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Distribution of hemagglutinin and neuraminidase on influenza virions as revealed by immunoelectron microscopy
149,36-43 (1986)
VIROLOGY
Distribution
of Hemagglutinin and Neuraminidase on Influenza Virions as Revealed by lmmunoelectron Microscopy K. G. MURTI’
Division
AND
R. G. WEBSTER
of Virology and Molecular Biology, St. Jude Children’s Research Hospital, P. 0. Box 318, Memphis, Tennessee 38101 Received August 26, 1985;accepted October 30, 1985
Monoclonal antibodies specific for the hemagglutinin (HA) or the neuraminidase (NA) of influenza viruses were used in immunoelectron microscopic studies to determine the distribution of the two surface spikes on the virion. Indirect immunogold staining revealed that the HA is uniformly distributed on the virion while the NA occurs in discrete areas. Crosslinking and low temperature studies argue against redistribution of the HA and NA after antibody attachment and indicate that the NA on influenza virus occurs in patches. 0 1986 Academic
Press, Inc.
INTRODUCTION
ative staining. Their observations suggested “that neuraminidase may be localized in patches on the viral envelope rather than being distributed in a regular manner on the virus surface.” This important observation has been overlooked, for all interpretations of influenza virus continue to show uniform distribution of both molecules of the surface of the virion. Information on the distribution of the HA and NA is relevant to our understanding of the function of these molecules in virus attachment, release, and perhaps the neutralization of infectivity. The availability of monoclonal antibodies specific for the HA and NA and improvement in immunoelectron microscopic techniques have permitted us to establish the distribution of the hemagglutinin and neuraminidase molecules on influenza virions. The results demonstrate that neuraminidase molecules are clustered on the virion while HA molecules are distributed uniformly over the virus surface.
The two glycoproteins of influenza viruses, the hemagglutinin (HA) and the neuraminidase (NA) that form the “spikes” on the surface of the virion are the best characterized of all virus proteins. Both molecules have been crystallized and their three-dimensional structures established by x-ray crystallography (Wilson et al., 1981; Wiley et al., 1981; Varghese et ah, 1983; and Colman et aZ., 1983). The antigenic areas and some of the functional domains on the molecules have been established (for reviews see Colman and Ward, 1985; Webster et ah, 1983). There are approximately 500 HA molecules and 100 NA molecules per virion (Lamb, 1983). Despite all of this information, we know very little concerning the distribution of these two glycoproteins on the virion. Are the molecules uniformly distributed or segregated over the virion surface? While nothing is known concerning distribution of the HA on the virion surface, some information is available on the distribution of NA. Compans et al. (1969) have treated influenza virions with antiserum specific for neuraminidase and observed them in the electron microscope after neg1 Author addressed.
to whom requests for reprints
0042-6822/86 $3.00 Copyright All rights
0 1986 by Academic Press, Inc. of reproduction in any form reserved.
MATERIALS
AND
METHODS
Vimses. The influenza viruses used in this study included A/NWS/33 (HlNl), A/Aichi/2/68 (H3N2), A/Seal/Mass/l/80 (H’7N7), A/Chick/Penn/8/25/83 (H5N2), and a number of reassortant viruses in-
should be
36
INFLUENZA
37
NEURAMINIDASE
eluding X-31 (H3N2), X-7 (HlN2), and X7Fl (HlN2) (Kilbourne et al, 1967). Other human and avian influenza viruses from the repository at St. Jude Children’s Research Hospital were also utilized and are given in the text. The viruses were grown in 11-day-old chicken embryos and purified by adsorption to and elution from chicken erythrocytes followed by differential centrifugation and sedimentation through a sucrose gradient (lo-40% sucrose, 0.15M NaCl) (Laver, 1969). Monoclonal antibodies. Hybrid cell lines producing antibodies to the HA or NA of influenza viruses have been described (Webster et aZ., 1979). The cell lines were selected following fusion of myeloma cells SP2/0 Ag14 (Shulman et ah, 1978) with immune spleen cells according to the method of Kohler and Milstein (1976). Hybridomas producing antibody were cloned in soft agar and injected intraperitoneally into pristane-treated mice. Ascitic fluid was collected 7 to 10 days later and used in the assays. Immunoelectron microscopy. The procedure was similar to the one described (Murti et ah, 1985) with minor modifications. The virus sample was adsorbed to parlodion-coated, 300-mesh copper grids and the excess sample was removed by touching the grids with the edge of a filter paper. The grid was floated for 5 min on Tris-buffered saline (TBS; 500 mM NaCl and 25 mM Tris, pH 7.6) contained in a plastic petri dish. The excess TBS was removed with a filter paper and the grid was floated for 1 hr on TBS containing 3% gelatin. The gelatin was used as a blocking agent to prevent nonspecific binding of antibodies to the parlodion surface. The grid was washed in TBS and floated on a drop of diluted antibody solution (20- to 40-fold diluted with TBS) placed on a parafilm strip. The incubation was for 1 hr at 21”. At the end of incubation, the grid was washed thoroughly with TBS and floated on colloidal gold (5 nm diameter)-conjugated goat anti-mouse antibodies (GAMG5 diluted 20-fold with TBS; Janssen Pharmacia, Belgium). After 1 hr, the grid was removed and rinsed thoroughly and
