Chapter 11 Fusion Activity of the Hemagglutinin of Influenza Virus

CURRENT TOPICS I N MEMBRANES AND TRANSPORT, VOLUME 32

Chapter I I

Fusion Activity of the Hemagglutinin of Influenza Virus MARY-JANE GETHING,**tJEAN HENNEBERRY," A N D JOE SAMBROOK* * Drpartnzent of Biochemistry and f Hmwrd Hughes Medical Institute University of Texas Soirthwestern Medical Center Drillas, Texas 75235

I.

11

t

111. IV. V.

v1. VI1. VIlI.

Introduction Influenza Virus-Mediated Fusion: Role of the Hemagglutinin Assays for the Fusion Activity of HA Expression of HA in Cultured Cells from Cloned HA cDNAs Genetic Approaches to Studies of HA-Mediated Membrane Fusion A. Studies on Variant Influenza Viruses That Induce Fusion at Elevated pH B. Site-Directed Mutagenesis of the Fusion Peptide of HA C. Possible Involvement of Other Regions of HA in the Process of Fusion Characterization of the Low pH-Induced Conformational Change in HA Studies on the Cleavage Activation of HA Conclusion References

1.

INTRODUCTION

The ability of influenza virus to change its antigenic properties presents a major obstacle to controlling the disease by herd immunity or vaccination, so that influenza continues to be one of the major unconquered pathogens of man (Kendal and Patriarca, 1986). The major antigen of the 337 Copyright 0 I988 by Academic Press. Inc. All rights of reproduction in any form reserved.

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virus particle is the hemagglutinin (HA) glycoprotein which, like the less abundant neuraminidase (NA), is inserted into the lipid membrane that envelops the virion (Laver and Kilbourne, 1966; Drzenick et al., 1966). Influenza viruses with the potential to cause new pandemics or epidemics in an immune population carry antigenically novel hemagglutinins (Laver and Webster, 1979). Apart from its importance to man as a mutable antigen, HA plays the major role in the penetration of the host cell by the virus (Lazarowitz and Choppin, 1975; Klenk et al., 1975). HA is responsible not only for the initial attachment of the virus to receptors on the surface of the cell (Hirst, 1941) but also for the fusion of viral and cellular membranes that marks the onset of infection (White et ul., 1981; Huang et al., 1981). Because of its importance in all these processes, much work has focused on elucidating the molecular details underlying the structure, function, and biological activities of HA. Initially this work depended almost exclusively on the techniques of classical protein chemistry (Laver, 1963, 1964, 1973; Laver and Webster, 1968; Brand and Skehel, 1972; Skehel and Waterfield, 1975; Waterfield et al., 1979, 1980; Ward and Dopheide, 1980; Ward, 1981) and on the study of temperature-sensitive mutants of HA (Ueda and Kilbourne, 1976; Scholtissek and Bowles, 1975; Klenk et al., 1981). Since the advent of recombinant DNA technology, the genes encoding the HAS of many strains of influenza have been cloned and sequenced (reviewed in Lamb, 1983). Knowledge of the amino acid sequence of the HA from the X-31 influenza strain, together with X-ray diffraction studies of crystals of the protein led to elucidation of the three-dimensional structure of the ectodomain of the HA molecule (Wilson et al., 1981). Monoclonal antibodies raised against influenza viruses have allowed a detailed analysis of the antigenic structure of the protein (Wiley et al., 1981), and from studies of the recognition of HA and other viral polypeptides by cytotoxic T lymphocytes is emerging a complex picture of the cellular immune response to influenza virus (reviewed by Braciale et al., 1986; Mills, 1986). Recently, assays have been developed to monitor the state of folding and oligomerization of nascent and mature forms of HA (Bachi et al., 1985; Gething et ul., 1986b; Copeland et ul., 1986). The culmination of work from these and many other sources has been a detailed description of the physical domains of the molecule, the locations of its antigenic sites, the points at which it is glycosylated, its organization into trimeric structures, and its orientation with respect to the membrane. HA is therefore the best characterized of all eukaryotic membrane proteins, providing a superb model system for studies of the mechanism of low pH-mediated membrane fusion.

