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The structural proteins of Newcastle disease virus
VIROLOGY
61, 229-239
(1974)
The Structural
Proteins
I. Identification MASAO Institute
of Microbiology,
Rutgers
of a Minor
IINUMA’ University,
of Newcastle
AND
Internal
ROBERT
The State Uniuersity 089032 Accepted
May
Disease
Virus
Protein
W. SIMPSON of New
Jersey,
New Brunswick,
New Jersey
29, 1974
Purified Newcastle disease virus (NDV), Miyadera strain, was dissociated and its proteins were analyzed by SDS polyacrylamide gel electrophoresis. Polypeptide VP 9 was consistently found to be the fastest migrating viral protein with an estimated molecular weight of approximately 26,500 when compared to known protein markers. VP 9 comprises approximately 3% of the total viral protein of virions grown in either chick embryo or HeLa cells. VP 91 was also identified by immunological procedures. Hyperimmune Ab-9 serum prepared against the purified VP 9 protein (eluted from polyacrylamide gels), was incapable of neutralizing the hemagglutinin or neuraminidase activities of NDV. In addition, this antibody did not react with the S-antigen (nucleoprotein) of the virus. Complement-fixation (CF) tests revealed that this antibody could react with solubilized NDV, but not intact virions. Immunofluorescent staining of NDV-infected HeLa cells with FITC-labeled Ab-9 resulted in cytoplasmic fluorescence which was not blocked by pretreatment with either unlabeled virus-specific antibody or anti-S antibody. To determine the relative proportion of VP 9 antigen in NDV stocks grown in various cell types, CF tests were performed with detergent-dissociated NDV grown in different cells. If the amount of test virus used in these CF reactions was standardized for S-antigen units, the CF titers for VP 9 antigen were the same for virus preparations grown in various cell types, whereas up to an &fold variation in titer was observed if standardization was based on hemagglutinin or neuraminidase units. It is concluded that VP 9 is an internal polypeptide of NDV virions which does not constitute part of the nucleoprotein complex and which does not exhibit significant quantitative variations as influenced by host factors. INTRODUCTION
Recent studies in several laboratories have established the identity of several of the polypeptides of influenza virus (Kilbourne et al., 1972). In contrast to influenza virus, relatively little information is available on the structural proteins of the paramyxovirus, Newcastle disease virus (NDV), even though these components have been extensively studied in several laboratories (Evans and Kingsbury, 1969; Haslam et al., 1969; Bike1 and Duesberg, I Permanent address: Department of Cancer Research Institute, Nagoya University of Medicine, Nagoya, Japan. *Reprints are available at this address.
Virology, School
1969; Mountcastle et- al., 1970, 1971; Iinuma et al., 1971b; Scheid and Choppin, 1973). Although it has been shown that purified NDV contains three major polypeptides, the exact number of minor polypeptides remains unknown. In addition, the structure-function relationships for most of these proteins within the virion are not yet clearly understood. One of the major polypeptides, however, has already been identified as the nucleocapsid protein of the virus (Evans and Kingsbury, 1969; Haslam et al., 1969; Bike1 and Duesburg, 1969; Mountcastle et al., 1970; Scheid and Choppin, 1973). Recently, Mountcastle et al. (1971) and Scheid and Choppin (1973)
230
IINUMA
AND
have suggested that the smallest known protein of NDV is a major protein which has a molecular weight of approximately 41,000. During our studies on the protein components of NDV, we discovered a new minor protein, VP 9, which is described in this paper utilizing analysis by polyacrylamide gel electrophoresis (PAGE) and immunological techniques for its identification. It will be shown that VP 9 is an internal protein representing approximately 3% of the total viral protein. MATERIALS
AND
METHODS
Virus and virus purification. The Miyadera strain of Newcastle disease virus (NDV) was used throughout this study. The method for purification of virus has previously been described (Iinuma et al., 1971a, b; 1973). Cell cultures. Primary chick embryo fibroblasts (CEF) were prepared by the method of Simpson and Hirst (1961). Primary cultures of bovine embryonic kidney (BEK) cells were obtained from 10 to 50-g embryos by digesting with 0.25% trypsin (Nutritional Biochemicals Corp., Ohio) and 0.01% collagenase (Worthington Biochemical Corp.) after whole kidney was minced. A more detailed account of the technique used will be given elsewhere (Iinuma and Simpson, in progress). Continuous lines of HeLa, HEp-2, and BHK-21 cells were propagated in reinforced Eagle’s MEM (Bablanian et al., 1965) supplemented with 10% fetal calf serum. Virus growth. In eggs: NDV stocks were diluted 1: 1000 with cold phosphate-buffered saline (PBS) and inoculated into the allantois of ll-day-old embryonated eggs. After incubation at 37” for 48 hr, the eggs were chilled, and allantoic fluids were harvested and subjected to purification. In cell culture: monolayer cultures (CEF, BEK, HeLa, HEp-2, or BHK-21) were infected with egg-grown NDV at an input multiplicity of approximately 8 PFU per cell after they were washed with modified Hanks’ balanced salts solution (BSS). Virus was allowed to adsorb for 1 hr at 37” after which the inoculum was removed by
SIMPSON
washing three times with BSS. Maintenance medium (reinforced MEM supplemented with 3% fetal calf serum) was added and the cultures were incubated for 24 hr at 37”. Cells and culture fluids were harvested and centrifuged at 8000 rpm for 10 min (4”) after three cycles of freezing and thawing. The resulting supernatants were subjected to purification. Isotopically labeled virus: isotopically labeled virus was prepared in CEF or HeLa cells. To NDVinfected cells, 0.5 &i/ml of [‘4C]glucosamine or 14C-labeled amino acid mixture, or 5 pCi/ml of [3H]leucine, or combinations of these isotopes, was added after 1 hr postinfection. Incubation was continued for 24 hr and culture medium was harvested. Neuraminidase assay. Neuraminidase assay was performed with neuraminlactose substrate by a modification of the Warren technique (Laver and Kilbourne, 1966; Maeno and Kilbourne, 1970). N-Acetylneuraminic acid-n-lactose (neuraminlactose) prepared from bovine colostrum was kindly supplied by Dr. M. Pons. An 0.2-ml volume of test material diluted with saline was added to 0.1 ml of substrate and adjusted to pH 5.0 (optimum pH for NDV enzyme) with 0.1 ml of 0.4 M sodium acetate buffer. After 30 min of incubation in a water bath at 37”, 0.2 ml of the reaction mixture was used for the assay of free N-acetylneuraminic acid (NANA). Optical density was read at 549 nm (OD,,,) against a blank tube containing neuraminlactose plus saline. Enzyme activity was measured by OD readings taken from the part of the slope where they vary linearly with the enzyme concentration. Enqme inhibition (EI) tests. One unit of neuraminidase was defined as the amount of enzyme required to yield sufficient NANA to produce an OD reading of 0.1 at 549 nm. Serial 2-fold dilutions of antibody in 0.2-ml volumes were combined with 0.2 ml of 5-7 enzyme units (virus particles) and incubated for 1 hr at 37” before the neuraminidase assay was performed. EI titer was expressed as the highest dilution of antibody which gave a 50% decrease in OD reading. EI-antibody blocking tests. An 0.2-ml
