Isolation and structural analysis of influenza C virion glycoproteins

VIROLOGY

113,

439-451

Isolation GEORG

(1981)

and Structural

Analysis of Influenza C Virion Glycoproteins

HERRLER,**t ARNO NAGELE,* HERBERT MEIER-EWERT,* AJIT S. BHOWN,t AND RICHARD W. COMPANS?’

* Virologische Abteilung, Znstitut ftir Med. Mikrobio&ie o!er Techn. Universit& Mtinchen, Biedersteinerstr. 29, 8 Munich 40, W. Germany, and TLlepartment of Microbiology, University of Alabama in Birmingham, Birmin&am, Alabama 35294 Received January 5, 1981; accepted April 10, 1981 Influenza C virions possess a single glycoprotein that is cleaved into two disulfidelinked subunits, gp65 and gp30. When analyzed under nonreducing conditions, the uncleaved (gp1) and cleaved (gpI1) glycoproteins differ significantly in apparent molecular weight; however, we observed no difference in their tryptic peptide patterns. We have isolated the glycoproteins by selective solubilization with Triton X-166 or octylglucoside; only preparations obtained with the latter detergent showed hemagglutinating activity. In purified glycoprotein samples, gp65 was routinely observed as a doublet on SDS-polyacrylamide gels. Analysis of tryptic peptides by ion-exchange chromatography demonstrated that the two gp65 bands have indistinguishable polypeptide backbones; they appear to differ, however, in carbohydrate content. The uncleaved glycoprotein as well as gp65 were resistant to Edman degradation indicating the presence of blocked amino termini, whereas gp30 was observed to have the N-terminal tripeptide sequence NH*Ile-Phe-Gly. This sequence is homologous to a sequence at the N termini of influenza A and B HA2 glycoproteins, except for the presence of an additional terminal glycine residue in these viruses. The influenza C glycoproteins form a regular hexagonal lattice on the viral envelope. This arrangement is sometimes maintained in disrupted virus preparations and in glycoprotein subunits released from the envelope by limited proteolysis, indicating that direct interactions between the glycoprotein molecules are responsible, at least in part, for the observed arrangement. Observations of clustered surface projections on plasma membranes of infected cells, and of released virus particles apparently devoid of internal nucleoproteins, are consistent with the suggestion that lateral interactions between the influenza C glycoproteins may be important in virus assembly.

INTRODUCTION

Influenza C virus shares most of the biochemical properties of other members of the orthomyxovirus group, including a segmented RNA genome, a virion RNA polymerase, and the requirement for a host cell function in virus replication (Cox and Kendal, 1976; Compans et al., 197’7; Petri et aZ., 1979b, c; Palese et al., 1980; Meier-Ewert et al., 1980). However, a significant difference exists between influenza C virus and other orthomyxoviruses in the nature and function of the viral glycoproteins. In contrast to influenza A and ’ To whom reprint requests should be addressed. 439

B viruses, the receptor-destroying enzyme (RDE) activity shown by influenza C viruses is not an a-neuraminidase, since it does not liberate N-acetylneuraminic acid (NANA) from any of a variety of substrates for such enzymes (Hirst, 1950; Kendal, 1975). Further, influenza C virions appear to possess only a single type of glycoprotein, which resembles the hemagglutinin (HA) glycoprotein of influenza A viruses in several biochemical properties. We previously reported that two species of glycosylated polypeptides (gp65 and gp30) observed in purified influenza C virions grown in eggs or chick kidney (CK) cells are held together by disulfide bonds (Meier-Ewert et aZ., 1978) like the HA1 and 0042-6822/81/120439-13$02.00/O Copyright All rights

0 1981 by Academic Press. Inc. of reproduction in any form resewed.