stained for electron microscopy. For the simultaneous localization of HA and NA, the grids were sequentially incubated in anti-HA antibodies, GAM-G5 antibodies, anti-NA antibodies, and GAM-G20 (20-nmdiameter gold particles) antibodies. To stain the grids for electron microscopy, two procedures were used. In the first procedure, the samples were positively stained with ethanolic uranyl acetate. The grids (still wet from the TBS wash) were first dipped in 50% ethyl alcohol for 10 see, swirled in ethyl alcohol:uranyl acetate (3:1, 100% ethyl alcohol and 2% aqueous uranyl acetate) for 30 see and dried from 95% ethyl alcohol. For negative staining, a drop of 2% phosphotungstic acid was placed on the specimen surface of the grid, the excess stain was removed with a filter paper, and the grid was dried. The positive staining procedure stains the ribonucleoprotein of the virion and does not resolve the structure of the virion. However, we used this method routinely in our immunoelectron microscopy because the positively stained samples provided a higher resolution and a lower background than the negatively stained samples (Murti et ab, 1985). The stained grids were viewed in a Philips 301 electron microscope operated at 80 kV.
RESULTS
Localization
of HA
To localize HA on the virus membrane, the virions [(X-31) (H3N2)] were incubated with monoclonal antibody to HA (HK 30/ 1 or MEM 123/4) followed by goat-antimouse antibodies conjugated with colloidal gold (GAM-G5). The samples were positively stained with uranyl acetate to resolve the outline of the virions. The results with X-31 virus using HK 30/l antibody are presented in Fig. 1. The HA molecules, as indicated by the gold particles, are distributed uniformly over the virion surface in all of the virions (-400) observed. Examination of A/Aichi/2/68 (H3N2), A/ Chick/Penn/l825 (H5N2), A/Seal/Mass/l/ 80 (H7N7), as well as the reassortants X-7
38
MURTI
AND
WEBSTER
FIG. 1. Immunogold labeling of X-31 influenza virus treated with antibody to hemagglutinin (HK 30/l). Note the uniform distribution of hemagglutinin molecules over the v&ion surface. X184,500.
(HlN2) and X-7Fl (HlN2) with monoclonal antibodies to the HA also showed uniform distribution of HA on the virion (results
not shown) suggesting that the findings are generally applicable. No binding was observed when the experiments were done
INFLUENZA
NEURAMINIDASE
FIG. 2. Immunogold labeling of X-31 influenza virus treated with antibody to the neuraminidase of A/Tokyo/l/67 (25/4). The neuraminidase is found in discrete patches over the virion surface. X254.800.
39
40
MURTI
AND
with monoclonal antibodies to Sendai HN glycoprotein. Localization
of NA
Localization of NA on the virion was done with X-31 (H3N2) virus and three different monoclonal antibodies to N2 neuraminidase (X-7F1129/2, A/Tokyo/l/G’I, 16/ 8,25/4). The results with X-31 virus using anti N2 to A/Tokyo/l/67 preparation 25/ 4 are shown in Fig. 2. In contrast to the HA molecules, the NA molecules in most virions (164/212) were found in one to three patches on the virion surface. A few (48/ 212) virions showed a less distinct segregation of the NA molecules but even in these, the distribution of NA was never uniform as found with the HA. The phenomenon was not confined to influenza virions from human hosts since a similar distribution of NA was observed when the studies were done with virions from other mammalian and avian hosts. The experiment was also done with X-7Fl virus which is known to possess a higher ratio of NA to HA. The results were similar to those of other strains except that the patches of neuraminidase were larger. To obtain morphological information on the neuraminidase-containing spikes, we have negatively stained some of the samples that have been incubated with antibodies to NA and GAM-G5 antibody. Figure 3 illustrates the results. The virus structure was better resolved in these preparations and the distribution of NA was similar to that described above. However, the quality of the preparations was not adequate to distinguish the NA spikes. Simultaneous
Localization
WEBSTER
with goat-anti-mouse antibody conjugated with 5-nm gold particles (GAM-G5). Thus, in these preparations, the large (20 nm) gold particles are attached to NA molecules while the small (5 nm) gold particles are attached to HA molecules. The results of the double immunogold staining of HA and NA are shown in Fig. 4. Overall, the pattern is similar to that observed with individual staining of HA and NA, i.e., the HA molecules are distributed all over the virion surface while the NA molecules are confined to discrete patches. Is Clustering of NA on the Virion ‘Capping ”
Due to
The clustering of neuraminidase detected may reflect the native distribution of this glycoprotein on the virion or it could be an artifact induced by the procedures. It is well established that viral antigens expressed over the cell surface move toward one pole of the cell (capping) when a live cell is treated with antiviral antibodies (Oldstone et al., 1980). The phenomenon of capping occurs on live cell surfaces at 37” (not at 4 or 46”) and requires intact mi-
of HA and NA
To determine the location of HA and NA relative to each other, the following experiment was done. The virions were first treated with monoclonal antibodies to NA followed by incubation with high concentration of goat-anti-mouse antibody conjugated with large (20 nm) gold particles (GAM-GBO). After extensive washing, the virions were incubated with monoclonal antibodies to HA followed by incubation
FIG. 3. Electron micrograph of a negatively stained virion prepared as in Fig. 2. Note the clustering of NA molecules. X380,000.