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II. INFLUENZA VIRUS-MEDIATED FUSION: ROLE OF THE HEMAGGLUTIN IN

Influenza viruses, like many other enveloped viruses, enter and infect cells by a process involving binding to receptors on the cell surface, endocytosis of the virions, and low pHmediated fusion of the viral membrane with the membranes of acidic intracellular vesicles or endosomes (Matlin et al., 1981, 1982: Miller and Lenard, 1981: Marsh r t u l . , 1982; reviewed by White er d . , 1983). This pH-dependent fusion activity has been extensively studied in uitro using as targets cultured cells (White r t e l l . , 1980, 1981; Huang et cil., 1981), erythrocytes (Vaanfinen and Kfiariainen, 1980; Huang et [ I / . , 1981), and liposomes (White et d . , 1980, 1982a; Maeda er al., 1981). It has been shown that it is the viral surface glycoproteins that mediate the lipid bilayer fusion with specific pH dependences that are characteristic of each virus species and strain (White et a/., 1981, 1983). Early studies with influenza virus suggested that H A plays the key role in both the infectivity and the fusion activity of the virus (Lazarowitz and Choppin, 1975: Klenk et ( I / . , 1975: Maeda and Onishi, 1980; Huang et a/., 1981; Maeda c’t N / . . 1981). By expressing the protein in simian CV-1 cells infected with SV40-HA vectors, we have demonstrated that the HA molecule displays low pH-mediated fusion activity in the absence of any other influenza virus-encoded components (White rf al., 1982b). The HA molecule in its neutral form is a trimer that projects from the viral envelope as a rod-shaped structure 135 A in length (Wilson et [ I / . , 1981). To be active in fusion. the HA precursor, which is synthesized as a single polypeptide chain, must be processed by a posttranslational proteolytic cleavage into disulfide-bonded HA I and HA2 subunits (Laver, 1971: Klenk et a / . , 1975; Lazarowitz and Choppin, 1975; White et a / . , 1981, 1982b). A new hydrophobic amino terminus. the fusion peptide, is generated on the HA2 subunit. This peptide is highly conserved in HAS from different virus strains (reviewed by Lamb, 1983) and in many studies has been implicated as being intimately involved in the fusion process (Gething ef a/., 1978, 1986a; Richardson e f ul., 1980; Garten er al., 1981: White et a/., 1982b). In the native structure of HA, the hydrophobic fusion peptide in each monomer is tucked into the interface between the subunits of the trimer, approximately 30 A from the site of insertion of the polypeptide chain into the lipid bilayer of the virus envelope or the plasma membrane (Wilson et al., 1981). It has been proposed (Skehel et ul., 1982; White er al., 1982a; Daniels er al., 1983, 1985; Doms et al., 1985) that protonation of one or more amino acid side chains results in partial disso-

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MARY-JANE GETHING ET AL.

ciation of the HA trimer and exposure of the fusion peptide which then inserts into the lipid bilayer. HA would then become an integral component of both the viral and target membranes, presumably bringing them close enough together to fuse. 111.

ASSAYS

FOR THE FUSION ACTIVITY OF HA

The fusion activity of HA can be manifested experimentally as cell-cell fusion (i.e., polykaryon formation) when monolayers of cells displaying cleavage-activated HA on their plasma membranes are transiently exposed to low pH. This activity can be measured following infection of cultured cells with influenza viruses (Maeda and Ohnishi, 1980; Huang et al., 1981; White et al., 1981) or following expression of recombinant HA from eukaryotic vector systems (see Figs. I and 3, below, and White et al., 1982b; Sambrook et al., 1985; Gething et al., 1986a). Such experiments have shown that HA-induced cell-cell fusion displays a characteristic pH profile, with the threshold pH varying between 5 and 6 depending on the viral origin of the HA molecule (Huang et al., 1981; White et al., 1983). In a second manifestation of the fusion activity of HA, molecules such as enzymes, antibodies, or oligonucleotides can be loaded into erythrocytes and delivered into cells that express cell surface HA by a process that involves binding of the loaded red cells via the hemagglutinating activity of HA, followed by HA-mediated fusion of the red cell and host cell membranes. Fusion can be measured quantitatively by delivery of horseradish peroxidase (HRP)followed by staining of the cell cytoplasm with diaminobenzidine (Figs. 1 and 3, below; Doxsey et al., 1985; Sambrook et al., 1985; Gething et al., 1986a). Finally, it is often desirable to follow the low pH-induced conformational change in HA that is necessary for initiation of the fusion reaction. This conformational change, which in wild-type HA closely parallels that of the fusion activity, has been monitored by following the acquisition of protease sensitivity by an ectodomain fragment of HA (BHA), released from the membrane by treatment with bromelain (Brand and Skehel, 1972), or by assessing the ability of this fragment to bind to lipid vesicles or detergents or to aggregate in lipid- or detergent-free solutions (Skehel et al., 1982; Doms et al.. 1985). As noted above, to undergo the low pH-induced confoFmational change or to be active in fusion, the HA precursor must have been cleaved into HA1 and HA2 subunits. During influenza virus infections, this cleavage is

11. HEMAGGLUTININ OF INFLUENZA VIRUS

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performed by an endogenous protease in the epithelial cells of the respiratory tract. However, the HAS from human virus strains are not cleaved when expressed in the great majority of cultured cell lines. Thus, to perform fusion experiments it is necessary to effect an exogenous proteolytic cleavage of the cell surface HA molecules. This can be readily achieved by treating intact monolayers of HA-expressing cells with low levels of trypsin (White et al., 1982b; see Fig. 2). IV.

EXPRESSION OF HA IN CULTURED CELLS FROM CLONED HA cDNAs

Key to our studies on HA-mediated fusion has been the ability to express wild-type and mutant forms of HA from a variety of eukaryotic expression vectors. We have developed a range of vector systems to express cDNAs encoding the HAS from the A/Japan/305/57 (H2 subtype) and A/Aichi/68 (H3 subtype) influenza virus strains. These vectors allow control of expression levels in a variety of cell types as well as choice between short-term, high level production in transient or lytic viral systems and constitutive expression in continuous cell lines. The expression systems that we have used most frequently for production and analysis of wild-type and mutant HAS are based on the doublestranded DNA virus, SV40. The characteristics of these transient or lytic viral vectors, from which HA cDNA is expressed in simian cells under the control of either the SV40 early or late promoters, have been reviewed in detail previously (Gething and Sambrook, 1981, 1983). Levels of expression of approximately lo8 molecules/cell/24 hr can routinely be achieved following infection of CV-I cells with high titer SV40-HA recombinant virus stocks. Figures I and 2 illustrate the techniques that are available to characterize the synthesis, cellular location, and functional activities of the wild-type Japan HA protein expressed in CV-1 cells. The protein, which can be shown using indirect immunofluorescence to be located at the cell surface as well as in the endoplasmic reticulum and Golgi regions of the cell, is active in hemagglutination, cell-cell fusion and red blood cell-mediated delivery. Although the high levels of HA expression obtained using the lytic SV40 viral vectors are very useful for the initial characterization of HA mutants, the ability to generate cell lines that continuously express wildtype or mutant forms of HA provides significant advantages for longer term studies and for experiments designed to investigate the function of

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MARYJANE GETHING ET AL.

