Amino Acid Sequence Requirements of the Transmembrane and Cytoplasmic Domains of Influenza Virus Hemagglutinin for Viable Membrane Fusion

Abstract

The amino acid sequence requirements of the transmembrane (TM) domain and cytoplasmic tail (CT) of the hemagglutinin (HA) of influenza virus in membrane fusion have been investigated. Fusion properties of wild-type HA were compared with those of chimeras consisting of the ectodomain of HA and the TM domain and/or CT of polyimmunoglobulin receptor, a nonviral integral membrane protein. The presence of a CT was not required for fusion. But when a TM domain and CT were present, fusion activity was greater when they were derived from the same protein than derived from different proteins. In fact, the chimera with a TM domain of HA and truncated CT of polyimmunoglobulin receptor did not support full fusion, indicating that the two regions are not functionally independent. Despite the fact that there is wide latitude in the sequence of the TM domain that supports fusion, a point mutation of a semiconserved residue within the TM domain of HA inhibited fusion. The ability of a foreign TM domain to support fusion contradicts the hypothesis that a pore is composed solely of fusion proteins and supports the theory that the TM domain creates fusion pores after a stage of hemifusion has been achieved. INTRODUCTIONMembrane fusion is common to many cellular processes, such as exocytosis and intracellular trafficking. But only in the case of viruses have the proteins responsible for fusion been unambiguously identified. A number of common features have been found among viral fusion proteins. All are oligomerized. The monomer of each oligomer always contains a critical stretch of nonpolar amino acids, known as the fusion peptide (Hernandez et al., 1996;Durell et al., 1997). As crystallographically determined, fusion proteins from several different virus families have the same backbone structure of extended coiled-coil ␣-helices (Bullough et al., 1994;Chan et al., 1997). Because viral fusion proteins of unrelated viruses have common structural patterns, it is logical to assume that their mechanisms of fusion are similar as well. Furthermore, a complex of SNARES, proteins thought to be responsible for the constitutive fusion of intracellular trafficking and the regulated fusion that occurs in exocytosis (Sü dhof, 1995), has been crystallographically determined (Poirier et al., 1998;Sutton et al., 1998). It too displays a long coiled-coil region. Therefore it may well be that the mechanisms of viral fusion pertain more generally to cellular fusion. Influenza virus has long been the prototypic virus for study of fusion mechanisms. It ‡ Corresponding author. E-mail address: fcohen@rush.edu. Abbreviations used: CF, carboxyfluorescein; CPZ, chlorpromazine; CT, cytoplasmic tail; DMEM, Dulbecco's modified Eagle's medium; GPI, glycosylphosphatidylinositol; HA, hemagglutinin; PBS-A-S, PBS supplemented with 0.1% sodium azide and 5% calf serum; p.i., postinfection; pIgR, polyimmunoglobulin receptor; R18, octadecylrhodamine B; RBC, red blood cell; RD, tetramethylrhodamine-tagged dextran; TM, transmembrane; VSV, vesicular stomatitis virus; WT,...

Membrane Structures of the Hemifusion-Inducing Fusion Peptide Mutant G1S and theFusion-Blocking Mutant G1V of Influenza Virus HemagglutininSuggest a Mechanism for Pore Opening in MembraneFusion

“…Perhaps the fusion domain interacts with the TM domain of HA in a similar fashion. This scenario is supported by the fact that the TM domain of influenza virus HA promotes conversion of the hemifused to the fully fused state (3,30,37,38). The finding that G1S and a mutant with a shortened TM domain exhibited the same hemifusion phenotype (3) is consistent with an interaction of these two domains at a late step in membrane fusion.…”

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.…”

The Paramyxovirus Fusion Protein C-Terminal Region: Mutagenesis Indicates an Indivisible Protein Unit

“…Biochemical analyses of several viral fusion proteins have identified length requirements of individual regions of the C termini (2,3,16,49,55), as well as specific residues that affect activity (1, 4, 8, 18, 20, 26-28, 30, 38, 39, 42, 43, 50). Individual domain replacements have shown various phenotypes from none to significant effects on fusion activity (8,18,26,29,37,39,51). However, extensive studies of the individual contributions and the interrelationship of the various regions of the C termini, in the context of a functional fusogenic molecule, have not been described previously.…”

“…The TM domain of NDV F, when replaced with that of Sendai virus F, MeV F, or VSV G, results in lowered surface expression levels and reduced fusion activity of the chimeric proteins (18). On the other hand, the TM domain of influenza virus HA can be replaced with that of Sendai virus F, Rous sarcoma virus Env, or even the polyimmunoglobulin receptor, an unrelated protein, without losing fusion activity (26,37). Similarly, the TM domain of VSV G and HIV gp41 can be replaced with sequences from other membrane proteins (8,29,51).…”