INFLUENZA
NEURAMINIDASE
FIG. 4. Simultaneous labeling of hemagglutinin and neuraminidase on X-31 virions using antibodies described in the legends of Figs. 1 and 2. The hemagglutinin molecules were labeled with large gold particles, and the neuraminidase with small gold particles. Note the uniform distribution of HA and clustering of NA. X191,100.
41
42
MURTI
AND
crotubules and microfilaments (Oldstone et aZ.,1980). To determine if the observed distribution of NA molecules on virions was due to capping, we have performed the experiments at 4” and after fixation of virions with formaldehyde or glutaraldehyde. Regardless of the treatment, the NA molecules were found in patches over the virion surface. The above results, plus the fact that the virions do not contain elements of cytoskeleton, eliminate the possibility that the observed distribution of NA is due to capping. DISCUSSION
The studies reported here indicate that the HA and NA differ in their distribution on influenza viruses. Localization of the HA and NA using monoclonal antibodies to different antigenic determinants and detection by indirect immunogold staining in the electron microscope reveal that the HA is uniformly distributed on the virion while the NA occurs in discrete areas. These findings were applicable to influenza virus of humans and lower animals and also to reassortment influenza viruses. One explanation for the clustered distribution of NA is that the phenomenon is due to “capping” (antibody-induced movement of antigens-see Oldstone et al, 1980). However, this seems unlikely because the same result was obtained when the experiments were done either at 4’ or with aldehyde-fixed virions. These studies virtually eliminate capping as the mechanism for the clustered distribution of NA because capping does not occur at 4” or on fixed membranes. The biological significance remains to be understood, but we can envision the following. The clustered arrangement of NA may be advantageous to the virus in the attachment to the host cell membrane, fusion with the cell membrane, and release from the cell membrane. Influenza virus attaches to the host cell membrane by the binding of HA molecules to the sialic acidcontaining receptors on the cell surface. Since the major function of NA is to cleave the sialic acid residues, the localized dis-
WEBSTER
tribution of NA on virus surface may increase the chance of virus binding to the cell surface. The observation that antibodies to NA will not neutralize influenza virus infectivity by blocking attachment (Kilbourne et al, 1968) is consistent with the clustered distribution of NA described here. The clustered distribution of NA may also be advantageous in the fusion of viral and cell membranes. The fusion of the two membranes is believed to be brought about by a conformational change in the HA molecule which occurs at pH 5.0. The conformational change exposes the fusion peptide by the cleavage or folding of the globular HA head. In either case, it would be advantageous to have clustered NA molecules since they will not sterically block the exposure of fusion peptide. Fi-
FIG. 5. Schematic diagram of stages in influenza virus budding from the cell membrane. The region of membrane at which the virus buds out contains many HA spikes and a few NA spikes (A). As the virus evaginates, the HA spikes on the virus surface proximal to the cell membrane bind to sialic acid-containing receptors (B). The NA molecules localized on the virus surface near the cell membrane cleave the sialic acid residues thereby facilitating virus release (C). HA, hemagglutinin; N, neuraminidase; R, receptor.