FIG.1 . Analysis of HA expressed from SV40-HA recombinant vectors. Forty-eight hours after infection of CV-I cells with the SVEHA3 recombinant vector (Gething and Sambrook, 198I), the following assays were performed: immunofluorescence on permeabilized cells (A), hemagglutination (B), cell-cell fusion (C), and erythrocyte-cell fusion measured by delivery of HRP and staining with diaminobenzidine (D). The details of the experimental protocols have been described previously (Gething and Sambrook 1981, 1982, 1986a; Doyle et a/., 1985).

cellular components involved in the transport and maturation of HA. We have therefore constructed vectors based on bovine papilloma virus (BPV) and used them to develop lines of cells that constitutively express HA at levels suitable for biochemical, immunological, and functional analysis. In initial experiments, BPV-transformed cell lines expressing HA were identified by the labor-intensive method of screening

343

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large numbers of individual clones by radioimmune assay (Sambrook et d.,1985). This was feasible for cell lines such as C127 and NlH-3T3 that

were efficiently transfected by the BPV-HA vector but did not prove successful with other cell types. However, use of a fluorescence-activated cell sorter to identify cells that bind FITC-conjugated anti-HA antibodies (or hemagglutinate FITC-labeled red blood cells) has facilitated the rapid

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MARY-JANE GETHING ET AL.

selection of rare HA-expressing cells and has also allowed us to clone out those cells that display the greatest amounts of HA at the cell surface. The Japan or Aichi (X-31) HA genes have now been introduced into cell lines derived from a number of different species including mouse (C127, NIH-3T3, and MME cells), dog (MDCK cells), pig (PK cells), and hamster (CHO cells). The level of expression of HA varies between cell types: up to 10’ molecules of HA per cell can be obtained in C127, NIH-3T3, or CHO cell lines, while somewhat lower levels (104-106 molecules per cell) are obtained in the other, more differentiated cell types. Obviously, the development of an increasing variety of HA-transformed cell lines of different types and species coupled with the ability to deliver efficiently into the cytoplasm probes such as antibodies or oligonucleotides provides great opportunities for studies of intracellular events and architecture in living cells. Figures 2 and 3 characterize the synthesis, cellular location, and functional activities of the wild-type Japan HA protein expressed in a continuous line of NIH-3T3 cells transformed with the BPV-HA vector, pBVIMTHA (Sambrook et al., 1985). Comparison of HAS produced from SV40-HA and BPV-HA vectors in CV-1 cells and NIH-3T3 cells, respectively (Fig. 21, indicates that the nonglycosylated forms of HA synthesized in each cell type in the presence of the drug tunicamycin are identical in size, as are the core-glycosylated forms of the proteins synthesized during a 15-min pulse. However, variation between these cell types in the trimming and modification of the oligosaccharide side chains results in differences in the terminal glycosylation patterns of the HA molecules, and thus in differences in their mobilities on SDS-polyacrylamide gels. Figure 2 also illustrates the quantitative cleavage by exogenous trypsin of terminally glycosylated precursor HA0 to HA1 and HA2 subunits. Figure 3 indicates the cell surface location of the HA synthesized in NIH-3T3 cells and demonstrates that the protein is active in hemagglutination, cellcell fusion, and red blood cell-mediated delivery assays. The availability of cell lines that express HA at different levels has allowed us to estimate the level of expression required on a per cell basis for cell-cell fusion to occur. Table I compares, for six different transformed NIH-3T3 cell lines, the number of HA molecules per cell and the cell-cell fusion and hemagglutination activities. The results indicate that a minimum expression level of approximately 3 x 106molecules per cell is necessary for the manifestation of fusion activity. A similar result was obtained when red blood cell-mediated delivery experiments were performed (results not shown).

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FIG.3. Analysis of HA expressed from BPV-HA recombinant vectors. A continuous NIH-3T3 cell line transformed with the pBVI-MTHA vector (Sambrook ef n l . , 1985) was characterized using the following assays: immunofluorescence on nonpermeabilized cells (A), hemagglutination (B), cell-cell fusion (C), and erythrocyte-cell fusion measured by delivery of HRP and staining with diaminobenzidine (D). The details of the experimental protocols have been described previously (Gething and Sambrook, 1981. 1982: Sambrook et al., 1985; Doxsey et al., 1985).

V.

GENETIC APPROACHES TO STUDIES OF HA-MEDIATED MEMBRANE FUSION

Two genetic approaches have been employed to analyze the mechanism of the fusion reaction mediated by HA. The first involves studies of variant influenza viruses that induce fusion with raised pH thresholds. Sequence analyses of HAs from the variant viruses have identified altered

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MARY-JANE GETHING ET AL.

