Ruben M. Markosyan

IFITM Proteins Restrict Viral Membrane Hemifusion

The interferon-inducible transmembrane (IFITM) protein family represents a new class of cellular restriction factors that block early stages of viral replication; the underlying mechanism is currently not known. Here we provide evidence that IFITM proteins restrict membrane fusion induced by representatives of all three classes of viral membrane fusion proteins. IFITM1 profoundly suppressed syncytia formation and cell-cell fusion induced by almost all viral fusion proteins examined; IFITM2 and IFITM3 also strongly inhibited their fusion, with efficiency somewhat dependent on cell types. Furthermore, treatment of cells with IFN also markedly inhibited viral membrane fusion and entry. By using the Jaagsiekte sheep retrovirus envelope and influenza A virus hemagglutinin as models for study, we showed that IFITM-mediated restriction on membrane fusion is not at the steps of receptor- and/or low pH-mediated triggering; instead, the creation of hemifusion was essentially blocked by IFITMs. Chlorpromazine (CPZ), a chemical known to promote the transition from hemifusion to full fusion, was unable to rescue the IFITM-mediated restriction on fusion. In contrast, oleic acid (OA), a lipid analog that generates negative spontaneous curvature and thereby promotes hemifusion, virtually overcame the restriction. To explore the possible effect of IFITM proteins on membrane molecular order and fluidity, we performed fluorescence labeling with Laurdan, in conjunction with two-photon laser scanning and fluorescence-lifetime imaging microscopy (FLIM). We observed that the generalized polarizations (GPs) and fluorescence lifetimes of cell membranes expressing IFITM proteins were greatly enhanced, indicating higher molecularly ordered and less fluidized membranes. Collectively, our data demonstrated that IFITM proteins suppress viral membrane fusion before the creation of hemifusion, and suggested that they may do so by reducing membrane fluidity and conferring a positive spontaneous curvature in the outer leaflets of cell membranes. Our study provides novel insight into the understanding of how IFITM protein family restricts viral membrane fusion and infection.

Human Immunodeficiency Virus Type 1 Env with an Intersubunit Disulfide Bond Engages Coreceptors but Requires Bond Reduction after Engagement To Induce Fusion

ABSTRACT A mutant human immunodeficiency virus (HIV) envelope protein (Env) with an engineered disulfide bond between the gp120 and gp41 subunits (SOS-Env) was expressed on cell surfaces. With the disulfide bond intact, these cells did not fuse to target cells expressing CD4 and CCR5, but the fusion process did advance to an intermediate state: cleaving the disulfide bond with a reducing agent after but not before binding to target cells allowed fusion to occur. Through the use of an antibody directed against CCR5, it was found that at the intermediate stage, SOS-Env had associated with coreceptors. Reducing the disulfide bond after this intermediate had been reached resulted in hemifusion at low temperature and fusion at physiological temperature. The addition of C34 or N36, peptides that prevent six-helix bundle formation, at the hemifused state blocked the fusion that would have resulted after raising the temperature. Thus, Env has not yet folded into six-helix bundles after hemifusion has been achieved. Because SOS-Env binds CCR5, it is suggested that the conformational changes in wild-type Env that result from this binding cause disengagement of gp120 from gp41 in the region of the engineered bond. It is proposed that this disengagement is the event that directly frees gp41 to undergo the conformational changes that lead to fusion. The intermediate state achieved prior to reduction of the disulfide bond was stable. The capture of this configuration of Env could yield a suitable antigen for vaccine development, and it may also be a target for pharmacological intervention against HIV-1 entry.

Role of the Cytoplasmic Tail of Ecotropic Moloney Murine Leukemia Virus Env Protein in Fusion Pore Formation

ABSTRACT Fusion between cells expressing envelope protein (Env) of Moloney murine leukemia virus and target cells were studied by use of video fluorescence microscopy and electrical capacitance measurements. When the full-length 632-amino-acid residue Env was expressed, fusion did not occur at all for 3T3 cells as target and only somewhat for XC6 cells. Expression of Env 616*—a construct of Env with the last 16 amino acid residues (617 to 632; the R peptide) deleted from its C terminus to match the proteolytically cleaved Env produced during viral budding—resulted in high levels of fusion. Env 601*, lacking the entire cytoplasmic tail (CT) (identified by hydrophobicity), also led to fusion. Truncation of an additional six residues (Env 595*) abolished fusion. The kinetics of forming fusion pores did not depend on whether cells were first prebound at 4°C and the time until fusion measured after the temperature was raised to 37°C or whether cells were first brought into contact at 37°C and the time until fusion immediately measured. This similarity in kinetics indicates that binding is accomplished quickly compared to subsequent steps in fusion. The fusion pores formed by Env 601* and Env 616* had the same initial size and enlarged in similar manners. Thus, once the R peptide is removed, the CT is not needed for fusion and does not affect formed pores. However, residues 595 to 601 are required for fusion. It is suggested here that the ectodomain and membrane-spanning domain of Env are directly responsible for fusion and that the R peptide affects their configurations at some point during the fusion process, thereby indirectly controlling fusion.