INFLUENZA
NEURAMINIDASE
nally, the clustered arrangement of NA molecules may be needed (and in fact may occur) during virus release. It is known that NA is involved in virus release since antibodies to NA block the process and permit selection of variants (Webster et ab, 1982). It is not known how NA is involved in the release, but we can envision the following. As the virus is budding out from the cell membrane, the HA on the virus membrane may attach to the sialic acid-containing receptors on the cell membrane near the point of budding (Fig. 5), thus preventing virus release. One way to prevent this phenomenon is to have the NA molecules localized at the junction of viral and cell membranes to cleave the sialic acid residues. Thus, clustering of NA to a pole of the virus proximal to the cell membrane may occur during virus budding. Experiments are underway with infected cells to determine the relative distribution of HA and NA on the cell membrane during the course of virus maturation. ACKNOWLEDGMENTS This work was supported by U. S. Public Health Research Grants AI 08831, AI 52586, and AI 20591 from the National Institute of Allergy and Infectious Diseases; Cancer Center Support (CORE) Grant CA 21765; a grant from the American Cancer Society (#CD-253); and American Lebanese Syrian Associated Charities. Katherine Troughton and Lisa Newberry provided excellent technical assistance. REFERENCES COLMAN, P. M., VARGHESE, J. N., and LAVER, W. G. (1983). Structure of the catalytic and antigenic sites in influenza virus neuraminidase. Nature (London) 303,41-44. COLMAN, P. M., and WARD, C. W. (1985). Structure and diversity of the influenza virus neuraminidase. A&v. Virus Res., in press. COMPANS, R. W., DIMMOCK, N. J., and MEIER-EWERT, H. (1969). Effect of antibody to neuraminidase on the maturation and hemagglutinating activity of an influenza A2 virus. J. Viral. 4, 528-534. KILBOURNE, E. D., LAVER, W. G., SCHULMAN, J. L., and WEBSTER, R. G. (1968). Antiviral activity of antiserum specific for an influenza virus neuraminidase. J. Viral. 2, 281-288. KILBOURNE, E. D., LIEF, F. S., SCHULMAN, J. L., JAHIEL, R. I., and LAVER, W. G. (1967). Antigenic hybrids
43
of influenza viruses and their implications. Perspect. ViroL 5, 87-106. KOHLER, G., and MILSTEIN, C. (1976). Derivation of specific antibody-producing tissue culture and tumor lines by cell fusion. Eur. J. Immunol. 6, 511519. LAMB, R. A. (1983). The influenza virus RNA segments and their encoded proteins. In “Genetics of Influenza Viruses” (P. Palese and D. W. Kingsbury, eds.), pp. 21-69. Springer-Verlag, New York. LAVER, W. G. (1969). Purification of influenza viruses. In “Fundamental Techniques in Virology” (K. Habel and N. P. Salzman, eds.), pp. 82-86. Academic Press, New York. MURTI, K. G., PORTNER, A., TROUGHTON, K., and DESHPANDE, K. (1985). Localization of proteins on viral nucleocapsids using immuno-electron microscopy. J. Electron Microscope Tech. 2,139-146. OLDSTONE, M. B. A., FUJINAMI, R. S., and LAMPERT, P. W. (1980). Membrane and cytoplasmic changes in virus-infected cells induced by interactions of antiviral antibody with surface viral antigen. Prog. Med. ViroL 26,45-93. SHULMAN, M., WILDE, C. D., and KOHLER, G. (1978). A better cell line for making hybridomas secreting specific antibodies. Nature (London) 276,269-270. SKEHEL, J. J., BALEY, P. M., BROWN, E. B., MARTIN, S. R., WATERFIELD, M. D., WHITE, J. R., WILSON, I. A., and WILEY, D. C. (1982). Changes in the conformation of the influenza virus hemagglutinin at the pH optimum of virus-mediated membrane fusion. Proc. Natl. Acad. Sci. USA 79,968-972. VARGHESE, J. N., LAVER, W. G., and COLMAN, P. M. (1983). Structure of the influenza virus glycoprotein antigen neuraminidase at 2.9A resolution. Nature (London) 303,35-40. WEBSTER, R. G., HINSHAW, V. S., and LAVER, W. G. (1982). Selection and analysis of antigenic variants of the neuraminidase of N2 influenza viruses with monoclonal antibodies. Virology 117,93-104. WEBSTER, R. G., KENDAL, A. P., and GERHARD, W. (1979). Analysis of antigenic drift in recently isolated influenza (HlNl) viruses using monoclonal antibody preparations. Virology 96,258-264. WEBSTER, R. G., LAVER, W. G. and AIR, G. M. (1983). Antigenic variation among type A influenza viruses. In “Genetics of Influenza Viruses” (P. Palase and D. W. Kingsbury, eds.), pp. 127-168. Springer-Verlag, New York. WILEY, D. C., WILSON, I. A., and SKEHEL, J. J. (1981). Structural identification of the antibody-binding sites of Hong Kong influenza hemagglutinin and their involvement in antigenic variation. Nature (London) 289,373-378. WILSON, I. A., SKEHEL, J. J., and WILEY, D. C. (1981). Structure of the hemagglutinin membrane glycoprotein of influenza virus at 3A resolution. Nature (London) 289,366-373.