PROTEINS
volume of test material (VP 9 protein or virions) was added to the same volume of anti-NDV serum (1: 20), and incubated for 60 min at room temperature. After this, 0.2 ml of 5 units of enzyme (virus particles) was added to each mixture. After further incubation for 60 min at room temperature, neuraminidase activity of the final reaction mixtures was determined. Treatment of virus with detergents or proteolytic enzymes. Purified preparations of virus were treated with 0.01 M sodium deoxycholate (DOC) or sodium dodecyl sulfate (SDS) in 0.01 M Na,HPO, and 0.14 M NaCl, pH 7.5. The mixture was held at 37” for 2 hr with stirring and then used as a CF antigen after dilution with Kolmer’s solution. For enzymatic digestion of virus, purified NDV was mixed with either chymotrypsin (Sigma) or trypsin (Difco) at a final concentration of 2.0 mg/ml in 0.1 M phosphate buffer, pH 7.6, and incubated in a water bath at 37” for 2 hr. After chilling in an ice bath, these mixtures were centtrifuged twice at 22,500 rpm for 1 hr at 4” in a Spinco No. 40 rotor (before and after washing with PBS). The resulting pellets were resuspended to original volume in PBS and used as CF-antigens. Electrophoresis and preparation of VP 9. Virus samples were suspended in 1% SDS and 1% 2-mercaptoethanol in 0.01 M sodium phosphate buffer, pH 7.2, and incubated at room temperature for 2 hr. After dialysis overnight at room temperature (0.1% SDS, 0.1% mercaptoethanol, and 10% sucrose in 0.1 M sodium phosphate buffer, pH 7.2) 50-18 samples were layered on 7.5% polyacrylamide gels prepared as described previously (Iinuma et al., 1971b). Electrophoresis was performed at 5 mA per gel for 5 hr after which the gel was stained (0.5% Coomassie Brilliant Blue in 25% isopropyl alcohol and 10% acetic acid) and destained by repeated changes of 7% acetic acid. The regions of several unstained gels corresponding to the location of the VP 9 component of a stained reference gel run in parallel were collected by slicing and homogenized in 0.1 ml of water per gel. After standing overnight in an ice bath, these gel-water mixtures were
OF NDV
231
combined with an equal volume of PBS and clarified by low-speed centrifugation (3000 rpm for 30 min). The resulting supernatant (containing the gel-eluted protein) was stored at -78”. For coelectrophoresis of NDV virion polypeptides and isolated VP 9, VP 9 labeled with [3H]leucine was collected from 10 PAGE gels as described above. Yields of VP 9 protein eluted from PAGE were relatively low accounting for approximately 30% of the starting material. A 1.8-ml quantity of the pooled VP 9 gel eluate was mixed with the same volume of 10% trichloroacetic acid (TCA) and allowed to stand at 4” for 30 min. The TCA precipitate was mixed with 1.8 ml of concentrated NDV virus particles labeled in their proteins with 14C-labeled amino acids followed by addition of 5.4 ml of absolute ethanol. Finally, the resulting ethanol precipitate was subjected to coelectrophoresis. For counting, gels containing radioactive proteins were cut into 1.5-mm slices which were placed into scintillation vials with 0.2 ml of H,O, and stored at 50” for 3 days. Thereafter, 10 ml of modified Bray’s solution (without methanol and ethylene glycol) was added to each vial and radioactivity was measured in a Packard Tri-Carb spectrometer. The following proteins were treated with SDS and 2-mercaptoethanol as described by Shapiro et al. (1967) and used as reference markers for molecular weight determinations: bovine serum albumin (MW: 67,000); ovalbumin (MW: 45,000); trypsin (MW: 23,000). Preparation of antiserum against VP 9. Gel-eluted VP 9 protein (stored at -78”) was inoculated intravenously into rabbits for a total of 20 times at 2-day intervals, using 7.5 pg of protein per injection. Hyperimmune serum (Ab-9) was obtained from the rabbits bled 7 days after the last injection. Ab-9 serum was heated at 56” for 30 min before use. Immunofluorescent staining. A yglobulin fraction was prepared from immune rabbit serum by precipitation with cold, one-third-saturated ammonium sulfate. After conjugation with fluorescein isothiocyanate by the method of Shimojo et al. (1967), the conjugate was immedi-
232
IINUMA
AND
SIMPSON
ately passed through a Sephadex G-50 gel column and further purified by passage through a diethylaminoethyl (DEAE) cellulose column and absorption with ace> tone-precipitated chick embryo powder to reduce nonspecific staining. HeLa cells grown on cover slips were infected with NDV, and’ after 9 hr they were stained with the antibody with or without prior fixation. Examination of these preparations was carried out with a Reichert Zetopan microscope equipped for fluorescence microsCOPY.
Additional techniques. The techniques of hemagglutinin titration, hemagglutination-inhibition (HI) test, antibody-blocking tests for hemagglutination, complement-fixation (CF) test, and preparation of antibodies against viral envelope protein (V-antigen) or nucleoprotein (S-antigen) have been described (Maeno et al., 1970; Iinuma et al., 1971a, b, 1973). The protein content of different samples was measured by the method of Lowry et al. (1951). RESULTS
Electrophoretic pattern of NDVproteins and the fastest migrating protein. Experiments carried out by one of us (Iinuma et al., 1971b) suggested that purified radioactive NDV, Miyadera strain, subjected to analysis by PAGE, contained small amounts of a protein, corresponding to a minor band on the stained gel which migrated to a position near the anode. Further experiments were conducted in the present study to determine whether this component represented a virus-specific protein. Figure 1 illustrates the electrophoretie pattern of NDV Miyadera proteins labeled in the presence of [3H]leucine and [‘%]glucosamine. Three major peaks of 3H radioactivity (VGP 4, VP 6, and VP 8) and some minor peaks of 3H radioactivity (VP 1, VP 2-3, VGP 5, VP 7, and VP 9) were detected. Of these polypeptides, VGP 4 and VGP 5 were identified as glycoproteins based on their labeling with radioactive glucosamine. Of particular interest was the finding of a small peak, designated as VP 9, which consistently was observed as the fastest migrating protein in different gel
FRACTION
NUMEER
FIG. 1. Electrophoretic pattern of NDV virion polypeptides and standard reference proteins. Puritied CEF-NDV labeled both with [aH]leucine and [ “Clglucosamine was mixed with the standard proteins indicated, dissociated with SDS and mercaptoethanol and subjected to PAGE analysis. After the gel was stained with Coomassie Brilliant Blue and destained with acetic acid, the gel was sliced and the discs were counted for their radioactivity. The small peaks labeled VP 2 and VP 3 were frequently, but not always, observed as a single peak in repeat experiments. The distance migrated by each band was plotted against the logarithm of the known molecular weight of each marker (Shapiro et al., 1967). BSA; bovine serum albumin (MW: 67,000); OV; ovalbumin (M,: 45,000); TR; trypsin (M,: 23,800).