TABLE 1 CORRELATION OF THE LEVELOF EXPRESSION OF HA IN MURINE BVi-MTHA CELLLINESWITH THE CAPACITY TO UNDERGO LOW PH-MEDIATED CELL-CELLFUSION Cell line

TRI-I TRI-4 TR 1-5 NTRI-4 NTRI-5 NTRI-I I

HA rnolecules/cell" 6 I 1 3 I 8

x x x x x

lo0 I06 106

I@

106

x 106

Fusion activityh

+++

+ + +++ -

+++

Hemagglutination' ++t ++t

+++ +++ +++ +++

" Cell extacts were prepared from a known number of cells and assayed by solid-phase radioimmunoassay (Cething and Sambrook. 1981), Ir Fusion activity was measured by polykaryon formation following treatment of cell monolayers with trypsin and neurdminidase (Sambrook P I a / . . 1985) and repeated ( 2 x ) brief exposure to buffer at pH 5.0 (PBS containing 10 mM HEPES). Symbols: -, no polykaryons observed; +. small numbers of polykaryons containing 4-5 nuclei: + + +. massive formation of polykaryons containing large numbers of nuclei (see. for example. Fig. 3 0 . Hemagglutination assays were performed after treatment of cell monolayers with trypsin and neuraminidase (Sambrook el d.,1985) using a IW solution of guinea pig erythrocytes. ++ +. Erythrocytes bound to SO-IOO%, of cells in the monolayer (see, for example. Fig. 3 B ) . 1

amino acids that play a role in the pH dependence of fusion. The second approach ulitizes oligonucleotide-directed, site-specific mutagenesis of a cloned HA gene to alter the nucleotide sequence encoding selected amino acids in the HA molecule. Expression of the mutant genes in simian cells has confirmed the central role of the fusion peptide and provided insights into the mechanism of the fusion reaction. A. Studies on Variant Influenza Vlruses That Induce Fusion at Elevated pH

To gain insight into the molecular mechanism underlying the pH dependence of HA-mediated fusion, influenza virus variants have been isolated that induce fusion at threshold pH values higher than those of their parent viruses. Rott et al. (1984) showed that variants of the X-31 strain, selected for their ability to undergo activation cleavage and grow in MDCK cells, also displayed an elevated threshold pH for fusion that was approximately 0.7 pH units higher than the wild type. Variants of the X-31 and Weybridge virus strains have been selected for by growth in the presence of amantadine, a compound that raises endosomal pH (Daniels et af., 1985). Variant viruses were obtained that mediated fusion at pH values 0.1-0.7

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347

unit higher than the parental strain. Finally, we have isolated and analyzed a naturally occurring variant ofthe X-31 strain whose pH threshold for fusion was elevated by 0.2-0.3 units (Doms P t al., 1985). Sequence analysis of the HAS from these variant viruses has identified individual amino acids that appear to play a role in the low pHmediated conformational change in the molecule. Some of these residues are located along the interface between the subunits of the trimer. while others stabilize the unexposed location of the fusion peptide at the amino terminus of HA2 (see Fig. 4A). It has been proposed that these amino acids participate in interactions that stabilize the structure of the wild-type molecule at neu1983. Substitution of these residues would lower tral pH (Daniels et d., the energy barrier necessary for the low pH-induced conformational transition to the fusion-active state. All the results are consistent with the widely held theory that dissociation of the HA trimer at low pH is a necessary and early step in the fusion mechanism. 6. Site-Directed Mutagenesis of the Fusion Peptide of HA

Although the majority of the amino acids that were altered in the fusion variants were located along the trimer interface (Fig. 4A), three altered residues were located within the hydrophobic fusion peptide at the amino terminus of the HA2 subunit. The fact that these three residues have undergone conservative substitutions probably reflects the fact that mutant viruses unable to mediate the fusion reaction at any pH could not enter and infect cells and therefore could not be propagated. To obtain mutant HAS that might be inactive or disabled for fusion and to probe the consequences of altering the length and hydrophobicity of the fusion peptide. we have used oligonucleotide-directed mutagenesis of the Japan HA cDNA to introduce single base changes into the sequence that encodes this peptide (Gething ef ul., 1986a). Three mutants were constructed that introduce single, nonconservative amino acid changes in the fusion peptide (Fig. 4B). When the mutants were assayed for fusion activities and for the low pHmediated conformational change and acquisition of lipid binding capacity (Table 11), three fusion phenotypes were observed: 1 . Substitution of glutamic acid for the glycine residue at the amino terminus of HA2 abolished all fusion activity although the mutant HA could still undergo a conformational change (at lower pH than the wild type) that resulted in protease sensitivity and lipid binding capability. Study of this mutant has provided the first indication that the conformational change can be temporally separated from lipid binding, and also

FIG.4. Schematic diagrams of the ectodomain of the HA monomer showing the location of amino acids that have been altered in the fusion variants (A) or the site-directed mutants (B). The schematic diagram of the three-dimensional structure of the HA monomer is taken from Wilson et a / . (1981). The data summarized in A are drawn from Rott er al. (1984),

348

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Daniels et a / . (1989, and Doms et u / . (1986), while that in B is from Gething ct u / . (1986a). In A the residues marked with an asterisk are from the HA1 subunit; all other residues are from

the HA2 subunit.