VIROLOGY
Distribution
of Hemagglutinin and Neuraminidase on Influenza Virions as Revealed by lmmunoelectron Microscopy K. G. MURTI’
Division
AND
R. G. WEBSTER
of Virology and Molecular Biology, St. Jude Children’s Research Hospital, P. 0. Box 318, Memphis, Tennessee 38101 Received August 26, 1985;accepted October 30, 1985
Monoclonal antibodies specific for the hemagglutinin (HA) or the neuraminidase (NA) of influenza viruses were used in immunoelectron microscopic studies to determine the distribution of the two surface spikes on the virion. Indirect immunogold staining revealed that the HA is uniformly distributed on the virion while the NA occurs in discrete areas. Crosslinking and low temperature studies argue against redistribution of the HA and NA after antibody attachment and indicate that the NA on influenza virus occurs in patches. 0 1986 Academic
Press, Inc.
INTRODUCTION
ative staining. Their observations suggested “that neuraminidase may be localized in patches on the viral envelope rather than being distributed in a regular manner on the virus surface.” This important observation has been overlooked, for all interpretations of influenza virus continue to show uniform distribution of both molecules of the surface of the virion. Information on the distribution of the HA and NA is relevant to our understanding of the function of these molecules in virus attachment, release, and perhaps the neutralization of infectivity. The availability of monoclonal antibodies specific for the HA and NA and improvement in immunoelectron microscopic techniques have permitted us to establish the distribution of the hemagglutinin and neuraminidase molecules on influenza virions. The results demonstrate that neuraminidase molecules are clustered on the virion while HA molecules are distributed uniformly over the virus surface.
The two glycoproteins of influenza viruses, the hemagglutinin (HA) and the neuraminidase (NA) that form the “spikes” on the surface of the virion are the best characterized of all virus proteins. Both molecules have been crystallized and their three-dimensional structures established by x-ray crystallography (Wilson et al., 1981; Wiley et al., 1981; Varghese et ah, 1983; and Colman et aZ., 1983). The antigenic areas and some of the functional domains on the molecules have been established (for reviews see Colman and Ward, 1985; Webster et ah, 1983). There are approximately 500 HA molecules and 100 NA molecules per virion (Lamb, 1983). Despite all of this information, we know very little concerning the distribution of these two glycoproteins on the virion. Are the molecules uniformly distributed or segregated over the virion surface? While nothing is known concerning distribution of the HA on the virion surface, some information is available on the distribution of NA. Compans et al. (1969) have treated influenza virions with antiserum specific for neuraminidase and observed them in the electron microscope after neg1 Author addressed.
to whom requests for reprints
0042-6822/86 $3.00 Copyright All rights
0 1986 by Academic Press, Inc. of reproduction in any form reserved.
MATERIALS
AND
METHODS
Vimses. The influenza viruses used in this study included A/NWS/33 (HlNl), A/Aichi/2/68 (H3N2), A/Seal/Mass/l/80 (H’7N7), A/Chick/Penn/8/25/83 (H5N2), and a number of reassortant viruses in-
should be
36
INFLUENZA
37
NEURAMINIDASE
eluding X-31 (H3N2), X-7 (HlN2), and X7Fl (HlN2) (Kilbourne et al, 1967). Other human and avian influenza viruses from the repository at St. Jude Children’s Research Hospital were also utilized and are given in the text. The viruses were grown in 11-day-old chicken embryos and purified by adsorption to and elution from chicken erythrocytes followed by differential centrifugation and sedimentation through a sucrose gradient (lo-40% sucrose, 0.15M NaCl) (Laver, 1969). Monoclonal antibodies. Hybrid cell lines producing antibodies to the HA or NA of influenza viruses have been described (Webster et aZ., 1979). The cell lines were selected following fusion of myeloma cells SP2/0 Ag14 (Shulman et ah, 1978) with immune spleen cells according to the method of Kohler and Milstein (1976). Hybridomas producing antibody were cloned in soft agar and injected intraperitoneally into pristane-treated mice. Ascitic fluid was collected 7 to 10 days later and used in the assays. Immunoelectron microscopy. The procedure was similar to the one described (Murti et ah, 1985) with minor modifications. The virus sample was adsorbed to parlodion-coated, 300-mesh copper grids and the excess sample was removed by touching the grids with the edge of a filter paper. The grid was floated for 5 min on Tris-buffered saline (TBS; 500 mM NaCl and 25 mM Tris, pH 7.6) contained in a plastic petri dish. The excess TBS was removed with a filter paper and the grid was floated for 1 hr on TBS containing 3% gelatin. The gelatin was used as a blocking agent to prevent nonspecific binding of antibodies to the parlodion surface. The grid was washed in TBS and floated on a drop of diluted antibody solution (20- to 40-fold diluted with TBS) placed on a parafilm strip. The incubation was for 1 hr at 21”. At the end of incubation, the grid was washed thoroughly with TBS and floated on colloidal gold (5 nm diameter)-conjugated goat anti-mouse antibodies (GAMG5 diluted 20-fold with TBS; Janssen Pharmacia, Belgium). After 1 hr, the grid was removed and rinsed thoroughly and