runs. In order to characterize this constituent, VP 9 labeled with [3H]leucine was eluted from unstained PAGE gels (Materials and Methods) and coelectrophoresed with %-labeled virion proteins of NDV grown in HeLa cells (Fig. 2). The electropherogram of Fig. 2 demonstrates that VP 9 protein recovered in this way was free of contamination from all the other viral proteins and retained its original electrophoretic migration properties. Assuming that the [3H]leucine or “Clabeled amino acids were incorporated equally into all NDV proteins, VP 9 accounted for 2.9% of CEF-NDV and 3.1% of HeLa-NDV of the total viral protein, respectively. A value of 26,500 for the molecular weight of VP 9 was obtained by the method of Shapiro et al. (1967), as shown in Fig. 1 and Fig. 2. These prelimi-
PROTEINS
g 300
110
i L 0 0
100
a00
0 f 100
SO
10 FRACTION
30
30
233
OF NDV
Oroszlan, et al., 1971). This gel eluate of VP 9 protein did not show any detectable hemagglutinin or neuraminidase activities, nor did it react with anti-S (nucleocapsid protein) antibody in CF-tests or antibodies active in hemagglutination inhibition (HI) and neuraminidase inhibition (EI). Hyperimmune serum prepared against VP 9 (Ab-9) in rabbits lacked the capacity to neutralize hemagglutinin or neuraminidase activity of intact NDV particles and could not react with viral S-antigen in CF tests (Table 1). Complement-fixation tests were also performed to determine whether the Ab-9 serum could react with NDV
NUMBER
FIG. 2. Coelectrophoresis of NDV virion polypeptides labeled with “C-labeled amino acids and polypeptide, VP 9 labeled with [3H]leucine (isolated from PAGE gels). Purified CEF-NDV labeled with [*H]leutine was dissociated with SDS and mercaptoethanol, and subjected to PAGE. The fastest migrating protein (VP 9) was eluted from 10 PAGE gels as described in Materials and Methods. VP 9 thus obtained was mixed with HeLa-NDV proteins labeled with “Clabeled amino acids, dissociated as described above and coelectrophoresed on the same gel. Radioactivities of gel slices and the molecular weight of VP 9 were determined as described in Fig. 1. (Abbreviations: see footnote in Fig. 1.)
TABLE
Hemagglutination inhibition: Neuraminidase inhibition: Anti-S titration:”
nary results suggested that polypeptide VP 9 might be a viral-specific protein hithertofore not reported possibly because of difficulties in demonstrating it with other NDV strains. Antigenicity of VP 9 eluted from polyacrylamide gels. The possibility of whether the VP 9 protein eluted from PAGE gels is antigenic was tested by immunizing rabbits with this material (Materials and Methods). Pooled gel slices derived from multiple gels used for electrophoretic separation of VP 9 protein were eluted in water (Materials and Methods) and finally suspended in PBS to provide material for rabbit immunization and various in vitro tests. It was of interest to determine whether the VP 9 protein eluted from polyacrylamide gels had biological activity since other workers have recovered enzymatically or serologically reactive proteins by this technique (e.g., Anfinsen, 1962;
D NDV (Miyadera strain) grown in eggs was dissociated and run in polyacrylamide gels (Materials and Methods) to separate the minor polypeptide VP 9. Gel cuts of VP 9 protein from multiple gels were eluted to provide the purified material for preparing the rabbit antiserum (Ab-9). Anti-NDV serum was prepared by immunization of rabbits with whole virus particles (Materials and Methods). The anti-S serum was prepared by rabbit immunization with nucleocapsids of NDV obtained from virus disrupted with DOC and centrifuged through a 5% to 20% sucrose gradient as described previously (Iinuma et al., 1971b). The anti-S titer of the serum after absorption with virus particles to remove antibodies against envelope antigens was l/128. Complement-fixation tests were done according to a modification of Kolmer’s method. b All titers are expressed as the reciprocal of the highest dilution of serum showing positive activity. ‘The S-antigen was obtained from HeLa cells infected with NDV. Extracts of HeLa cells were subjected to sedimentation by CsCl gradient centrifugation according to Compans and Choppin (1967). The nucleocapsid band was harvested and dialyzed against PBS.
INHIBITORY
1
ACTIVITY OF ANTISERUM (Ab-9) PURIFIED VP 9 PROTEIN=
Rabbit Test
Preimmune serum
AGAINST
antiserum”
Ab-9
AntiNDV
AntiS
<5
<5
5120
<5
<5
<5
480
<5
<4
<4
<4
128
234
IINUMA
AND
structural proteins. As shown in Table 2, a high CF titer was obtained in the reaction only with detergent-solubilized NDV but not with intact NDV virions before or after digestion with either chymotrypsin or trypsin. In addition, this antibody could react neither with the DOC-solubilized heterologous paramyxovirus, Sendai, nor with the extracts of uninfected allantoic membranes. These results suggested that the VP 9 protein is a viral structural protein internally located in NDV virions. To confirm the observations described above using other approaches, immunofluorescent monitoring of NDV-infected cells with FITC-labeled Ab-9 was employed. HeLa cells grown on coverslips were infected with NDV at an input multiplicity of approximately 8 PFU/cell and stained with FITC-labeled Ab-9 at the 9th hr of infection with or without prior fixation. Large amounts of intracellular virus precursor proteins were previously found to accumulate at this time of infection (Iinuma et al., 1971a, 1973). The micrographs presented in Fig. 3 illustrate that when infected cells were stained after fixation, a TABLE COMPLEMENT-FIXATION
TESTS WITH VARIOUS
Antigens
NDV
SIMPSON
cytoplasmic fluorescence marked by strong staining in the perinuclear area was observed (Fig. 3 A). Conversely, staining without prior fixation resulted in no fluorescence (Fig. 3 B). Attempts to block virus-specific fluorescence by either unlabeled anti-S or anti-NDV globulin resulted in no significant difference in the extent or intensity of cytoplasmic fluorescence (Fig. 3 C and D). It is important to note that no fluorescence could be detected in noninfected cells by staining with FITC-labeled Ab-9 after fixation. These results supported the observations described above and suggested that the protein VP 9 was indeed incorporated into virions during maturation but not in the modified host cell membrane itself. Amount of VP 9 protein detected in NDV particles grown in various cell types. The results of experiments described in the preceding sections suggested that the protein VP 9 was a virus-specific protein situated within virions and internal to the envelope surface components. Further experiments were carried out to determine whether the proportion of VP 9 antigen in 2
PREPARATIONS PROTEIN“
Hemagglutination activity (HAW0.25 ml)
AND ANTISERUM
Neuraminidase activity
(OD,,,/O.l
ml,
AGAINST
PURIFIED
CF titer
of serum:
Preimmune
VP 9
Ab-9
1:600)
Purified NDV DOC-dissociated NDV SDS-dissociated NDV Chymotrypsinized NDV’ Trypsinized NDV’ Purified Sendai DOC-dissociated Sendai Allantoic membrane extract
4000 NTb NT 4000 4000 4000 NT <2
0.492 NT NT 0.283 0.508 NT NT 0
<4 <4 <4 <4 <4 <4 <4 <4
<4 256 256 <4 <4 <4 <4 <4
a The same rabbit Ab-9 antiserum used in the tests described for Table 1 was employed. Antigens were prepared as described in Materials and Methods. As controls, extracts of uninfected allantoic membranes or the heterologous paramyxovirus, Sendai, were used. Allantoic membranes were harvested from uninfected ll-dayembryonated eggs and washed throughly with PBS. The homogenized materials were subjected to three cycles of freezing and thawing and centrifugation at low speed. The resulting supernatant fluid was used as an extract of allantoic membranes. The CF titer was expressed as the reciprocal of the highest dilution of antigen showing more than 75% fixation of complement per 0.2ml volume. b NT = not tested. L Plaque-forming capacity of chymotrypsin-treated NDV was unaffected, even though the treatment reduced viral neuraminidase activity. Trypsin was without effect on the viral activities measured.