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HA2 subunits of influenza A viruses (Laver, moved by centrifugation for 30 min at 1971). These subunits are cleavage prod30,000 rpm in a Beckman SW50.1 rotor. ucts of a primary gene product designated The glycoproteins in the supernatant were gp1 (Herrler et al, 1979). It was also demprecipitated by the addition of n-butanol. onstrated that proteolytic cleavage of this Fractionation with octylglucoside. Puglycoprotein is necessary for full biologirified influenza C virions were suspended cal activity of the virus (Herrler et aZ., in 0.1 M NaCl, 0.1 M Tris-HCl, pH 7.3, 1979), as is the case with the hemaggluat a protein concentration of about 1 tinin glycoprotein of influenza A viruses mg/ml, and n-octyl-@-D-glucopyranoside (Lazarowitz and Choppin, 1975; Klenk et was added to a final concentration of 2%. aZ., 1975) and the F glycoprotein of paraAfter 30 min at room temperature, the myxoviruses (Homma and Ohuchi, 1973; suspension was centrifuged at 30,000 rpm Scheid and Choppin, 1974). for 30 min in a SW50.1 rotor. The superThe glycoproteins of a number of en- natant was dialyzed for 20-24 hr at 4’ veloped viruses have been isolated using against 0.01 M NaCl, 0.01 M Tris-HCl, nonionic detergents. We describe here pH 7.3. some biochemical and biological properHemagglutination titration. Hemaggluties of isolated glycoproteins of influenza tination titers were determined as deC virus obtained by such procedures, as scribed previously (Compans, 1974). well as some morphological aspects of the Polyacrylamide gel electrophoresis. organization of the glycoproteins on the SDS-polyacrylamide gel electrophoresis viral envelope and the plasma membranes was performed as described previously of infected cells. (Herrler et aZ., 1979; Nakamura et ab, 1979). For analysis under reducing conMATERIALS AND METHODS ditions, samples were boiled for 2 min in the presence of 1% mercaptoethanol Vim~s. Stocks of the Johannesburg/l/66 For analysis unstrain of influenza C virus were grown in prior to electrophoresis. der nonreducing conditions, mercaptoethembryonated eggs as described previously anol was omitted. (Herrler et al., 1979). GZycopeptide analysis. Purified 3H-gluCells. The conditions for growth of cosamine-labeled virus was fractionated MDCK canine kidney cells and preparawith Triton X-100. Polypeptides in the sution of primary CK cells are described elsewhere (Nakamura and Compans, 1978; pernatant fraction were precipitated with n-butanol and separated under reducing Petri et aZ., 1979a). Chick embryo fibroblasts (CEF) were prepared from lo- or conditions on a 10% SDS-polyacrylamide gel. After briefly staining with Coomassie 11-day-old chick embryos. brilliant blue and destaining, bands were Radiolabeling and pur$icatiwn of virus. cut out and eluted in 0.1 M Tris-HCl, pH In order to obtain labeled virions, medium 8.0, 10 mM CaClz. Samples were lyophicontaining 10 PCi of 3H-leucine or 3H-glucosamine per ml was added to CK cells at lized, digested with Pronase, and analyzed on a &o-Gel P-6 column as described pre2 hr pi. At 24 hr p.i., virus was harvested, and Compans, 1977). concentrated by pelleting, and purified by viously (Nakamura sucrose gradient centrifugation as deAnalysis of tryptic peptides. Glycoproteins of purified 3H-leucine-labeled influscribed previously (Herrler et al., 1979). T&on fractionation. Purified influenza enza C virions were separated by SDSpolyacrylamide gel electrophoresis as C virions were fractionated using Triton X-100 according to the procedure de- described above. The stained gel slices scribed by Scheid et aZ. (1972). Virus was were lyophilized to remove residual acetic suspended in 2% Triton X-100,0.1 M Trisacid. After elution in 0.05 M NH4HC03, HCl, pH 7.4. After 15 min the RNP com- 0.1% SDS at 37” for 3 days, glycoproteins together with 100 pg plexes and most of the M protein were re- were precipitated