stained for electron microscopy. For the simultaneous localization of HA and NA, the grids were sequentially incubated in anti-HA antibodies, GAM-G5 antibodies, anti-NA antibodies, and GAM-G20 (20-nmdiameter gold particles) antibodies. To stain the grids for electron microscopy, two procedures were used. In the first procedure, the samples were positively stained with ethanolic uranyl acetate. The grids (still wet from the TBS wash) were first dipped in 50% ethyl alcohol for 10 see, swirled in ethyl alcohol:uranyl acetate (3:1, 100% ethyl alcohol and 2% aqueous uranyl acetate) for 30 see and dried from 95% ethyl alcohol. For negative staining, a drop of 2% phosphotungstic acid was placed on the specimen surface of the grid, the excess stain was removed with a filter paper, and the grid was dried. The positive staining procedure stains the ribonucleoprotein of the virion and does not resolve the structure of the virion. However, we used this method routinely in our immunoelectron microscopy because the positively stained samples provided a higher resolution and a lower background than the negatively stained samples (Murti et ab, 1985). The stained grids were viewed in a Philips 301 electron microscope operated at 80 kV.
RESULTS
Localization
of HA
To localize HA on the virus membrane, the virions [(X-31) (H3N2)] were incubated with monoclonal antibody to HA (HK 30/ 1 or MEM 123/4) followed by goat-antimouse antibodies conjugated with colloidal gold (GAM-G5). The samples were positively stained with uranyl acetate to resolve the outline of the virions. The results with X-31 virus using HK 30/l antibody are presented in Fig. 1. The HA molecules, as indicated by the gold particles, are distributed uniformly over the virion surface in all of the virions (-400) observed. Examination of A/Aichi/2/68 (H3N2), A/ Chick/Penn/l825 (H5N2), A/Seal/Mass/l/ 80 (H7N7), as well as the reassortants X-7
38
MURTI
AND
WEBSTER
FIG. 1. Immunogold labeling of X-31 influenza virus treated with antibody to hemagglutinin (HK 30/l). Note the uniform distribution of hemagglutinin molecules over the v&ion surface. X184,500.
(HlN2) and X-7Fl (HlN2) with monoclonal antibodies to the HA also showed uniform distribution of HA on the virion (results
not shown) suggesting that the findings are generally applicable. No binding was observed when the experiments were done
INFLUENZA
NEURAMINIDASE
FIG. 2. Immunogold labeling of X-31 influenza virus treated with antibody to the neuraminidase of A/Tokyo/l/67 (25/4). The neuraminidase is found in discrete patches over the virion surface. X254.800.
39
40
MURTI
AND
with monoclonal antibodies to Sendai HN glycoprotein. Localization
of NA
Localization of NA on the virion was done with X-31 (H3N2) virus and three different monoclonal antibodies to N2 neuraminidase (X-7F1129/2, A/Tokyo/l/G’I, 16/ 8,25/4). The results with X-31 virus using anti N2 to A/Tokyo/l/67 preparation 25/ 4 are shown in Fig. 2. In contrast to the HA molecules, the NA molecules in most virions (164/212) were found in one to three patches on the virion surface. A few (48/ 212) virions showed a less distinct segregation of the NA molecules but even in these, the distribution of NA was never uniform as found with the HA. The phenomenon was not confined to influenza virions from human hosts since a similar distribution of NA was observed when the studies were done with virions from other mammalian and avian hosts. The experiment was also done with X-7Fl virus which is known to possess a higher ratio of NA to HA. The results were similar to those of other strains except that the patches of neuraminidase were larger. To obtain morphological information on the neuraminidase-containing spikes, we have negatively stained some of the samples that have been incubated with antibodies to NA and GAM-G5 antibody. Figure 3 illustrates the results. The virus structure was better resolved in these preparations and the distribution of NA was similar to that described above. However, the quality of the preparations was not adequate to distinguish the NA spikes. Simultaneous
Localization
WEBSTER
with goat-anti-mouse antibody conjugated with 5-nm gold particles (GAM-G5). Thus, in these preparations, the large (20 nm) gold particles are attached to NA molecules while the small (5 nm) gold particles are attached to HA molecules. The results of the double immunogold staining of HA and NA are shown in Fig. 4. Overall, the pattern is similar to that observed with individual staining of HA and NA, i.e., the HA molecules are distributed all over the virion surface while the NA molecules are confined to discrete patches. Is Clustering of NA on the Virion ‘Capping ”
Due to
The clustering of neuraminidase detected may reflect the native distribution of this glycoprotein on the virion or it could be an artifact induced by the procedures. It is well established that viral antigens expressed over the cell surface move toward one pole of the cell (capping) when a live cell is treated with antiviral antibodies (Oldstone et al., 1980). The phenomenon of capping occurs on live cell surfaces at 37” (not at 4 or 46”) and requires intact mi-
of HA and NA
To determine the location of HA and NA relative to each other, the following experiment was done. The virions were first treated with monoclonal antibodies to NA followed by incubation with high concentration of goat-anti-mouse antibody conjugated with large (20 nm) gold particles (GAM-GBO). After extensive washing, the virions were incubated with monoclonal antibodies to HA followed by incubation
FIG. 3. Electron micrograph of a negatively stained virion prepared as in Fig. 2. Note the clustering of NA molecules. X380,000.