PROTEINS
OF NDV
235
Fro. 3. Fluorescent-antibody staining of NDV-infected cells, using antibody against purified VP 9 protein. HeLa cells grown on coverslips were infected with NDV at an input multiplicity of 8 PFU/cell and incubated at 37” for 9 hr. After washing with PBS, they were allowed to react with FITC-labeled Ab-9 with (A, C, D, E) or without (B) prior fixation with cold acetone. Cytoplasmic fluorescence observed after fixation (A) or without prior fixation (B). Cells pretreated with either FITC-unlabeled anti-S serum (C) or anti-NDV serum (D) before staining. The titer of unlabeled anti-S serum (1:20) or anti-NDV serum (1: 40) was sufficient to block the cytoplasmic fluorescence obtained by either FITC-labeled anti-S or anti-NDV, respectively. Specific fluorescence was completely blocked by pretreatment with FITC-unlabeled Ab-9 (E). Noninfected HeLa cells with or without prior fixation showed no fluorescence by staining with FITC-labeled Ab-9 (not shown).
NDV particles grown in various cell types shows’ host-controlled variation with the assumption that if VP 9 was an internal protein of this virus, the amount of VP 9 might not be affected by the host cell factors (Stanley and Haslam, 1971; Lazorowitz et al., 1973). Viruses grown in various cell systems (HeLa, HEp-2, BHK-21, BEK, CEF cells, or embryonated eggs) were prepared and purified as described in
Materials and Methods. For preparing CF test antigen, purified viruses were dissociated with 0.01 M SDS after diluting each virus preparation with saline to the same concentration for hemagglutinin or for neuraminidase units. As illustrated in Tables 3 and 4, the apparent amount of VP 9 protein based on CF titer was found to vary in NDV grown in various cell types when standardized against these surface
IINUMA
236
AND SIMPSON TABLE
RELATIVE TITER OF COMPLEMENT-FIXATION
3
VP 9 ANTIGEN
IN NDV GROWN IN VARIOUS CELL TYPES AS DETECTED IN TESTS USING STANDARDIZED AMOUNTS OF HEMAGGLUTININ UNITS (HAU)O
NDV grown in
Hemagglutinin activity” (HAW0.25 ml)
S-antigen (CF-titer)
VP 9 antigen (CF-titer)
HEp-2 HeLa BHK-21 BEK Egg CEF
1280 1920 1920 1280 1920 1280
512 512 256 256 256 64
512 512 256 128 128 64
Calculated VP 9 CF titer per 1000 HA units 400 (8.O)C 267 (5.3) 133 (2.7) 100 (2.0) 67 (1.3) 50 (1.0)
a Viruses grown in various cell types were prepared and purified as described Purified viruses were dissociated with SDS at a final concentration of 0.01 A4 and units of antibody (anti-S Ab-9) were used. b Hemagglutinin activity was determined by both serial 2- and 3-fold dilutions with SDS. ‘The value given in parentheses is the ratio of the CF titer for a given NDV CEF-grown virus. RELATIVE TITER OF VP COMPLEMENT-FIXATION
NDV grown in HEp-2 HeLa BHK-21 BEK Egg CEF
Neuraminidase activityb (Ol++$;,ml,
9 ANTIGEN
OF TESTS USING
STANDARDIZED
AMOUNTS
VP 9 antigen (CF titer)
512 256 256 256 128 64
256 256 128 128 64 64
a See footnote to Table 3. b Neuraminidase activity was determined c See footnote to Table 3.
1.0 1.0 1.0 0.5 0.5 1.0
in Materials and Methods. subjected to CF tests. Four of viruses before treatment preparation
to that of the
TABLE 4 NDV GROWN IN VARIOUS CELL TYPES AS DETECTED IN
S-antigen (CF titer)
1.004 1.042 1.450 1.402 0.932 1.308
CFS-titer
OF NEURAMINIDASE
UNITP
Calculated VP 9 CF titer per 1000 Enzyme units 256 245 90 93 68 44
(5.8)c (5.6) (2.0) (2.1) (1.5) (1.0)
CFS-titer 0.5 1.0 0.5 0.5 0.5 1.0
before treatment with SDS.