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MARY-JANE GETHING ET AL.

TABLE I1 FUSION PHENOTYPES OF T H E WILD-TYPE A N D MUTANT HA

SUMMARY OF THE ~~

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Efficiency at pH 5 of cell-cell fusion (%)

90 0 50

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pH at which 50% of BHA is converted to protease sensitivity and lipid binding

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This table is adapted from and summarizes results described in Cething

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allows distinction of the stages of lipid binding and bilayer fusion, indicating that HA does more than simply bring the two membranes close together. 2. Substitution of glutamic acid for the glycine residue at position 4 in HA2, which decreased the length of the apolar stretch to 6 amino acids, raised the threshold pH both for the conformational change and for fusion and also reduced the efficiency of fusion. It appears that the mutation has destabilized the neutral conformation of the HA trimer in a similar fashion to the amino acid alterations identified in the variant viruses described above. The results suggest that the amino acid at position 4 in HA2 may play two roles: a structural role in maintaining the fusion peptide in its neutral conformation and another role in the stage of bilayer destabilization. 3 . Extension of the hydrophobic stretch by replacement of the glutamic acid at position 11 with glycine yielded a mutant protein that underwent the conformational change and induced fusion of erythrocytes with cells with the same efficiency and pH profile as the wild-type protein. However, the ability of this mutant to induce polykaryon formation was greatly impaired. This phenotype provides the evidence for a distinction between cell-cell fusion and erythrocyte-cell fusion. The mutant HA is competent to mediate fusion of the cell and erythrocyte membranes over the small area necessary for injection of HRP into the cytoplasm, but, except at very low pH, the mutant HA is unable to induce bilayer destabilization over areas sufficient to cause polykaryon formation. Analysis of these mutants has allowed us to delineate several stages of the mechanism of HA-mediated membrane fusion which were not separated in previous studies. These include (1) the low pH-induced conformational change in HA that exposes the fusion peptide; (2) the interaction

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of the fusion peptide and/or other regions of the HA molecule with the target lipid bilayer; ( 3 ) destabilization of the lipid bilayer and membrane coalescence over small areas; and (4) membrane coalescence and separation over large areas, resulting in polykaryon formation. The amino terminus of the HA2 subunit is the most highly conserved region of HA (Gething P I a / . . 1980; Kawaoka rt a / . , 19841, indicating that this region of the molecule must be of great importance for the structure and function of HA and the life cycle of the virus. Nevertheless, although the HAS of field strains never show amino acid changes in the fusion peptide, it is possible, either by selection of variant viruses by growth in amantadine or by in vitro mutagenesis, to introduce alterations into this peptide that do not inhibit the assembly, intracellular transport, or fusion activity of this protein. Presumably variant viruses carrying these altered HA molecules would be at a disadvantage in a competition with field strains, either because occurrence of the conformational change or the fusion reaction at a higher pH is not desirable or because the mutant HA proteins are more unstable or thermolabile. Whatever their defects would be in the real world, HA mutants generated in uitro provide the opportunity to study the role of this hydrophobic peptide in the fusion reaction. C. Possible Involvement of Other Regions of HA in the Process of Fusion

Although these studies have confirmed a function for the amino terminus of HA2 in the fusion mechanism, they have not ruled out a role for other regions of the molecule in the interaction with the target lipid bilayer. Very promising candidates for other structures that may be involved in fusion are the two amphipathic helices of the stalk domain, which are made up of sequences from the HA2 subunit (Wilson el ul., 1981). In the neutral trimer, hydrophobic amino acids which lie along one face of the top half of the long helix form extensive contacts between the individual subunits. Following the low pH-induced conformational change when the subunits ofthe trimer separate, the helices will no longer interact with each other and may well be available to interact instead with the lipid bilayer. The short helix is also amphipathic. Physiologically important interactions of amphipathic surfaces (Q helices or p sheets) with membranes have been described (Kaiser and Kezdy, 1984) with the amphilicity of the structure being more conserved than specific amino acid sequence. Design and analysis of mutations in this region of HA will be quite complex, since it is clear that the long helix in HA2 is intimately involved in the folding and final structure of the trimeric molecule (Wilson et ul.,

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1981). It will be necessary to design mutations that do not affect the folding and transport of the protein but that might interfere with the interaction with the target bilayer following the low pH-induced conformational change. Candidates for oligonucleotide-directed mutagenesis would include residues with aliphatic or aromatic side chains on the hydrophobic face of the helix, but which do not appear to be so intimately involved in the interactions that connect the subunits in the trimer. Conservative substitutions would test the role of individual amino acids in the fusion mechanism, while nonconservative substitutions would test the importance of amphilicity rather than specific sequence. Alteration of the pH threshold for fusion would indicate that the substituted amino acid plays a structural role in the conformational change, while alteration of the efficiency of fusion would suggest that the amphipathic helices might indeed interact with the target lipid bilayer during the fusion reaction. VI.