INFLUENZA
NEURAMINIDASE
FIG. 4. Simultaneous labeling of hemagglutinin and neuraminidase on X-31 virions using antibodies described in the legends of Figs. 1 and 2. The hemagglutinin molecules were labeled with large gold particles, and the neuraminidase with small gold particles. Note the uniform distribution of HA and clustering of NA. X191,100.
41
42
MURTI
AND
crotubules and microfilaments (Oldstone et aZ.,1980). To determine if the observed distribution of NA molecules on virions was due to capping, we have performed the experiments at 4” and after fixation of virions with formaldehyde or glutaraldehyde. Regardless of the treatment, the NA molecules were found in patches over the virion surface. The above results, plus the fact that the virions do not contain elements of cytoskeleton, eliminate the possibility that the observed distribution of NA is due to capping. DISCUSSION
The studies reported here indicate that the HA and NA differ in their distribution on influenza viruses. Localization of the HA and NA using monoclonal antibodies to different antigenic determinants and detection by indirect immunogold staining in the electron microscope reveal that the HA is uniformly distributed on the virion while the NA occurs in discrete areas. These findings were applicable to influenza virus of humans and lower animals and also to reassortment influenza viruses. One explanation for the clustered distribution of NA is that the phenomenon is due to “capping” (antibody-induced movement of antigens-see Oldstone et al, 1980). However, this seems unlikely because the same result was obtained when the experiments were done either at 4’ or with aldehyde-fixed virions. These studies virtually eliminate capping as the mechanism for the clustered distribution of NA because capping does not occur at 4” or on fixed membranes. The biological significance remains to be understood, but we can envision the following. The clustered arrangement of NA may be advantageous to the virus in the attachment to the host cell membrane, fusion with the cell membrane, and release from the cell membrane. Influenza virus attaches to the host cell membrane by the binding of HA molecules to the sialic acidcontaining receptors on the cell surface. Since the major function of NA is to cleave the sialic acid residues, the localized dis-
WEBSTER
tribution of NA on virus surface may increase the chance of virus binding to the cell surface. The observation that antibodies to NA will not neutralize influenza virus infectivity by blocking attachment (Kilbourne et al, 1968) is consistent with the clustered distribution of NA described here. The clustered distribution of NA may also be advantageous in the fusion of viral and cell membranes. The fusion of the two membranes is believed to be brought about by a conformational change in the HA molecule which occurs at pH 5.0. The conformational change exposes the fusion peptide by the cleavage or folding of the globular HA head. In either case, it would be advantageous to have clustered NA molecules since they will not sterically block the exposure of fusion peptide. Fi-
FIG. 5. Schematic diagram of stages in influenza virus budding from the cell membrane. The region of membrane at which the virus buds out contains many HA spikes and a few NA spikes (A). As the virus evaginates, the HA spikes on the virus surface proximal to the cell membrane bind to sialic acid-containing receptors (B). The NA molecules localized on the virus surface near the cell membrane cleave the sialic acid residues thereby facilitating virus release (C). HA, hemagglutinin; N, neuraminidase; R, receptor.