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bovine serum albumin by trichloroacetic acid (TCA) at a final concentration of 20%. Pellets were resuspended in 1 M NH,OH and precipitated once more with TCA and a third time with ethanol. The lyophilized samples were oxidized with performic acid (Crawford and Gesteland, 1973). The oxidized polypeptides were resuspended in 0.5 ml of 0.05 M NH4HC03 and digested with 25 pg of TPCK-trypsin at 37”. After 2 hr, an additional 25 pg of trypsin was added and incubation continued for 2 more hr. The tryptic peptides were lyophilized and dissolved in 1 ml of 0.05 M pyridine-acetic acid buffer, pH 3.0. After centrifugation for 5 min at 10,000 g, the supernatant was applied to a Durrum DC 1A cation-exchange column. The peptides were separated as described by Gentsch and Bishop (1978). Fractions of 3.5 ml were collected and dried at 80”. The radioactivity was determined in a liquid scintillation counter. Amino acid sequence determination. Sequential Edman degradation of tryptic peptides was achieved on a modified Beckman 890C automated sequenator (Bhown et al., 1980) using 0.5 M Quadrol containing AEAP glass beads (Bhown et aZ., 1981) and Polybrene as a carrier (Tarr et aZ., 1978). Polybrene (2 mg) in water was first applied to the cup and dried using the sample application subroutine (SAS) program of Beckman. It was then subjected to one complete cycle of Edman degradation. Following this initial treatment of the carrier, lyophilized peptide was dissolved in water and/or formic acid and applied to the cup. After a second SAS drying cycle was completed, the sample was subjected to regular sequential degradation. Phenylthiohydantoin (PTH) amino acids were identified spectrophotometrically at 254 nm by high-pressure liquid chromatography as described elsewhere (Bhown et aZ., 1981). PTH-Thr was detected at 313 nm as its dehydro product. Thin-layer chromatography (Summers et aZ., 1973) was employed as a second method of PTHamino acid identification. Electron microscopy. For negative staining, samples were applied to copper grids

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with carbon-coated Formvar films, stained with 2% sodium phosphotungstate, pH 7.2, and examined in a Philips 301 electron microscope. For examination of thin sections, cell monolayers grown on plastic dishes were fixed in situ with 1% glutaraldehyde for 1 hr and postfixed for 1 hr with 1% osmium tetroxide at 4”. The monolayers were dehydrated with 70%, 95%, and absolute ethanol, lifted intact off the plastic dishes with propylene oxide, and embedded in an epoxy resin. Thin sections were stained with uranyl acetate and lead citrate. Chemicals. Radioisotopes were obtained from Amersham-Buchler (England). Octylglucoside was purchased from Sigma Chemical Co., St. Louis, MO., and Triton X-100 from Merck (W. Germany). RESULTS

Isolation of glycoproteins with T&on X100. The glycoproteins of influenza C virions were isolated by selective solubilization with Triton X-100. After centrifugation of detergent-treated virions, the supernatant and pellet. fractions were recovered and analyzed on polyacrylamide gels. The supernatant fraction contained the purified glycoproteins, whereas the pellet fractions (not shown) contained the internal vjrion proteins. The Triton supernatants of virus preparations grown in CEF and CK cells are compared in Fig. 1. Under nonreducing conditions, only the gp1 glycoprotein is detected in CEF-grown virus, whereas two species of glycoproteins, gp1 and gpI1, are observed in CK cell-grown virus (lanes 1 and 2). We showed previously that the gpI1 glycoprotein is produced by proteolytic cleavage of gp1 into two subunits, which remain linked by disulfide bonds under nonreducing conditions (Herrler et aZ., 1979). Since a significant difference is found in the apparent molecular weights of the cleaved (gpI1) and uncleaved (gp1) glycoproteins of influenza C virions when analyzed in nonreducing gels, we compared their tryptic peptide patterns to determine whether a

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ClPl

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-

CIPII -c

,gp 88

FIG. 1. Isolation of the glycoprotein of influenza C virions with Triton X-100. Purified ‘H-leucine-labeled influenza C/JHB/66 virions grown in CK or in CEF cells were disrupted with 2% Triton X-100 and centrifuged at 100,000 g for 30 min. The polypeptides of the supernatant were precipitated with n-butanol, analyzed by SDS-polyacrylamide gel electrophoresis, and detected by fluorography. Lanes 1 and 4, supernatant fraction, CEF-grown virus; lanes 2 and 3, supernatant fraction, CK-grown virus. The samples in lanes 1 and 2 were eleetrophoresed under nonreducing conditions, and those in lanes 3 and 4, under reducing conditions.