CHARACTERIZATION OF THE LOW-pH INDUCED CONFORMATIONAL CHANGE IN HA

A current view of the low pH-induced conformational change in HA is that protonation of one or more amino acid side chains in the subunit interface causes the dissociation of the HA trimer. Because the quaternary interactions between the three globular domains are few in number in the neutral conformation of the trimer (Wilson et al., 1981), dissociation of the subunits may simply occur by separation of the globular domains at the top of the molecule. This would then be followed by breaking of the interactions between the amphipathic helices in the stalk which leads to exposure of the previously buried fusion peptide. Recent experiments, however, have suggested that a more complicated series of conformational changes may occur. White and Wilson (1987) have used antipeptide antibodies, directed against individual sequences located near the fusion peptide and along the subunit interface in both the globular and stalk domains, to probe the temporal sequence of separate conformational alterations that take place following treatment at low pH. Surprisingly, their results indicated that the conformational change begins with exposure of a loop (HA1 residues 14-52) which in the neutral structure is embedded in the trimer interface, followed by exposure of the fusion peptide and then the carboxy terminus of HAl. The separation of the subunits, and in particular of the globular domains, appears to be a relatively late event in the process. Thus, although these results must be interpreted with the caveat that the experiments were carried out using HA solubilized in

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detergent, it appears that conformational changes around the stem of the molecule may precede dissociation of the subunits. Neither scenario is contradicted by the data from the fusion variant studies which identified amino acids important in stabilizing the neutral positions of both the HA1 carboxy terminus and the fusion peptide, as well as the intersubunit connections, but could not define any order of events in the conformational change. Whichever model is the more correct, we still need to understand better the events that lead to disruption and reestablishment of the lipid bilayer. It is to be hoped that elucidation of the role of individual amino acids of HA in the fusion reaction might lead to a physiochemical model for the rearrangements that occur within the fusing membranes.

VII. STUDIES ON THE CLEAVAGE ACTIVATION OF HA

As described above, the fusion activity of HA and thus the infectivity of influenza virus requires that the HA0 precursor be processed by a posttranslational proteolytic cleavage to an active form of the molecule. The HAS from human type A influenza viruses contain a single arginine residue at the processing site, and activation involves cleavage on the carboxyl side of this amino acid by a trypsinlike protease, followed by remove1 of the arginine by a carboxypeptidase activity (reviewed by Rott and Klenk, 1986). Cellular proteases that carry out these cleavages are expressed only in primary epithelial cells, a situation which normally restricts viral replication to the respiratory epithelium during influenza virus infections of humans. Virulent avian influenza viruses encode HAS that have several arginine and lysine residues at the processing site, so that the HA is cleaved in most cell types (Rott and Klenk, 1986), leading to systemic infections. The pathogenicity of these viruses tends to correlate with the numbers of basic amino acids at the cleavage site of HA, although other structural features of HA such as the positioning of carbohydrate side chains can modulate the virulence of the infection (Kawaoka et al.. 1984). In addition, pathogenicity has proven to be a multigenic trait (Schulman, 1983). In cells in culture, HAS derived from human viruses are not cleaved so that it is the precursor HA molecule that is displayed at the cell surface; treatment with exogenously added trypsin is required to activate the protein for fusion. HAS derived from pathogenic avian viruses are activated by cellular proteases in cultured cells; it is as yet uncertain whether cleavage takes place before or after arrival of the protein at the cell surface.

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To study the structural requirements for activation cleavage of HA we have used oligonucleotide-directedmutagenesis to alter the amino acid sequence at the processing site of X-31 HA. A single nucleotide change, AGA- ACA, wasintroduced togenerate the cleavage-minus mutant, C-T, in which the arginine residue at the cleavage site is substituted by a threonine residue. Loop-in mutagenesis of 3 or 15 nucleotides was performed to generate the other two mutants. The first, C+RR, contains an additional arginine residue at the cleavage site. The second, C+RRKKR, contains two extra arginines and two extra lysine residues at the cleavage site (reproducing the cleavage site in the HA from the A/Turkey/Ontario/ 66 strain of influenza virus). Figure 5 shows the results of DNA sequence analysis to confirm the nucleotide sequences encoding the region around the cleavage sites in the wild-type and mutant HA cDNAs. The nucleic acid and corresponding amino acid sequences of the relevant segment of the wild-type and mutant proteins are shown in Fig. 5B.Following confirmation of the desired mutant sequences, the mutated X-31 cDNAs were used to replace the corresponding wild-type fragments in the SVEXHA recombinant viral genome (Doyle ef d.,1986).The recombinant genomes were transfected into CV-I cells, and high titer virus stocks were developed (Doyle et al., 1985) and used to infect fresh monolayers of CV-I cells for analysis of the biosynthesis, intracellular transport, and functional activities of the wild-type and mutant HA proteins. An analysis of the transport and proteolytic cleavage of these proteins is shown in Fig. 6. Fifteen minutes after synthesis, the wild-type HA is present predominately as the core-glycosylated HA0 species which appears as a sharper, faster migrating band. Following a 2-hr chase period, most of the protein has been converted to the slightly slower migrating, terminally glycosylated form of the precursor and has been transported to the cell surface where it is available for cleavage by exogenously added trypsin. The C-T protein is also quantitatively converted to the terminally glycosylated form during the 2-hr chase period. However, although this protein can be demonstrated to be at the cell surface by immunofluorescence and hemagglutination assays (results not shown), the mutant protein cannot be cleaved by exogenously added trypsin. The C+RR mutant is indistinguishable from the wild-type protein in this experiment, demonstrating that the presence of an additional arginine residue at the cleavage site does not result in endogenous cleavage of HA in CV-I cells. Finally, the C+RRKKR mutant is cleaved into HA1 and HA2 subunits in the absence of any exogenous protease. The addition of trypsin to the medium above the cells does not result in any further cleavage of the remaining (core-glycosylated) precursor protein. This result demonstrates that the presence of multiple arginine and lysine residues (five total) at the