INFLUENZA
NEURAMINIDASE
nally, the clustered arrangement of NA molecules may be needed (and in fact may occur) during virus release. It is known that NA is involved in virus release since antibodies to NA block the process and permit selection of variants (Webster et ab, 1982). It is not known how NA is involved in the release, but we can envision the following. As the virus is budding out from the cell membrane, the HA on the virus membrane may attach to the sialic acid-containing receptors on the cell membrane near the point of budding (Fig. 5), thus preventing virus release. One way to prevent this phenomenon is to have the NA molecules localized at the junction of viral and cell membranes to cleave the sialic acid residues. Thus, clustering of NA to a pole of the virus proximal to the cell membrane may occur during virus budding. Experiments are underway with infected cells to determine the relative distribution of HA and NA on the cell membrane during the course of virus maturation. ACKNOWLEDGMENTS This work was supported by U. S. Public Health Research Grants AI 08831, AI 52586, and AI 20591 from the National Institute of Allergy and Infectious Diseases; Cancer Center Support (CORE) Grant CA 21765; a grant from the American Cancer Society (#CD-253); and American Lebanese Syrian Associated Charities. Katherine Troughton and Lisa Newberry provided excellent technical assistance. REFERENCES COLMAN, P. M., VARGHESE, J. N., and LAVER, W. G. (1983). Structure of the catalytic and antigenic sites in influenza virus neuraminidase. Nature (London) 303,41-44. COLMAN, P. M., and WARD, C. W. (1985). Structure and diversity of the influenza virus neuraminidase. A&v. Virus Res., in press. COMPANS, R. W., DIMMOCK, N. J., and MEIER-EWERT, H. (1969). Effect of antibody to neuraminidase on the maturation and hemagglutinating activity of an influenza A2 virus. J. Viral. 4, 528-534. KILBOURNE, E. D., LAVER, W. G., SCHULMAN, J. L., and WEBSTER, R. G. (1968). Antiviral activity of antiserum specific for an influenza virus neuraminidase. J. Viral. 2, 281-288. KILBOURNE, E. D., LIEF, F. S., SCHULMAN, J. L., JAHIEL, R. I., and LAVER, W. G. (1967). Antigenic hybrids
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of influenza viruses and their implications. Perspect. ViroL 5, 87-106. KOHLER, G., and MILSTEIN, C. (1976). Derivation of specific antibody-producing tissue culture and tumor lines by cell fusion. Eur. J. Immunol. 6, 511519. LAMB, R. A. (1983). The influenza virus RNA segments and their encoded proteins. In “Genetics of Influenza Viruses” (P. Palese and D. W. Kingsbury, eds.), pp. 21-69. Springer-Verlag, New York. LAVER, W. G. (1969). Purification of influenza viruses. In “Fundamental Techniques in Virology” (K. Habel and N. P. Salzman, eds.), pp. 82-86. Academic Press, New York. MURTI, K. G., PORTNER, A., TROUGHTON, K., and DESHPANDE, K. (1985). Localization of proteins on viral nucleocapsids using immuno-electron microscopy. J. Electron Microscope Tech. 2,139-146. OLDSTONE, M. B. A., FUJINAMI, R. S., and LAMPERT, P. W. (1980). Membrane and cytoplasmic changes in virus-infected cells induced by interactions of antiviral antibody with surface viral antigen. Prog. Med. ViroL 26,45-93. SHULMAN, M., WILDE, C. D., and KOHLER, G. (1978). A better cell line for making hybridomas secreting specific antibodies. Nature (London) 276,269-270. SKEHEL, J. J., BALEY, P. M., BROWN, E. B., MARTIN, S. R., WATERFIELD, M. D., WHITE, J. R., WILSON, I. A., and WILEY, D. C. (1982). Changes in the conformation of the influenza virus hemagglutinin at the pH optimum of virus-mediated membrane fusion. Proc. Natl. Acad. Sci. USA 79,968-972. VARGHESE, J. N., LAVER, W. G., and COLMAN, P. M. (1983). Structure of the influenza virus glycoprotein antigen neuraminidase at 2.9A resolution. Nature (London) 303,35-40. WEBSTER, R. G., HINSHAW, V. S., and LAVER, W. G. (1982). Selection and analysis of antigenic variants of the neuraminidase of N2 influenza viruses with monoclonal antibodies. Virology 117,93-104. WEBSTER, R. G., KENDAL, A. P., and GERHARD, W. (1979). Analysis of antigenic drift in recently isolated influenza (HlNl) viruses using monoclonal antibody preparations. Virology 96,258-264. WEBSTER, R. G., LAVER, W. G. and AIR, G. M. (1983). Antigenic variation among type A influenza viruses. In “Genetics of Influenza Viruses” (P. Palase and D. W. Kingsbury, eds.), pp. 127-168. Springer-Verlag, New York. WILEY, D. C., WILSON, I. A., and SKEHEL, J. J. (1981). Structural identification of the antibody-binding sites of Hong Kong influenza hemagglutinin and their involvement in antigenic variation. Nature (London) 289,373-378. WILSON, I. A., SKEHEL, J. J., and WILEY, D. C. (1981). Structure of the hemagglutinin membrane glycoprotein of influenza virus at 3A resolution. Nature (London) 289,366-373.