segment of the polypeptide might be removed during cleavage. As seen in Fig. 2, the patterns obtained from gp1 and gpI1 were indistinguishable. However, since other data indicated that some carbohydrate components are lost upon cleavage (Herrler et aZ., 1979), it is likely that a segment of the molecule not labeled with 3Hleucine is removed upon cleavage. When the Triton supernatant was analyzed under reducing conditions (Fig. 1, lanes 3 and 4), gp1 was observed to migrate as a single glycoprotein, whereas gpI1 was split into the two subunits, gp65 and gp30. Though the majority of the M protein was found in the pellet fraction (not shown), traces of this polypeptide were also detectable in the supernatant fraction. The larger cleavage product gp65 was regularly observed as a doublet (Fig. 1, lane 3). This difference in electrophoretic mobility was not due to varying amounts of sialic

Fraction

No

FIG. 2. Comparison of tryptic peptides of the gp1, gpI1, and gp63 glycoproteins of influenza C/JHB/l/ 66 virus. The isolation of the glycoproteins and preparation and analysis of tryptic peptides was carried out as described in Materials and Methods.

acid residues in the two bands, since after neuraminidase treatment, the doublet band of gp65 was retained and the elec-

b

I I-

50 Fraction

100 No

150

FIG. 3. Tryptic peptide analysis of the doublet band of influenza C/JHB/1/66 gp65 glycoprotein. The virus was grown in CK cells and labeled with 3H-leucine. The upper (a) and the lower (b) bands of gp65 were cut from polyacrylamide slab gels, and tryptic peptides were prepared and analyzed as described in Materials and Methods.

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trophoretic mobility of the two bands was only marginally faster (not shown). To investigate whether the two gp65 bands differed in amino acid sequence, we isolated each band and compared their tryptic peptide patterns by ion-exchange chromatography. As seen in Fig. 3, the patterns obtained were virtually identical, demonstrating that the polypeptides share common amino acid sequences. In contrast, the tryptic peptide patterns obtained from gp65 and gp30 were completely different (Fig. 4). The pattern obtained from gpI1, the disulfide-linked complex of gp65 and gp30, corresponds to that obtained if the separate patterns of the two subunits are superimposed. When the extent of glycosylation was estimated by determining the 3H/14C ratios of each band in sliced SDS-polyacrylamide gels of preparations of isolated glycoproteins that were doubly labeled with 3H-glucosamine and 14C-amino acids, it was found that the upper band had a ratio of 3.5, compared to 2.6 for the lower band. This result suggests that the two bands may differ in carbohydrate content. However, no signif-

FIG. 4. Tryptic peptide analysis of influenza C/ JHB/1/66 virus glycoproteins gp30, gp65, and gpI1. The isolation of the proteins and preparation and analysis of tryptic peptides were carried out as described in Materials and Methods.

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C GLYCOPROTEINS

gP’ gp”

FIG. 5. Isolation of influenza C glycoproteins with octylglucoside. Influenza C/JHB/1/66 was grown in embryonated eggs or in chick kidney cells. Glycoproteins of purified virions were isolated using octylglucoside as described in Materials and Methods. SDSpolyacrylamide gel electrophoresis was performed under nonreducing conditions. The protein bands were stained with Coomassie brilliant blue G250. Lane 1, glycoproteins of virions grown in CK cells, isolated with 1% octylglucoside. Lanes 2-4, glycoproteins of egg-grown virions, isolated with 0.2% (lane 2), 1% (lane 3), or 5% (lane 4) octylglucoside.

icant differences were apparent in the elution profiles of glycopeptides derived from the two gp65 bands after extensive pronase digestion (not shown). Isolation of glycoproteim with octylglucoside. In contrast to results obtained with glycoproteins of paramyxoviruses isolated by solubilization with Triton X-100 (Scheid et al., 1972; Scheid and Choppin, 1974), we found that the ability to agglutinate chicken red blood cells had been lost in the influenza C glycoprotein preparations isolated using this detergent. It is not clear