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cleavage site facilitates the cleavage of the HA precursor by a protease endogenous to CV-I cells. The data shown in Fig. 6 do not provide evidence that this mutant protein reaches the cell surface; however, immunofluorescence and erythrocyte binding experiments reveal the presence of this protein at the plasma membrane. Each mutant protein was then tested for a requirement for in uirro proteolytic activation for mediation of low pH-induced cell-cell fusion. Figure 7 shows that (as previously reported; Gething c't a / . , 1986a) cells expressing the wild-type HA protein can be fused by treatment at low pH only after the HA0 precursor at the cell surface has been treated with exogenous trypsin, thereby cleaving the molecule into disulfide-bonded HA1 and HA2 subunits. However, the C T mutant cannot mediate low pH-induced fusion even after treatment with trypsin, presumably because alteration of the cleavage site prevents cleavage activation of the molecule. The phenotype of the C'RR mutant is identical to that of the wildtype protein, requiring pretreatment with trypsin to activate the molecule for fusion. The C+RRKKR mutant is active in low pH-mediated cell fusion without prior treatment with exogenous trypsin because the HA molecules at the cell surface have already been activated for fusion via cleavage by an endogenous protease. In summary, a mutant in which the single arginine residue at the cleavage site was altered to threonine was not cleaved under any circumstances, even after treatment with exogenous protease. This cleavageminus mutant was transported normally to the cell surface and displayed hemagglutinating activity although it could not mediate low pH-induced fusion. The second mutant contained two arginine residues at the processing site; this protein was indistinguishable from the wild-type HA in its transport and biological activities and was cleaved only after treatment with external trypsin. The final mutant reproduced the processing site of the HA from a pathogenic avian virus, i.e., . . . Arg.Arg.Lys.Lys.Arg. . . . This mutant HA was cleaved by a cellular protease in CV- I cells and mediated low pH-induced fusion without prior treatment with trypsin. Further experiments are planned to probe the precise structural requirements for cleavage activation. In addition to testing whether endogenous processing occurs when the site contains three or four basic amino acids, it will be of interest to probe any differences betwen arginine and lysine as protease substrates and to study the role of the conserved nonpolar residues that both precede and follow the processing site. The processing site mutants that are already available will be useful for a number of purposes. First, the cleavage-minus mutant will provide an opportunity to produce precursor HA0 for crystallographic studies. A significant, although possibly localized, conformational change must take

WT

C'T

WT

C+RR

C+RRKKR

FIG. 5. Nucleic acid and corresponding amino acid sequences around the cleavage activation sites of wild-type and mutant HA molecules. The Clal-BarnHI restriction fragment that encompasses the entire coding sequence of X-31 HA was cloned into an M13 mp18 vector. Single-stranded phage DNA was used as the template for oligonucleotide-directed mutagenesis to generate mutants that encode altered sequences at the activation cleavage site of HA. The autoradiographs (A) show the results of DNA sequence analysis to confirm the nucleotide sequences encoding the region around the cleavage sites in the wild-type and mutant HA cDNAs. The arrowheads and dashed lines show the positions and extent of nucleotide changes or insertions. The amino acids corresponding to each codon are shown in single letter code: R, Arg; K, Lys. (B) The nucleic acid and corresponding amino acid sequences of the relevant segment of the wild-type and mutant proteins. The vertical arrows show potential cleavage sites.

357

11. HEMAGGLUTININ OF INFLUENZA VIRUS

B

1HA2

XHA-WT

..Asn.Val.Pro.Glu.Lys.Gln.Thr Arg.Gly.Leu.Phe.Gly. AAT GTA CCA GAG AAA CAA ACT AGA GGC CTA TTC GGC

XHA-C-T

..Asn.Val.Pro.Glu.Lys.Gln.Thr

XHA-C'RR

,

XHA-C'RRKKR

. . A m .Val .Pro.Glu.Lys.Gln.Thr.Arg.Arg. 1 1Lys. Lys.Arg.Gly.Leu.Phe.Gly.. I IHA2

ihr.Gly.Leu.Phe.Gly. ACA

1

.Asn.Val .Pro.Glu. Lys.Gln.Thr.Arg.Arg. /HA* .Gly.Leu.Phe.Gly.. AGG AGA

I

AGG AGA AAG AAG AGA

FIG.5 .

(Con~in~ed)

place on cleavage activation since the carboxy terminus of the HA1 chain and the amino terminus of the HA2 chain, which are separated by only one amino acid in the precursor polypeptide, end up separated by a gap corresponding to approximately 10 residues in the cleaved molecule (Wilson PI a/.. 1981). The only satisfactory way to analyze this conformational change is by comparison of the known three-dimensional structure of the cleaved molecule with that of the precursor form. Uncleaved HA produced in tissue culture systems has not been a suitable source of precursor because it undergoes activation cleavage during harvesting from the cell surface with bromelain. Furthermore, the anchor-minus precursor HA that is secreted in large amounts from CV-1 cells that have been infected with the SVEHA20-A- recombinant virus (Gething and Sambrook, 1982) also is not useful for structural studies. Although the anchor-minus HA is initially assembled into trimeric structures, the oligomers are not stable and fall apart into monomers over time. By contrast, the solublized trimers of wild-type HA (BHA) that can be released from the virion or cell surface with bromelain (Brand and Skehel, 1972) are very stable. Preliminary studies indicate that soluble BHAO trimers can be prepared from the cleavage-minus variant of X-3 1 HA. A second application involves the C+RRKKR mutant which undergoes endogenous activation cleavage in cultured cells. We have constructed a composite mutant X-31 HA cDNA that encodes a protein containing both the cleavage site insertion and the substitution of asparagine for aspartic acid at residue 132 (Dams et al., 1985), which results in a higher pH threshold for fusion activity. When this cDNA was expressed in CV-I

