane bilayers using stacked glass plate samples

The human immunodeficiency virus (HIV) and influenza virus fusion peptides are approximately 20-residue sequences which catalyze the fusion of viral and host cell membranes. The orientations of these peptides in lipid bilayers have been probed with 15N solid-state nuclear magnetic resonance (NMR) spectroscopy of samples containing membranes oriented between stacked glass plates. Each of the peptides adopts at least two distinct conformations in membranes (predominantly helical or beta strand) and the conformational distribution is determined in part by the membrane headgroup and cholesterol composition. In the helical conformation, the 15N spectra suggest that the influenza peptide adopts an orientation approximately parallel to the membrane surface while the HIV peptide adopts an orientation closer to the membrane bilayer normal. For the beta strand conformation, there appears to be a broader peptide orientational distribution. Overall, the data suggest that the solid-state NMR experiments can test models which correlate peptide orientation with their fusogenic function.     

Changes in the transmembrane region of the human immunodeficiency virus type 1 gp41 envelope glycoprotein affect membrane fusion.

The charged amino acids near or within the membrane-spanning region of the human immunodeficiency virus type 1 gp41 envelope glycoprotein were altered. Two mutants were defective for syncytium formation and virus replication even though levels of envelope glycoproteins on the cell or virion surface and CD4 binding were comparable to those of the wild-type proteins. Thus, in addition to anchoring the envelope glycoproteins, sequences proximal to the membrane-spanning gp41 region are important for the membrane fusion process.     

Destabilization exerted by peptides derived from the membrane-proximal external region of HIV-1 gp41 in lipid vesicles supporting fluid phase coexistence

The human immunodeficiency virus (HIV) envelope is enriched in cholesterol and sphingomyelin, two lipids that sustain the formation of laterally segregated liquid-ordered fluid domains in model systems. Several evidences indicate that the high lipid order existing at the envelope may play a role in HIV pathogenesis. A putative mechanism might involve the modulation of the membrane-perturbing function of the gp41 membrane-proximal external region (MPER). To test such hypothesis, we investigate here the effect of lipid phase coexistence on the membrane-restructuring properties of NpreTM and CpreTM, two peptides based on the amino- and carboxy-terminal MPER sequences, respectively. Fluid phase coexistence elicited the fusogenic activity of NpreTM at high membrane doses and stimulated "graded" leakage at low doses. By comparison, the effect on CpreTM was restricted to an enhancement of "all-or-none" leakage that was consistent with the promotion of its surface aggregation. Confocal microscopy of single vesicles revealed the preference of both peptides for liquid-disordered domains. Accordingly, we speculate that confinement into envelope fluid nanodomains might boost the distinct capacities of HIV MPER hydrophobic modules for inducing membrane defects during fusion.     

Infectious HIV-1 assembles in late endosomes in primary macrophages

Although human immunodeficiency virus type 1 (HIV-1) is generally thought to assemble at the plasma membrane of infected cells, virions have been observed in intracellular compartments in macrophages. Here, we investigated virus assembly in HIV-1-infected primary human monocyte-derived macrophages (MDM). Electron microscopy of cryosections showed virus particles, identified by their morphology and positive labeling with antibodies to the viral p17, p24, and envelope proteins, in intracellular vacuoles. Immunolabeling demonstrated that these compartments contained the late endosomal marker CD63, which was enriched on vesicles within these structures and incorporated into the envelope of budding virions. The virus-containing vacuoles were also labeled with antibodies against LAMP-1, CD81, and CD82, which were also incorporated into the viral envelope. To assess the cellular source of infectious viruses derived from MDM, virus-containing media from infected cells were precipitated with specific antibodies. Only antibodies against antigens found in late endosomes precipitated infectious virus, whereas antibodies against proteins located primarily on the cell surface did not. Our data indicate that most of the infectious HIV produced by primary macrophages is assembled on late endocytic membranes and acquires antigens characteristic of this compartment. This notion has significant implications for understanding the biology of HIV and its cell-cell transmission.     

P-glycoprotein-overexpressing multidrug-resistant cells are resistant to infection by enveloped viruses that enter via the plasma membrane.

The multidrug resistance gene product P-glycoprotein confers drug resistance to tumor cells by acting as a transporter that blocks the entry into the cell of a great variety of drugs and hydrophobic peptides. In this study we find that in drug-resistant cells, the insertion of the influenza virus fusion protein (hemagglutinin-2) into the plasma membrane is blocked and that the fusion of the viral envelope with the plasma membrane of these cells is impaired. Multidrug-resistant cells display significant resistance to infection by envelope viruses that invade cells by fusion with the plasma membrane, but not to infection by pH-dependent viruses that penetrate cells by fusion with endocytic vesicles. These observations suggest that multidrug resistance phenomena may protect cells from infection by a large group of disease-causing viruses that includes human immunodeficiency virus, herpes simplex virus, and some cancer-inducing retroviruses.     