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whether this is due to the butanol precipitation which was necessary to remove the detergent or whether the Triton-isolated glycoproteins of influenza C virus do not bind to red cells for other reasons. Therefore, octylglucoside was selected as another detergent, because it can be removed from the glycoproteins by dialysis. Analysis of the solubilized glycoproteins by polyacrylamide gel electrophoresis shows that this detergent is equally suitable for the isolation of the viral glycoproteins (Fig. 5). Further, when the isolated glycoproteins were tested for hemagglutination activity after extensive dialysis, a titer of 49,100 HAU/ml was observed. However, the isolated glycoprotein preparation did not show elution from erythrocytes after 1 hr at 20”, as is routinely observed with intact influenza C virions. This result may indicate that the isolated glycoproteins do not possess the capacity to destroy the virus receptor on the red cell membrane. N-Terminal amino acid sequences. To investigate further the structure of the influenza C glycoproteins, we attempted to determine the N-terminal amino acid sequences of gp1 and its cleavage products. Also, it was of interest to know whether any homology existed between the sequence observed at the cleavage site of the influenza C glycoprotein and the highly conserved sequence that has been observed at the N termini of the HA2 subunits of the influenza A and B hemagglutinin glycoproteins (Skehel and Waterfield, 1975). The gp1, gp65, and gp30 glycoproteins were isolated from SDS-polyacrylamide gels, then ethanol precipitated, and 500-1000 pmol was analyzed for N-terminal amino acid sequence by sequential Edman degradation using a liquid phase sequencer. Whereas a partial N-terminal sequence was obtained for gp30, it was observed that the N termini of gp1 as well as gp65 were resistant to sequential degradation, suggesting the presence of blocked N termini. These results suggest that gp65 is derived from the N-terminal portion of the precursor glycoprotein, and gp30 from the C-terminal portion. The gp30 terminal sequence contains a pre-

ET AL.

ponderance of hydrophobic residues and shows regions of sequence homology with the corresponding sequence of the HA2 subunit of influenza A viruses. In particular, the N-terminal tripeptide sequence Ile-Phe-Gly of influenza C gp30 corresponds exactly to a sequence observed in the HA2 glycoprotein of A/Victoria/3/75 (H,Nz) virus, but the latter contains an additional N-terminal glycine residue (Min Jou et al., 1980). However, a homologous sequence is also present at the N termini of the F1 subunits of paramyxovirus F glycoproteins, and in these glycoproteins the terminal glycine residue is absent (Gething et aZ., 19’78; Scheid et aZ., 1978), thus resembling the terminal sequence found in influenza C gp30. Morphology of viral glycoproteins. The glycoproteins on the surfaces of spherical as well as filamentous influenza C virus particles are usually found in regular hexagonal arrays (Figs. 6a and b). At high magnification, a single spike subunit is observed on each of the six vertices of the hexagons (Fig. 6~). The hexagonal organization appears to involve lateral interactions between the glycoprotein molecules themselves, since the spikes are sometimes observed to maintain their arrangement in a network upon release from the viral membrane by limited proteolytic digestion (Fig. 6d) or upon spontaneous disruption of the viral membrane (Fig. 6e). When the spikes are viewed end-on, as in Figs. 6c and e, they appear to have a rounded end, as opposed to the triangular shapes of the HA spikes of influenza A viruses in end-on views (Wrigley, 1979). The influenza C spikes have a length of 810 nm and a diameter of 4-5 nm; the center-to-center distance between spikes in hexagonal networks was determined to be 7.5 nm. The glycoproteins isolated by octylglucoside treatment were observed to form rosettes after removal of the detergent by dialysis (Fig. 6f), as expected for amphipathic integral membrane proteins. Maturation of in&enza C virus in MDCK cells. To study the assembly process of influenza C virions, we examined thin sections of infected MDCK cells, in which the virus undergoes a productive

FIG. 6. Electron micrographs of influenza C virions and glycoprotein clusters. (a) Filamentous particle completely covered with glycoproteins in a regular hexagonal arrangement. X215000. (b) Spherical particle showing the hexagonal arrangement of surface glycoproteins. X500,000. (c) Highmagnification view of a portion of a pleomorphic virus particle, showing the individual subunits (arrow) in a hexagonal pattern. X700,000. (d) Release of a network of subunits (arrow) from the surface of a virion upon treatment for 1 hr with 1 mg/ml of trypsin. X250,000. (e) Hexagonal pattern of subunits observed in a preparation containing spontaneously disrupted influenza C/ JHB/1/66 virions. X450,000. (f) Rosette structure observed in preparation of influenza C glycoproteins isolated with octylglucoside. X225,000.