Ternary Complex Formation of Human Immunodeficiency Virus Type 1 Env, CD4, and Chemokine Receptor Captured as an Intermediate of Membrane Fusion

ABSTRACT Human immunodeficiency virus (HIV) Env-induced fusion is highly temperature dependent. When effector and target cells were coincubated at 37°C, there was a kinetic delay before fusion commenced. When effector and target cells were coincubated for varied times at 23°C, a temperature that does not permit fusion, a temperature-arrested stage was created. Raising temperature to 37°C from the 23°C intermediate eliminated the kinetic delay. Inhibitors (T22, AMD3100, and Sch-C) that block fusion by binding chemokine receptors were added after creating the intermediate so as to assess the extent of engagement between gp120 and chemokine receptors at that stage. For both CXCR4 and CCR5 as coreceptors, increasingly long times of coincubation at 23°C reduced the efficacy of the coreceptor-binding inhibitors in blocking fusion. This implies that an increasing number of ternary Env/CD4/coreceptor complexes form over time at 23°C. It also shows that ternary complex formation has a lower temperature threshold than the downstream steps that include Env folding into a six-helix bundle; this provides an experimental means to separate coreceptor binding by gp120 from the subsequent refolding of gp41 into a six-helix bundle structure. As the time of cell coincubation at 23°C was prolonged, more cells quickly fused upon the raising of the temperature to 37°C, and the increase quantitatively correlated with the greater percentage of fusion that was resistant to drugs. Therefore the pronounced kinetic delay in HIV Env-induced fusion is caused predominantly by the time needed for ternary complexes to form.

Low pH Is Required for Avian Sarcoma and Leukosis Virus Env-Induced Hemifusion and Fusion Pore Formation but Not for Pore Growth

ABSTRACT Binding of avian sarcoma and leukosis virus (ASLV) to its cognate receptor on the cell surface causes conformational changes in its envelope protein (Env). It is currently debated whether low pH is required for ASLV infection. To elucidate the role of low pH, we studied the association between ASLV subgroup B (ASLV-B) and liposomes and fusion between effector cells expressing Env from ASLV-A and ASLV-B and target cells expressing cognate receptors. Neither EnvA nor EnvB promoted cell-cell fusion at neutral pH, but lowering the pH resulted in quick and extensive fusion. As expected for a low-pH-triggered reaction, fusion was a steep function of pH. Steps that required low pH were identified. Binding a soluble form of the receptor caused ASLV-B to hydrophobically associate with liposome membranes at neutral pH, indicating that low pH is not required for insertion of Envs fusion peptides into membranes. But both cell-cell hemifusion and fusion pore formation were pH dependent. It is proposed that fusion peptide insertion stabilizes the conformation of ASLV Env into a form that can be acted upon by low pH. At this point, but not before, low pH can induce fusion and is in fact required for fusion to occur. However, low pH is no longer necessary after formation of the initial fusion pore: pore enlargement does not require low pH.

Evolution of Intermediates of Influenza Virus Hemagglutinin-Mediated Fusion Revealed by Kinetic Measurements of Pore Formation

Cells expressing wild-type influenza virus hemagglutinin (HA) or HA with a point mutation within the transmembrane domain (G520L) were bound to red blood cells and exposed to low pH for short times at suboptimal temperatures followed by reneutralization. This produced intermediate states of fusion. The ability of intermediate states to proceed on to fusion when temperature was raised was compared kinetically. In general, for wild-type HA, fusion occurred more quickly by directly lowering pH at 37 degrees C in the bound state than by raising temperature at the intermediate stage. When pH was lowered for 1-2 min, kinetics of fusion upon raising temperature of an intermediate slowed the longer the intermediate was maintained at neutral pH. But for a more sustained (10 min) acidification, kinetics was independent of the time the intermediate was held at neutral pH before triggering fusion by raising temperature. In contrast, generating intermediates in the same way with G520L yielded kinetics of fusion that did not depend on the time intermediates were maintained after reneutralization. For both HA and G520L, the extents of fusion did not depend on the temperature at which pH was lowered, but fusion from the intermediate was extremely sensitive to the temperature to which the cells were raised. The measured kinetics and temperature dependencies suggest that the rate-limiting step of fusion occurs subsequent to formation of any of the intermediates; the conformational change of HA into its final configuration may be the rate-limiting step.

Tension of membranes expressing the hemagglutinin of influenza virus inhibits fusion.

The effects of membrane tension on fusion between cells expressing the hemagglutinin (HA) of influenza virus and red blood cells were studied by capacitance measurements. Inflation of an HA-expressing cell was achieved by applying a positive hydrostatic pressure to its interior through a patch-clamp pipette in the whole-cell configuration. Inflating cells to the maximum extent possible without lysis created a membrane tension and completely inhibited low-pH-induced fusion at room temperature. Fully inflated cells that were subsequently deflated to normal size resumed the ability to fuse in response to low pH. At the higher temperature of 32 degrees C, fusion conditions were sufficiently optimal that full inflation did not hinder fusion, and once formed, pores enlarged more rapidly than those of never inflated cells. It is suggested that under fusogenic conditions HA causes the formation of a dimple within the membrane in which it resides, and that membrane tension hinders fusion by preventing the formation of dimples. Because dimpling bends the bilayer portion of bound membranes so that they come into intimate contact, the damping of dimpling would suppress this initial step in the fusion process.