glycoproteins, the highest titers being found for VP 9 of virus grown in HEp-2 cells. Conversely, the amount of VP 9 antigen detected in these CF tests did not show this marked variation if the tests were standardized for the internal S-antigen of disrupted virions (Tables 3 and 4). These results suggested that VP 9 is an internal protein of NDV virions (Miyadera strain), which, unlike surface glycoproteins, does not appear to exhibit host-controlled variation. DISCUSSION
The data described in this communication suggest that the Miyadera strain of
Newcastle disease virus contains a minor internal protein, designated here as VP 9, with a molecular weight of approximately 26,500. While a similar constituent has not been reported in several earlier studies concerned with the structural proteins of various other strains of this paramyxovirus (Evans and Kingsbury, 1969; Haslam et al., 1969; Bike1 and Duesberg, 1969; Mountcastle et al., 1970, 1971; Iinuma et al., 1969; Scheid and Choppin, 1973), it is possible that strain differences per se could account for this discrepancy. An examination of published electropherograms of NDV structural proteins in some cases shows minor distributions of radioactive
PROTEINS
counts in a position near the anode approximating the location of the VP 9 protein of this study (see Bike1 and Duesberg, 1969; Haslam et al., 1969; Mountcastle et al., 1970, 1971). To resolve this question, we plan to test for the presence of VP 9 antigen in other NDV strains using the CF test with detergent-dissociated virus and antiserum monospecific for the Miyadera VP 9 protein. Yields of VP 9 eluted from PAGE gels were relatively low, an observation in agreement with the findings of other workers (Laver, 1970) who have attempted recovery of viral polypeptides from gel bands. Despite this limitation, however, VP 9 protein was extractable from PAGE gels in an antigenically active form and apparently free of other viral structural polypeptides. It is not surprising that VP 9 protein derived from these gels exhibits antigenicity since it is recognized that viral proteins extracted from virions and dissociated by a variety of methods retain their capacity to elicit formation of antibodies specific for individual polypeptide chains (see Horwitz and Scharff, 1969). The Ab-9 antiserum of this study, which our data indicate contains monospecific antibody for the VP 9 protein, could not react with viral envelope protein(s) or the internal S-antigen associated with viral nucleocapsids (Tables 1 and 2). These observations are consistent with the finding that cytoplasmic fluorescence obtained with FITC-conjugated Ab-9 could not be blocked by pretreating cells with either anti-NDV or anti-S antibody. Furthermore, immunofluorescent monitoring of NDV-infected cells with the Ab-9 antibody gave negative staining of the plasma membrane with unfixed cells and positive staining for cytoplasmic deposits of VP 9 antigen with fixed cells, indicating that this protein does not become incorporated into host cell membranes during viral budding. Collectively, these results suggest that VP 9 is an internal component of the NDV virion although not necessarily an integral part of the nucleoprotein complex per se. Further work will be required to establish the precise location of this virion constituent.
OF NDV
237
The possible functional role of the VP 9 polypeptide and its relationship to other structural components of NDV Miyadera is also unknown. It is entirely conceivable that VP 9 represents a monomer of one of the larger NDV structural polypeptides. In this connection, the VP 9 protein of Miyadera virus particles dissociated with deoxycholate can be separated in the upper fractions of sucrose-DOC gradients (Iinuma et al., 1971b) which also contain the polypeptides VGP 4, VGP 5, and VP 8 (Fig. 1). The hemagglutinin but not the neuraminidase activity associated with these gradient fractions can be specifically removed by absorption with red blood cells with concomitant loss of VGP 4 protein as judged by PAGE analysis. Additionally, recent experiments (Iinuma, unpublished) have demonstrated that neuraminidaseactive fractions of enzymatically dissociated NDV Miyadera virus separated by column chromatography contain no detectable VP 9 protein but only VGP 5 and VP 8. While these findings would suggest that VGP 4 is associated with the hemagglutinin activity of this strain whereas VGP 5 and VP 8 are functionally linked with its neuraminidase activity, they do not exclude the possibility that the minor component VP 9 derives from one of these polypeptides. Such postulated structure-function relationships for the proteins of NDV Miyadera must be further substantiated in light of the recent report that the hemagglutinin and neuraminidase activities of NDV (Hickman strain) reside in the same glycoprotein (Scheid and Choppin, 1973). Work now in progress indicates that the VP 9 protein is synthesized and incorporated into mature virions late in the infectious cycle. We have also found that VP 9 is rich in arginine and that this amino acid is required for the synthesis of this polypeptide as well as viral progeny. These and other findings lend further credence to the earlier hypothesis that VP 9 may be an essential protein for the NDV assembly process (Iinuma et al., 1971a; Iinuma et al., 1973). An interesting aspect of this work was the finding that the relative amount of VP 9 protein present in NDV Miyadera grown
238
IINUMA
AND SIMPSON
in various cell types is constant when the test antigen (dissociated virus) used for its determination in CF tests was standardized against the internal S-antigen of these preparations (Tables 3 and 4). Conversely, standardization of these CF titrations using constant hemagglutinin or neuraminidase units of the test viruses gave up to an g-fold variation in the amount of VP 9 antigen detected in virus from various cell types. Such a variation most likely reflects host-controlled modifications of these glycoproteins of the type previously documented for various myxoviruses and paramyxoviruses (Ishida and Homma, 1961; Kates et al., 1961; Drake and Lay, 1962; Matsumoto and Maeno, 1962; Stenbeck and Durand, 1963; Simpson and Hauser, 1966; Klenk and Choppin, 1969; Compans et al., 1970; Homma, 1971, 1972). ACKNOWLEDGMENTS We thank Miss Janeen E. Dougherty for her excellent technical assistance throughout this investigation. The initial phase of this work was conducted at the Cancer Research Institute (Department of Virology), Nagoya University School of Medicine, Nagoya, Japan. This study was supported, in part, by funds from Research Contract NIH-NIAID-72-2504 and USPH Grant AI-09124 from the National Institute of Allergy and Infectious Diseases. Additional support was provided by funds from Research Contract NIH-NCI-E-71-2077 (The Virus Cancer Program) from the National Cancer Institute. REFERENCES C. B., SELA, M., and COOKE, J. P. (1962). The reversible reduction of disulfide bonds in polyalanyl ribonuclease. J. Biol. Chem. 237, 182551831. BAEILANIAN,R., EGGERS, H. J., and TAMM, I. (1965). Studies on the mechanism of poliovirus-induced cell damage. I. The relation between poliovirusinduced metabolic and morphological alterations in cultured cells. Virology 26, 100-113. BIKEL, I., and DUESBERG, P. H. (1969). Proteins of Newcastle disease virus and of the viral nucleocapsid. J. Virol. 4, 388-393. COMPANS, R. W., and CHOPPIN, P. W. (1967). The length of the helical nucleocapsid of Newcastle disease virus. Virology 33, 344-346. COMPANS, R. W., KLENK, H., CALXXJIRI, L. A., and CHOPPIN, P. W. (1970). Influenza virus proteins. 1. Analysis of polypeptides of the virion and identification of specific glycoproteins. Virology 42,
ANFINSEN,
880-889.
DRAKE, J. W., and LAY, P. A. (1962). Host-controlled variation in NDV. Virology 17, 56-64. EVANS, M. J., and KINGSBURY,D. M. (1969). Separation of Newcastle disease virus proteins by polyacrylamide gel electrophoresis. Virology 37, 597-604.
