Modification of the Cytoplasmic Domain of Influenza Virus Hemagglutinin Affects Enlargement of the Fusion Pore

Abstract

The fusion activity of chimeras of influenza virus hemagglutinin (HA) (from A/fpv/Rostock/34; subtype H7) with the transmembrane domain (TM) and/or cytoplasmic tail (CT) either from the nonviral, nonfusogenic T-cell surface protein CD4 or from the fusogenic Sendai virus F-protein was studied. Wild-type or chimeric HA was expressed in CV-1 cells by the transient T7-RNA-polymerase vaccinia virus expression system. Subsequently, the fusion activity of the expression products was monitored with red blood cells or ghosts as target cells. To assess the different steps of fusion, target cells were labeled with the fluorescent membrane label octadecyl rhodamine B-chloride (R18) (membrane fusion) and with the cytoplasmic fluorophores calcein (molecular weight [MW], 623; formation of small aqueous fusion pore) and tetramethylrhodamine-dextran (MW, 10,000; enlargement of fusion pore). All chimeric HA/F-proteins, as well as the chimera with the TM of CD4 and the CT of HA, were able to mediate the different steps of fusion very similarly to wild-type HA. Quite differently, chimeric proteins with the CT of CD4 were strongly impaired in mediating pore enlargement. However, membrane fusion and formation of small pores were similar to those of wild-type HA, indicating that the conformational change of the ectodomain and earlier fusion steps were not inhibited. Various properties of the CT which may affect pore enlargement are considered. We surmise that the hydrophobicity of the sequence adjacent to the transmembrane domain is important for pore dilation.The fusion of influenza viruses with target membranes is mediated by the spike membrane glycoprotein, HA. In the viral membrane, HA is organized as a homotrimer, each monomer consisting of two disulfide-linked subunits, HA 1 and HA 2 (14, 38). The HA-mediated fusion is triggered by acidic pH, which causes the HA ectodomain to be converted to a fusogenic conformation, thereby exposing the hydrophobic N terminus of the HA 2 subunit (37), the so-called fusion sequence. X-ray crystallographic studies of a fragment of the ectodomain suggest that the loop of the HA 2 subunit connecting two ␣-helices in the neutral form (39) becomes a part of an extended trimeric coiled coil at low pH (5,6,30,36). Recent reconstruction of the three-dimensional structure of the complete HA ectodomain at low pH by cryoelectron microscopy revealed that the preserved trimeric shape of the ectodomain may serve to guide the orientation of the coiled coil, thereby moving the fusion sequence to the tip of HA toward the target membrane (3). Destabilization of the target membrane by this sequence is then believed to initiate membrane fusion (11,19).Expression of HA in the plasma membrane of various cell lines preserves its fusion activity. In studies with these systems, it was revealed that the HA ectodomain and its low-pH-triggered conformational change is sufficient to induce membrane mixing (17,(21)(22)(23)(24). Indeed, the HA ectodomain membrane anchored by GPI mediates the merger between the outer layers of...

The Final Conformation of the Complete Ectodomain of the HA2 Subunit of Influenza Hemagglutinin Can by Itself Drive Low pH-dependent Fusion

“…Note that many of the curvature-generating proteins involved in membrane remodeling lack transmembrane domains (66). The finding that HA2* does not produce fusion pores large enough to allow passage of 10-kDa fluorescent dextran (Stokes radius of 2.4 nm) and does not produce syncytia substantiates the hypothesis that the transmembrane domain (and perhaps its interactions with the fusion peptide) as well as the cytoplasmic domain of HA can be especially important for later fusion stages including fusion pore expansion (26,57,67,68).…”

Acylation-Mediated Membrane Anchoring of Avian Influenza Virus Hemagglutinin Is Essential for Fusion Pore Formation and Virus Infectivity

“…This HA, which is anchored only in the outer leaflet of the membrane, proved capable of inducing hemifusion but failed to mediate complete fusion with the opening and dilation of a fusion pore (16). Other studies supported the concept that the transmembrane and the cytoplasmic portions of HA are crucial for the formation and enlargement of the fusion pore (2,18,26,35). From our results it is obvious that fatty acids attached to the cytoplasmic tail represent important structural determinants ruling the infectivity of influenza viruses by specifically promoting the transition from hemifusion to pore formation.…”

Peptide Mimics of the Vesicular Stomatitis Virus G-protein Transmembrane Segment Drive Membrane Fusion in Vitro

“…without added peptide, was reduced to an average of 60% upon outer leaflet bleaching. 3 This suggests that a substantial fraction of peptideindependent spontaneous liposome fusion does not proceed beyond hemifusion. We speculate, therefore, that the hemifusion diaphragm may form spontaneously from randomly colliding liposomes.…”

“…These viral fusion proteins consist of an ectodomain harboring an amphipathic fusion peptide, a single transmembrane segment (TMS), 1 and a cytoplasmic domain. All these domains appear to cooperate in fusion protein function (2,3). In the case of influenza hemagglutinin (HA), a pH-driven global conformational change of the ectodomain is thought to eject the fusion peptide toward the target bilayer to establish initial contact between both membranes (4).…”