445

FIG. 7. Electron micrographs of thin sections of MDCK cells 41 hr after infection with influenza C virus. (a) Low-magnification view showing virus particles at the apical surface of an infected MDCK cell; the basal surface is free of virus. X12,500. (b) Part of an apical cell surface showing virus particles, several of which appear devoid of nucleocapsids (arrows). Some crescent-shaped clusters of projections are found on the cell surface. X53,000. (c) High-magnification view of segment of the cell surface with clusters of projections on the cell surface. The small arrows indicate a particle in the process of budding, with electron-dense strands in the subadjacent cytoplasm of a 446

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replication cycle (Nerome and Ishida, 1978). These cells form monolayers in which tight junctions separate the plasma membrane into two domains, and maturation of several enveloped viruses has been reported to occur exclusively at one domain or the other. Influenza and parainfluenza viruses are assembled at the free apical surface, whereas vesicular stomatitis virus is formed at the basolateral membrane (Rodriguez Boulan and Sabatini, 19’78; Roth et cd., 1979). As seen in Fig. 7a, influenza C virus particles were observed on the apical surface of MDCK cells; no examples were observed of virus budding at the basolateral membranes. In addition to virus particles with typical dense internal cores (Figs. 7b, c, and e), numerous particles apparently devoid of nucleocapsids were observed in the process of budding or apparently released (Figs. 7b, c, and d). These particles possessed an electron-dense membrane covered with tightly packed surface projections, enclosing an area with an appearance similar to the cytoplasmic matrix. Frequently, tightly packed clusters of surface projections were also observed on crescent shaped outfoldings of the plasma membrane on cell surfaces where virus maturation was occurring (Figs. 7c and d); such areas were apparently devoid of underlying electron dense nucleocapsid structures. These observations suggest that nucleocapsids may not be required to initiate the budding process of influenza C virions. DISCUSSION

In order to unambiguously assign biological functions to the influenza C glycoprotein, it was necessary to obtain the glycoprotein in pure form. Triton X-100 fractionation resulted in an almost complete separation between the surface protein and the internal unglycosylated polypeptides. However, in contrast to the

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hemagglutinin of paramyxoviruses (Scheid et aZ., 1972), the isolated influenza C glycoprotein did not show any hemagglutinating activity. Hemagglutination was observed, however, when the glycoprotein was isolated by solubilization with octylglucoside. Since this detergent was removed by dialysis, lipids were still present in the glycoprotein fraction, whereas in the Triton X-100 fractionation, lipids were separated during the butanol precipitation step. Thus it is possible that hemagglutination requires the presence of lipids in addition to the influenza C glycoproteins. It is possible that during dialysis, lipid vesicles are formed which incorporate glycoproteins, thus producing the conformation required for hemagglutinating activity; liposome-like structures were sometimes observed when the glycoprotein preparations obtained with octylglucoside were examined in the electron microscope. It was not possible to assign the receptor-destroying activity to the influenza C virus glycop-rotein. The glycoprotein isolated by octylglucoside caused hemagglutination, but the pattern observed was unusual in that it was stable even after several hours, whereas the hemagglutination pattern caused by intact influenza C virions starts to disappear after 1 hr at room temperature. Thus there is still uncertainty about the protein responsible for the receptor destroying activity. It seems unlikely that a minor unidentified polypeptide has this function. The genome of influenza C virus apparently consists only of seven RNA segments (Petri et a!., 1979b, 1980; Palese et aZ., 1980) in contrast to the eight segments of influenza A and B viruses. Six structural and two nonstructural polypeptides have been described for influenza C viruses: P1, Pz, P3, gp1, NP, M, NS1, and NSz (Petri et cd., 1980). Even if the two NS proteins are products of a sin-

size (9-10 nm in diameter) corresponding to viral nucleoprotein complexes. An extracellular particle apparently devoid of nucleocapsids is indicated by the large arrow. X95,000. (d) Clusters of projections on a region of the plasma membrane (arrows), and an extracellular virus particle, both apparently lacking associated nucleocapsids. X109,090. (e) High-magnification view of a virus particle with nucleocapsids cut in cross section, and a tightly packed layer of surface projections. x180.900