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FIG.6. Analysis of the transport and proteolytic cleavage of wild-type and mutant HA proteins. CV-1 cells infected for 36 hr with SV40-HA viruses encoding the wild-type or mutant HA proteins were labeled for I5 min with [Y5]methionine. Cell extracts were prepared immediately (A) or following further incubation for 2 hr in medium containing an excess of nonradioactive methionine (B,C). In C trypsin at a concentration of 10 j&ml was included in the medium above the cells for the final I5 min of the 2-hr period. Proteins were precipitated from the cell extracts with anti-HA serum, separated by SDS-PAGE, and autoradiographed. The positions of the precursor H A 0 molecules and of the HA1 and HA2 cleavage products are shown to the right.

cells from an SV40-HA vector, the expected phenotype was obtained of fusion activity at pH 6.0 in the absence of prior treatment with trypsin. The double-mutant cDNA has also been inserted into a BPV-based vector, and continous cell lines (NIH-3T3and CHO) that express the variant HA are being selected by fluorescence-activated cell sorting. Because these cell lines will undergo cell-cell or erythrocyte-cell fusion under very mild and simple conditions (only moderately low pH and no protease treatment), they should provide excellent experimental systems for studies of fusion or as universal recipients for any materials of biological interest (antibodies, enzymes, oligonucleotides, etc.) that can be loaded into erythrocyte ghosts (Doxsey et al., 1985).

FIG.7. Low-pH-induced cell-cell fusion of CV-I cells expressing wild-type or mutant HA proteins: requirement for proteolytic cleavage activation. CV-I cells were infected with SV40-HA vectors containing the wild-type or mutant HA genes. Fifty hours after infection. the medium was aspirated from the monolayers which were then washed 3 times with medium lacking serum. Following incubation at 37°C for IS min with medium alone (top row) or with medium containing trypsin (10 &ml) (bottom row). the cells were briefly treated with buffer at pH 5.3 and then incubated further in medium containing serum for 8 hr to permit visualization of the formation of polykaryons with clustered nuclei. The cell monolayers were then fixed with formaldehyde, stained with Giemsa. and photographed.

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VIII.

CONCLUSION

The studies described in this chapter illustrate how the use of recombinant DNA techniques can advance our knowledge of the mechanism of membrane fusion. Cloning of cDNA copies of genes encoding wild-type and variant fusogenic proteins provides information on their primary amino acid sequences. Site-directed mutagenesis of the cloned genes can be used to probe the importance of chosen domains of a protein in the fusion mechanism. Expression of wild-type and mutant genes in mammalian cells using various types of eukaryotic vectors facilitates the analysis of their fusion phenotypes and in addition provides cell lines that can be used for cell fusion experiments or for delivery of macromolecules into the cell cytoplasm. Future experiments that combine the power of recombinant DNA techniques with the elegance of the model system provided by influenza hemagglutinin should reveal further details of the molecular mechanism of the fusion reaction induced by this particular membrane protein. The availability of cloned copies of a number of other viral fusion proteins (for references, see Gething et al., 1986a) will now permit similar analyses in other systems. Such studies should reveal any common or unique features of the mechanisms by which these different fusogenic proteins mediate membrane fusion. ACKNOWLEDGMENTS and This work was supported by grants from the National Institutes of Health to M.J.G. J.S. REFERENCES Bachi, T.,Gerhard, W., and Yewdell, J. W. (1985). Monoclonal antibodies detect different forms of influenza virus during viral penetration and biosynthesis. J. Virol. 55,307-313. Braciale, T. J., Lukacher, A. E., Morrison, L., Braciale, V. J., Smith, G., Moss, B., Gething, M. J., and Sambrook, J. (1986). Influenza viral antigen recognition by Class I and Class 11 MHC restricted cytolytic T lymphocytes. In "Options for the Control of Influenza Virus" (P. A. Kendal and P. A. Patriarca, eds.), pp. 407-421. Liss, New York. Brand, C. M., and Skehel, J. 3. (1972). Crystalline antigen from the influenza virus envelope. Nature (London) New Biol. 238, 145-147. Copeland, C., Doms, R. W., Bolzau. E. M., Webster, R. G., and Helenius, A, (1986). Assembly of influenza hemagglutinin trimers and its role in intracellular transport. J. Cell Biol. 103, 1179-1 191. Daniels, R. S., Downie, J. C., Hay, A. J., Knossow, M., Skehel, J. I., Wang, M. L., and Wiiey. D. C. (1985). Fusion mutants of the influenza virus hemagglutinin glycoprotein. Cell 40,431-439. Doms, R. W., Helenius, A., and White, J. M. (1985). Membrane fusion activity of the

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