HASLAM, E. A., CHYENE, I. M., and WHITE, D. 0. (1969). The structural proteins of Newcastle disease virus. Virology 39, 118-129. HOMMA, M. (1971). Trypsin action on the growth of Sendai virus in tissue culture cells. I. Restoration of the infectivity for L cells by direct action of trypsin on L cell-borne Sendai virus. J. Virol. 8,619-629. HOMMA, M. (1972). Trypsin action on the growth of Sendai virus in tissue culture cells. II. Restoration of the hemolytic activity of L cell-borne Sendai virus by trypsin. J. Virol. 9,829-835. HORWITZ, M. S., and SCHARFF,M. D. (1969). The production of antiserum against viral antigens. In “Fundamental Techniques in Virology” (K. Habel and N. P. Salzman, eds), pp. 253-262. Academic Press, New York. IINUMA, M., MAENO, K., and MATSUMOTO, T. (1973). Studies on the assembly of Newcastle disease virus: An arginine-dependent step in virus replication. Virology 51, 205-215. IINUMA, M., NAGAI, Y., MAENO, K., YOSHIDA,T., and MATSUMOTO, T. (1971a). Studies on the assembly of Newcastle disease virus: Incorporation of structural proteins into virus particles. J. Gen. Viral. 12, 239-247. IINUMA, M., YOSHIDA, T., NAGAI, Y., MAENO, K., MATSUMOTO, T., and HOSHINO, M. (1971b). Subunits of NDV. Hemagglutinin and neuraminidase subunits of Newcastle disease virus. Virology 46, 663-677. ISHIDA, N., and HOMMA, M. (1961). Host-controlled variation observed with Sendai virus grown in mouse fibroblast (L) cells. Virology 14, 486-488. KATES, M., ALLISON, A. C., TYRREL, A. J., and JAMES, A. T. (1961). Lipids of influenza virus and their relation to those of the host cell. Biochim. Biophys. Acta
52,455-466.
KILBOURNE, E. D., CHOPPIN, P. W., SCHULZE,I. T., SCHOLTISSEK,C., and BUCHER, D. L. (1972). Influenza virus polypeptides and antigens. J. Infect. Dis. 125, 447-455. KLENK, H. D., and CHOPPIN, P. W. (1969). Lipids of plasma membrane of monkey and human kidney cells and of parainfluenza virus grown in these cells. Virology
38, 225-268.
LAVER, W. G. (1970). Isolation of arginine-rich protein from particles of adenovirus type 2. Virology 41, 488-500. LAVER, W. G., and KILBOURNE, E. D. (1966). Identification in a recombinant influenza virus structural proteins derived from both parents. Virology 30, 493-501.
PROTEINS LAZAROWITZ, S. G., COMPANS, R. W., and CHOPPIN, P. W. (1973). Proteolytic cleavage of the hemagglutinin polypeptide of influenza virus. Function of the uncleaved polypeptide HA. Virology 52, 199-212. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., and RANDALL, R. J. (1951). Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265-275. MAENO, K., and KILBOURNE, E. D. (1970). Developmental sequence and intracellular sites of synthesis of three structural protein antigens of influenza A, virus. J. Virol. 5, 153-164. MAENO, K., YOSHIDA, T., IINUMA, M., NAGAI, Y., MATSUMOTO, T., and ASAI, J. (1970). Isolation of hemagglutinin and neuraminidase subunits of hemagglutinating virus of Japan. J. Viral. 6.492-499. MATSUMOTO, T., and MAENO, K. (1962). A hostinduced modification of hemagglutinating virus of Japan (HVJ, Sendai virus) in its hemolytic and cytopathic activity. Virology 17, 563-570. MOUNTCASTLE, W. E., COMPANS, R. W., CALIGUIRI, L. A., and CHOPPIN, P. W. (1970). Nucleocapsid protein subunits of Simian virus 5, Newcastle disease virus and Sendai virus. J. Virol. 6, 677-684. MOUNTCASTLE, W. E., COMPANS, R. W., and CHOPPIN, P. W. (1971). Proteins and glycoproteins of paramyxoviruses: A comparison of Simian virus 5, Newcastle disease virus, and Sendai virus. J. Virol. 7, 47-52. OROSZLAN, S., FOREMAN, C., KELLOFF, G., and GILDEN, R. V. (1971). The group-specific antigen and other
OF NDV
239
structural proteins of hamster and mouse C-type viruses. Virology 43, 665-674. SCHEID, A., and CHOPPIN, P. W. (1973). Isolation and purification of the envelope proteins of Newcastle disease virus. J. Virol. 11. 263-271. SHAPIRO, A. L., VIN~ELA, E., and MAIZEL, J. V., JR. (1967). Molecular weight estimation of polypeptide chains by electrophoresis in SDS-polyacrylamide gels. Biochem. Biophys. Res. Commun. 28, 815-820. SHIMOJO, H., YAMAMOTO, H., and ABE, C. (1967). Differentiation of adenovirus 12 antigens in cultured cells with immunofluorescent analysis. Virology 31, 748-752. SIMPSON, R. W., and HAUSER, R. E. (1966). Influence of lipids on the viral phenotype I. Interaction of myxoviruses and their lipid constituents with phospholipases. Virology 30, 684-697. SIMPSON, R. W., and HIRST, G. K. (1961). Genetic recombination among influenza viruses. I. Cross reactivation of plaque-forming capacity as a method for selecting recombinants from the progeny of crosses between influenza A strains. Virology 15, 436-451. STANLEY, P., and HASLAM, E. A. (1971). The. polypeptide of influenza virus. V. Localization of polypeptides in the virion by iodination techniques. Virology 46, 764-773. STENBACK, W. A., and DURAND, D. P. (1963). Host influence on the density of Newcastle disease virus (NDV). Virology 20, 545-551.
61, 229-239
(1974)
The Structural
Proteins
I. Identification MASAO Institute
of Microbiology,
Rutgers
of a Minor
IINUMA’ University,
of Newcastle
AND
Internal
ROBERT
The State Uniuersity 089032 Accepted
May
Disease
Virus
Protein
W. SIMPSON of New
Jersey,
New Brunswick,
New Jersey
29, 1974
Purified Newcastle disease virus (NDV), Miyadera strain, was dissociated and its proteins were analyzed by SDS polyacrylamide gel electrophoresis. Polypeptide VP 9 was consistently found to be the fastest migrating viral protein with an estimated molecular weight of approximately 26,500 when compared to known protein markers. VP 9 comprises approximately 3% of the total viral protein of virions grown in either chick embryo or HeLa cells. VP 91 was also identified by immunological procedures. Hyperimmune Ab-9 serum prepared against the purified VP 9 protein (eluted from polyacrylamide gels), was incapable of neutralizing the hemagglutinin or neuraminidase activities of NDV. In addition, this antibody did not react with the S-antigen (nucleoprotein) of the virus. Complement-fixation (CF) tests revealed that this antibody could react with solubilized NDV, but not intact virions. Immunofluorescent staining of NDV-infected HeLa cells with FITC-labeled Ab-9 resulted in cytoplasmic fluorescence which was not blocked by pretreatment with either unlabeled virus-specific antibody or anti-S antibody. To determine the relative proportion of VP 9 antigen in NDV stocks grown in various cell types, CF tests were performed with detergent-dissociated NDV grown in different cells. If the amount of test virus used in these CF reactions was standardized for S-antigen units, the CF titers for VP 9 antigen were the same for virus preparations grown in various cell types, whereas up to an &fold variation in titer was observed if standardization was based on hemagglutinin or neuraminidase units. It is concluded that VP 9 is an internal polypeptide of NDV virions which does not constitute part of the nucleoprotein complex and which does not exhibit significant quantitative variations as influenced by host factors. INTRODUCTION
Recent studies in several laboratories have established the identity of several of the polypeptides of influenza virus (Kilbourne et al., 1972). In contrast to influenza virus, relatively little information is available on the structural proteins of the paramyxovirus, Newcastle disease virus (NDV), even though these components have been extensively studied in several laboratories (Evans and Kingsbury, 1969; Haslam et al., 1969; Bike1 and Duesberg, I Permanent address: Department of Cancer Research Institute, Nagoya University of Medicine, Nagoya, Japan. *Reprints are available at this address.