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gle gene as was found with influenza A viruses (Inglis et aZ., 1979; Lamb and Choppin, 1979), there would be no genome segment available to code for an additional polypeptide, unless another segment also codes for two gene products. Thus, the possibility still exists that one influenza C virus glycoprotein could be responsible for three biological functions: hemagglutination, receptor-destroying activity, and virus penetration (see below). The lack of receptor-destroying activity in isolated glycoproteins might be due to a conformational change during isolation. Also, the lack of a sensitive assay for the receptor-destroying activity might contribute to our inability to demonstrate this activity in isolated glycoproteins. Previous protein and nucleic acid sequence analysis has shown that the N terminus of the HA2 polypeptide of influenza A and B viruses is highly conserved and consists of hydrophobic residues (Waterfield et al, 1979, Ward and Dopheide, 1979; Porter et aZ., 1979; Min Jou et al., 1980). A related sequence was also found at the N termini of the F1 polypeptide of several paramyxoviruses, and it has been proposed that this sequence might be important for the penetration of these viruses into cells (Gething et aZ., 1978; Scheid et aZ., 1978). Recently evidence supporting this proposal was obtained in a study of the effect of oligopeptides on the replication of paramyxoviruses and influenza viruses (Richardson et al, 1980). It was found that oligopeptides with amino acid sequences that resemble those of the N termini of the F1 polypeptide or the HA2 polypeptide are potent inhibitors of the infectivity of the corresponding virus. Those oligopeptides inhibiting the replication of paramyxoviruses were shown also to be inhibitors of cell fusion and hemolysis induced by these viruses. In the present study, we found that gp30, one of the two cleavage products of the influenza C virus glycoprotein (Herrler et al., 1979), also has a homologous amino acid sequence at its N terminus. Although the Nterminal tripeptide of gp30 corresponds to a sequence in the HA2 polypeptide of influenza A/Victoria/3/75 (Min Jou et al.,

ET AL.

1980), it was found not to contain an Nterminal glycine residue, in contrast to the HA2 polypeptides of all influenza A and B strains so far analyzed (Laver and Air, 1980). As a similar sequence lacking an Nterminal glycine was observed in the paramyxovirus F1 polypeptide, the influenza C glycoprotein more closely resembles the paramyxovirus F glycoprotein with respect to the N-terminal sequence exposed by proteolytic cleavage. The N-terminal amino acid sequence of gp37 of Rous sarcoma virus is also generated by cleavage of a precursor and is characterized by a preponderance of hydrophobic residues (E. Hunter, personal communication) but shows less homology than can be observed among influenza A, B, and C viruses and paramyxoviruses. The N termini of gp65, the other cleavage product, as well as the uncleaved glycoprotein gp1, were both found to be resistant to sequential Edman degradation. This finding indicates that gp65 is located at the N-terminal and gp30 at the C-terminal portion of gp1. This result is also consistent with the previous finding that gp30 is glycosylated to a lesser extent than gp65 (Herrler et al., 1979). In paramyxoviruses as well as in influenza A and B viruses, the N-terminal cleavage products of the F and HA polypeptides contain more carbohydrate than the C-terminal parts (Laver, 1971, Scheid and Choppin, 1977), and in influenza A and B viruses, HA2 is the membrane bound part of the HA polypeptide (Compans et al., 1970; Skehe1 and Waterfield, 1975). By analogy, it is likely that the gp30 portion binds the influenza C virus glycoprotein to the viral membrane. The glycoproteins on intact influenza C virions are arranged in a characteristic pattern, forming a reticular structure consisting mainly of hexagons. Our results indicate that these structures are held together at least in part by lateral interactions between glycoprotein subunits, because they were also found after removal from the membrane upon protease treatment or after spontaneous release. Such a reticular pattern was observed previously in the envelope of influenza C vi-