A hunter virus that targets both infected cells and HIV free virions: Implications for therapy

The design of ‘hunter’ viruses aimed at destroying human immunodeficiency virus (HIV) infected cells is an active area of research that has produced promising results in vitro. Hunters are designed to target exposed viral envelope proteins in the membranes of infected cells, but there is evidence that the hunter may also target envelope proteins of free HIV, inducing virus-virus fusion. In order to predict the effects of this fusion on therapy outcomes and determine whether fusion ability is advantageous for hunter virus design, we have constructed a model to account for the possibility of hunter-HIV fusion. The study was based on a target cell-limited model of HIV infection and it examined the hunter therapeutic effect on recovering the HIV main target cells, the activated CD4⁺ T lymphocytes. These cells assist in setting up an immune response to opportunistic infections. The study analyzed the hunter dual mechanisms to control infection and because of diverse estimates for viral production and clearance of HIV, simulations were examined at rates spanning an order of magnitude. Results indicate that without hunter-HIV fusion ability, hunters that kill HIV-infected cells lead to a substantial recovery of healthy cell population at both low and high HIV turnover rates. When hunter-HIV fusion is included, cell recovery was particularly enhanced at lower HIV turnover rates. This study shows that the fusion ability, in addition to hunter infection ability, could be a favorable attribute for improving the efficacy of hunter-viral therapy. These results provide support for the potential use of engineered viruses to control HIV and other viral infections.     

Bilayer conformation of fusion peptide of influenza virus hemagglutinin: a molecular dynamics simulation study

Unraveling the conformation of membrane-bound viral fusion peptides is essential for understanding how those peptides destabilize the bilayer topology of lipids that is important for virus-cell membrane fusion. Here, molecular dynamics (MD) simulations were performed to investigate the conformation of the 20 amino acids long fusion peptide of influenza hemagglutinin of strain X31 bound to a dimyristoyl phosphatidylcholine (DMPC) bilayer. The simulations revealed that the peptide adopts a kinked conformation, in agreement with the NMR structures of a related peptide in detergent micelles. The peptide is located at the amphipathic interface between the headgroups and hydrocarbon chains of the lipid by an energetically favorable arrangement: The hydrophobic side chains of the peptides are embedded into the hydrophobic region and the hydrophilic side chains are in the headgroup region. The N-terminus of the peptide is localized close to the amphipathic interface. The molecular dynamics simulations also revealed that the peptide affects the surrounding bilayer structure. The average hydrophobic thickness of the lipid phase close to the N-terminus is reduced in comparison with the average hydrophobic thickness of a pure dimyristoyl phosphatidylcholine bilayer.     

Syncytium-inducing capacity of human immunodeficiency virus (HIV): analysis by the use of cloned viruses

The marked cytopathic effects of human immunodeficiency virus HIV for susceptible cells are caused mainly by fusion between cells expressing viral envelope glycoproteins and cells expressing CD4 molecule. In this study, we tested the ability of different clones of HIV to induce syncytia in CD4-positive cells. We have reported marked difference in syncytium-inducing capacity of 2 clones of human T lymphotropic virus type III (HTLV-IIIB) isolate despite no detectable difference in expression of viral glycoprotein (gp120). This difference in syncytium induction could be explained by the difference detected in their infectivity and binding activities to CD4-positive cells. Meanwhile we reported difference in syncytium-inducing capacity of 2 clones of lymphadenopathy associated virus (LAV1) isolate parallel to the different amounts of gp120 and other viral proteins expressed by these 2 clones. These results suggest that viral factors like infectivity and binding affinity of the virus to the susceptible cells and the amount of viral gp120 expressed by the infected cells may interact in a complex manner affecting fusion activity and syncytium induction in CD4-positive cells.     

The most highly amphiphilic alpha-helices include two amino acid segments in human immunodeficiency virus glycoprotein 41

A search for highly amphiphilic alpha-helices has been made in a data base of protein sequences, using the helical hydrophobic moment as a criterion of amphiphilicity. The protein segments of largest hydrophobic moment have been analyzed. For the segments whose structures are known, they are in fact alpha-helices. Two of the segments having very large hydrophobic moments are from the smaller C-terminal portion of the human immunodeficiency virus (HIV) envelope glycoprotein gp41. Also, among segments having large hydrophobic moments, but not among the most extreme, are lytic peptides such as melittin. Melittin seeks surfaces between polar and apolar phases, including the membrane-water interface. It is conceivable that the gp41 segments of extreme hydrophobic moment may participate in one of the membrane-related functions of the HIV virus.