Virology, School
1969; Mountcastle et- al., 1970, 1971; Iinuma et al., 1971b; Scheid and Choppin, 1973). Although it has been shown that purified NDV contains three major polypeptides, the exact number of minor polypeptides remains unknown. In addition, the structure-function relationships for most of these proteins within the virion are not yet clearly understood. One of the major polypeptides, however, has already been identified as the nucleocapsid protein of the virus (Evans and Kingsbury, 1969; Haslam et al., 1969; Bike1 and Duesburg, 1969; Mountcastle et al., 1970; Scheid and Choppin, 1973). Recently, Mountcastle et al. (1971) and Scheid and Choppin (1973)
230
IINUMA
AND
have suggested that the smallest known protein of NDV is a major protein which has a molecular weight of approximately 41,000. During our studies on the protein components of NDV, we discovered a new minor protein, VP 9, which is described in this paper utilizing analysis by polyacrylamide gel electrophoresis (PAGE) and immunological techniques for its identification. It will be shown that VP 9 is an internal protein representing approximately 3% of the total viral protein. MATERIALS
AND
METHODS
Virus and virus purification. The Miyadera strain of Newcastle disease virus (NDV) was used throughout this study. The method for purification of virus has previously been described (Iinuma et al., 1971a, b; 1973). Cell cultures. Primary chick embryo fibroblasts (CEF) were prepared by the method of Simpson and Hirst (1961). Primary cultures of bovine embryonic kidney (BEK) cells were obtained from 10 to 50-g embryos by digesting with 0.25% trypsin (Nutritional Biochemicals Corp., Ohio) and 0.01% collagenase (Worthington Biochemical Corp.) after whole kidney was minced. A more detailed account of the technique used will be given elsewhere (Iinuma and Simpson, in progress). Continuous lines of HeLa, HEp-2, and BHK-21 cells were propagated in reinforced Eagle’s MEM (Bablanian et al., 1965) supplemented with 10% fetal calf serum. Virus growth. In eggs: NDV stocks were diluted 1: 1000 with cold phosphate-buffered saline (PBS) and inoculated into the allantois of ll-day-old embryonated eggs. After incubation at 37” for 48 hr, the eggs were chilled, and allantoic fluids were harvested and subjected to purification. In cell culture: monolayer cultures (CEF, BEK, HeLa, HEp-2, or BHK-21) were infected with egg-grown NDV at an input multiplicity of approximately 8 PFU per cell after they were washed with modified Hanks’ balanced salts solution (BSS). Virus was allowed to adsorb for 1 hr at 37” after which the inoculum was removed by
SIMPSON
washing three times with BSS. Maintenance medium (reinforced MEM supplemented with 3% fetal calf serum) was added and the cultures were incubated for 24 hr at 37”. Cells and culture fluids were harvested and centrifuged at 8000 rpm for 10 min (4”) after three cycles of freezing and thawing. The resulting supernatants were subjected to purification. Isotopically labeled virus: isotopically labeled virus was prepared in CEF or HeLa cells. To NDVinfected cells, 0.5 &i/ml of [‘4C]glucosamine or 14C-labeled amino acid mixture, or 5 pCi/ml of [3H]leucine, or combinations of these isotopes, was added after 1 hr postinfection. Incubation was continued for 24 hr and culture medium was harvested. Neuraminidase assay. Neuraminidase assay was performed with neuraminlactose substrate by a modification of the Warren technique (Laver and Kilbourne, 1966; Maeno and Kilbourne, 1970). N-Acetylneuraminic acid-n-lactose (neuraminlactose) prepared from bovine colostrum was kindly supplied by Dr. M. Pons. An 0.2-ml volume of test material diluted with saline was added to 0.1 ml of substrate and adjusted to pH 5.0 (optimum pH for NDV enzyme) with 0.1 ml of 0.4 M sodium acetate buffer. After 30 min of incubation in a water bath at 37”, 0.2 ml of the reaction mixture was used for the assay of free N-acetylneuraminic acid (NANA). Optical density was read at 549 nm (OD,,,) against a blank tube containing neuraminlactose plus saline. Enzyme activity was measured by OD readings taken from the part of the slope where they vary linearly with the enzyme concentration. Enqme inhibition (EI) tests. One unit of neuraminidase was defined as the amount of enzyme required to yield sufficient NANA to produce an OD reading of 0.1 at 549 nm. Serial 2-fold dilutions of antibody in 0.2-ml volumes were combined with 0.2 ml of 5-7 enzyme units (virus particles) and incubated for 1 hr at 37” before the neuraminidase assay was performed. EI titer was expressed as the highest dilution of antibody which gave a 50% decrease in OD reading. EI-antibody blocking tests. An 0.2-ml
PROTEINS
volume of test material (VP 9 protein or virions) was added to the same volume of anti-NDV serum (1: 20), and incubated for 60 min at room temperature. After this, 0.2 ml of 5 units of enzyme (virus particles) was added to each mixture. After further incubation for 60 min at room temperature, neuraminidase activity of the final reaction mixtures was determined. Treatment of virus with detergents or proteolytic enzymes. Purified preparations of virus were treated with 0.01 M sodium deoxycholate (DOC) or sodium dodecyl sulfate (SDS) in 0.01 M Na,HPO, and 0.14 M NaCl, pH 7.5. The mixture was held at 37” for 2 hr with stirring and then used as a CF antigen after dilution with Kolmer’s solution. For enzymatic digestion of virus, purified NDV was mixed with either chymotrypsin (Sigma) or trypsin (Difco) at a final concentration of 2.0 mg/ml in 0.1 M phosphate buffer, pH 7.6, and incubated in a water bath at 37” for 2 hr. After chilling in an ice bath, these mixtures were centtrifuged twice at 22,500 rpm for 1 hr at 4” in a Spinco No. 40 rotor (before and after washing with PBS). The resulting pellets were resuspended to original volume in PBS and used as CF-antigens. Electrophoresis and preparation of VP 9. Virus samples were suspended in 1% SDS and 1% 2-mercaptoethanol in 0.01 M sodium phosphate buffer, pH 7.2, and incubated at room temperature for 2 hr. After dialysis overnight at room temperature (0.1% SDS, 0.1% mercaptoethanol, and 10% sucrose in 0.1 M sodium phosphate buffer, pH 7.2) 50-18 samples were layered on 7.5% polyacrylamide gels prepared as described previously (Iinuma et al., 1971b). Electrophoresis was performed at 5 mA per gel for 5 hr after which the gel was stained (0.5% Coomassie Brilliant Blue in 25% isopropyl alcohol and 10% acetic acid) and destained by repeated changes of 7% acetic acid. The regions of several unstained gels corresponding to the location of the VP 9 component of a stained reference gel run in parallel were collected by slicing and homogenized in 0.1 ml of water per gel. After standing overnight in an ice bath, these gel-water mixtures were