STRUCTURE

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rions, and it was suggested that an underlying structure in the viral membrane may be responsible for the observed arrangement (Waterson et aZ., 1963; Flewett and Apostolov, 1967). No convincing evidence for such a regular arrangement of spikes has been found on the surfaces of most influenza A viruses; the presence of morphologically distinct hemagglutinin and neuraminidase spikes with different elements of symmetry may preclude the formation of a regular surface lattice. If the hexagonal arrangement of influenza C glycoproteins is determined by lateral interactions between the glycoprotein spikes, as suggested in some electron micrographs, it would follow that the spike structure exhibits threefold symmetry as does the influenza A hemagglutinin spike (Wrigley, 1979). Influenza C particles apparently devoid of internal nucleocapsid structures were frequently observed in studies of thin sections of infected cells. In addition, tightly packed clusters of surface projections were observed on crescent-shaped outfoldings of the plasma membrane of infected cells; such outfoldings could presumably give rise to the empty particles. In contrast to these observations, empty particles were only rarely observed for influenza A viruses (Compans and Dimmock, 1969). The formation of apparently empty particles at a high frequency suggests that interactions between envelope proteins of influenza C virus may be sufficient to lead to the process of virus budding; nucleocapsids are apparently not required to initiate outfolding of the membrane. The present results do not enable us to determine whether a layer of M protein is present in influenza C particles devoid of nucleocapsids or whether interactions between glycoproteins alone are sufficient to lead to budding at the cell surface. Taken together, the present results and previous findings show that influenza C virus possesses several properties which are significantly different from influenza A and B virus. As a consequence, influenza C virus could be established as a separate genus within the family orthomyxoviridae.

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ACKNOWLEDGMENTS We thank Gisela Foerst for excellent technical assistance, L. R. Melsen for assistance with electron microscopy, and S. Basak for analyses of glycopeptides. This research was supported by the Deutsche Forschungsgemeinschaft, USPHS Grants AI12680 and CA13148, and NSF Grant PCM 80-06498. REFERENCES A. S., CORNELIUS, T. W., MOLE, J. E., LYNN, J. D., TIDWELL, W. A., and BENNET& J. C. (1980). A simple modification on the vacuum system of the Beckman automated sequencer to improve the efficiency of Edman degradation. Anal. B&hem. 102, 35-38. BHOWN, A. S., MOLE, J. E., and BENNETT, J. C. (1981). An improved procedure for high sensitivity microsequencing: Use of aminoethyl aminopropyl glass beads in the Beckman sequencer and their ultrasphere ODS column for PTH amino acid identification. Anal. Biochem. 110,355-359. COMPANS, R. W. (1974). Hemagglutination-inhibition: rapid assay for neuraminic acid-containing viruses. J. Viral. 14, 1307-1309. COMPANS, R. W., and DIMMOCK, N. J. (1969). An electron microscopic study of single-cycle infection of chick embryo fibroblasts by influenza virus. Virology 39,499-515. COMPANS, R. W., KLENK, H.-D., CALIGUIRI, L. A., and CHOPPIN, P. W. (1970). Influenza virus proteins. I. Analysis of polypeptides of the virion and identification of spike glycoproteins. Virology 42, 880889. COMPANS, R. W., BISHOP, D. H. L., and MEIER-EWERT, H. (19’77). Structural components of influenza C virions. J. Viral. 21, 658-665. COX, N. J., and KENDAL, A. P. (1976). Presence of a segmented single-stranded RNA genome in influenza C virions. Virology 74, 239-241. CRAWFORD,L. V., and GESTELAND, R. F. (1973). Synthesis of polyoma proteins in vitro. J. 1MoZ.Biol. 74. 627-634. FLEWETT, T. H., and APOSTOLOV, K. (1967). A reticular structure in the wall of influenza C virus. J. Gen. Viral. 1, 297-304. GENTSCH, J. R., and BISHOP, D. H. L. (1978). Small viral RNA segment of bunyaviruses codes for viral nucleocapsid protein. J. Viral. 28, 417-419. GETHING, M. J., WHITE, J. M., and WATERFIELD, M. D. (1978). Purification of the fusion protein of Sendai virus. Analysis of the NH,-terminal sequence generated during precursor activation. Proc. Nat. Acad. Sci. USA 75,2737-2740. HERRLER, G., COMPANS, R. W., and MEIER-EWERT, H. (1979). A precursor glycoprotein in influenza C virus. Virology 99, 49-56. BHOWN,

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