The Six-Helix Bundle of Human Immunodeficiency Virus Env Controls Pore Formation and Enlargement and Is Initiated at Residues Proximal to the Hairpin Turn

ABSTRACT Residues that create the grooves of the human immunodeficiency virus type 1 (HIV-1) Env triple-stranded coiled coil (HR1) and the residues that pack into the grooves (HR2) to complete the formation of the six-helix bundle (6HB) were mutated. The extent and kinetics of fusion as well as pore enlargement were measured for each mutant. Mutations near the hairpin turns of each monomer of the 6HB were more important than those far from the turn, for both HR1 and HR2. This result is consistent with the idea that binding of HR2 to the HR1 grooves is initiated near the hairpin turn of each monomer. Mutations at the distal portions also reduced fusion, albeit to a smaller extent. An intermediate of fusion (temperature-arrested state [TAS]) was formed, and the consequences of mutation were compared; a mutant that exhibited less fusion also showed slower kinetics from TAS. This suggests that formation of the bundle is a rate-limiting step downstream of the intermediate state. The rate of enlargement of a fusion pore also correlated with the extent and kinetics of fusion. The rate of pore enlargement was severely reduced by mutation. This supports our prior conclusion that formation of the 6HB occurs after pore creation and strongly suggests that the free energy released by bundle formation is directly used to promote pore growth.

The process of membrane fusion: Nipples, hemifusion, pores, and pore growth

Abstract In this chapter we discuss the current understanding of the process of membrane fusion and the contributions of proteins and lipids to this process. Many viral fusion proteins and proteins probably responsible for intracellular fusion fold into a bundle of coiled-coils. Formation of these coiled-coils is thought to be responsible for bringing membranes close together and may also cause the fusion event. Based on studies using the hemagglutinin (HA) of influenza virus, a prototypic fusion protein, the most likely sequence for fusion is as follows: Membranes make local contact by bending toward each other; the contacting lipid monolayers merge at these sites, yielding a state of hemifusion; with the subsequent merger of distal leaflets, fusion is completed. Many properties of lipids contribute to the fusion process. Important among these is spontaneous monolayer curvature. The contribution of spontaneous curvature to fusion is the best-understood aspect of the process in quantitative biophysical terms. Positive spontaneous curvature of contacting leaflets inhibits hemifusion, negative spontaneous curvature promotes it. Intermediate states that exhibit properties of hemifusion and can proceed on to full fusion have been captured and characterized. The transmembrane domain of HA greatly augments the transition from hemifusion to full fusion, but is not essential. The amino acid sequence of this domain is not critical, indicating that the conversion of hemifused to fused membranes occurs by a physical process. The growth of fusion pores is regulated by fusion proteins and affected by the lipid composition of the fused membranes.

Induction of Cell-Cell Fusion by Ebola Virus Glycoprotein: Low pH Is Not a Trigger

Ebola virus (EBOV) is a highly pathogenic filovirus that causes hemorrhagic fever in humans and animals. Currently, how EBOV fuses its envelope membrane within an endosomal membrane to cause infection is poorly understood. We successfully measure cell-cell fusion mediated by the EBOV fusion protein, GP, assayed by the transfer of both cytoplasmic and membrane dyes. A small molecule fusion inhibitor, a neutralizing antibody, as well as mutations in EBOV GP known to reduce viral infection, all greatly reduce fusion. By monitoring redistribution of small aqueous dyes between cells and by electrical capacitance measurements, we discovered that EBOV GP-mediated fusion pores do not readily enlarge—a marked difference from the behavior of other viral fusion proteins. EBOV GP must be cleaved by late endosome-resident cathepsins B or L in order to become fusion-competent. Cleavage of cell surface-expressed GP appears to occur in endosomes, as evidenced by the fusion block imposed by cathepsin inhibitors, agents that raise endosomal pH, or an inhibitor of anterograde trafficking. Treating effector cells with a recombinant soluble cathepsin B or thermolysin, which cleaves GP into an active form, increases the extent of fusion, suggesting that a fraction of surface-expressed GP is not cleaved. Whereas the rate of fusion is increased by a brief exposure to acidic pH, fusion does occur at neutral pH. Importantly, the extent of fusion is independent of external pH in experiments in which cathepsin activity is blocked and EBOV GP is cleaved by thermolysin. These results imply that low pH promotes fusion through the well-known pH-dependent activity of cathepsins; fusion induced by cleaved EBOV GP is a process that is fundamentally independent of pH. The cell-cell fusion system has revealed some previously unappreciated features of EBOV entry, which could not be readily elucidated in the context of endosomal entry.