OF NDV
231
combined with an equal volume of PBS and clarified by low-speed centrifugation (3000 rpm for 30 min). The resulting supernatant (containing the gel-eluted protein) was stored at -78”. For coelectrophoresis of NDV virion polypeptides and isolated VP 9, VP 9 labeled with [3H]leucine was collected from 10 PAGE gels as described above. Yields of VP 9 protein eluted from PAGE were relatively low accounting for approximately 30% of the starting material. A 1.8-ml quantity of the pooled VP 9 gel eluate was mixed with the same volume of 10% trichloroacetic acid (TCA) and allowed to stand at 4” for 30 min. The TCA precipitate was mixed with 1.8 ml of concentrated NDV virus particles labeled in their proteins with 14C-labeled amino acids followed by addition of 5.4 ml of absolute ethanol. Finally, the resulting ethanol precipitate was subjected to coelectrophoresis. For counting, gels containing radioactive proteins were cut into 1.5-mm slices which were placed into scintillation vials with 0.2 ml of H,O, and stored at 50” for 3 days. Thereafter, 10 ml of modified Bray’s solution (without methanol and ethylene glycol) was added to each vial and radioactivity was measured in a Packard Tri-Carb spectrometer. The following proteins were treated with SDS and 2-mercaptoethanol as described by Shapiro et al. (1967) and used as reference markers for molecular weight determinations: bovine serum albumin (MW: 67,000); ovalbumin (MW: 45,000); trypsin (MW: 23,000). Preparation of antiserum against VP 9. Gel-eluted VP 9 protein (stored at -78”) was inoculated intravenously into rabbits for a total of 20 times at 2-day intervals, using 7.5 pg of protein per injection. Hyperimmune serum (Ab-9) was obtained from the rabbits bled 7 days after the last injection. Ab-9 serum was heated at 56” for 30 min before use. Immunofluorescent staining. A yglobulin fraction was prepared from immune rabbit serum by precipitation with cold, one-third-saturated ammonium sulfate. After conjugation with fluorescein isothiocyanate by the method of Shimojo et al. (1967), the conjugate was immedi-
232
IINUMA
AND
SIMPSON
ately passed through a Sephadex G-50 gel column and further purified by passage through a diethylaminoethyl (DEAE) cellulose column and absorption with ace> tone-precipitated chick embryo powder to reduce nonspecific staining. HeLa cells grown on cover slips were infected with NDV, and’ after 9 hr they were stained with the antibody with or without prior fixation. Examination of these preparations was carried out with a Reichert Zetopan microscope equipped for fluorescence microsCOPY.
Additional techniques. The techniques of hemagglutinin titration, hemagglutination-inhibition (HI) test, antibody-blocking tests for hemagglutination, complement-fixation (CF) test, and preparation of antibodies against viral envelope protein (V-antigen) or nucleoprotein (S-antigen) have been described (Maeno et al., 1970; Iinuma et al., 1971a, b, 1973). The protein content of different samples was measured by the method of Lowry et al. (1951). RESULTS
Electrophoretic pattern of NDVproteins and the fastest migrating protein. Experiments carried out by one of us (Iinuma et al., 1971b) suggested that purified radioactive NDV, Miyadera strain, subjected to analysis by PAGE, contained small amounts of a protein, corresponding to a minor band on the stained gel which migrated to a position near the anode. Further experiments were conducted in the present study to determine whether this component represented a virus-specific protein. Figure 1 illustrates the electrophoretie pattern of NDV Miyadera proteins labeled in the presence of [3H]leucine and [‘%]glucosamine. Three major peaks of 3H radioactivity (VGP 4, VP 6, and VP 8) and some minor peaks of 3H radioactivity (VP 1, VP 2-3, VGP 5, VP 7, and VP 9) were detected. Of these polypeptides, VGP 4 and VGP 5 were identified as glycoproteins based on their labeling with radioactive glucosamine. Of particular interest was the finding of a small peak, designated as VP 9, which consistently was observed as the fastest migrating protein in different gel
FRACTION
NUMEER
FIG. 1. Electrophoretic pattern of NDV virion polypeptides and standard reference proteins. Puritied CEF-NDV labeled both with [aH]leucine and [ “Clglucosamine was mixed with the standard proteins indicated, dissociated with SDS and mercaptoethanol and subjected to PAGE analysis. After the gel was stained with Coomassie Brilliant Blue and destained with acetic acid, the gel was sliced and the discs were counted for their radioactivity. The small peaks labeled VP 2 and VP 3 were frequently, but not always, observed as a single peak in repeat experiments. The distance migrated by each band was plotted against the logarithm of the known molecular weight of each marker (Shapiro et al., 1967). BSA; bovine serum albumin (MW: 67,000); OV; ovalbumin (M,: 45,000); TR; trypsin (M,: 23,800).
runs. In order to characterize this constituent, VP 9 labeled with [3H]leucine was eluted from unstained PAGE gels (Materials and Methods) and coelectrophoresed with %-labeled virion proteins of NDV grown in HeLa cells (Fig. 2). The electropherogram of Fig. 2 demonstrates that VP 9 protein recovered in this way was free of contamination from all the other viral proteins and retained its original electrophoretic migration properties. Assuming that the [3H]leucine or “Clabeled amino acids were incorporated equally into all NDV proteins, VP 9 accounted for 2.9% of CEF-NDV and 3.1% of HeLa-NDV of the total viral protein, respectively. A value of 26,500 for the molecular weight of VP 9 was obtained by the method of Shapiro et al. (1967), as shown in Fig. 1 and Fig. 2. These prelimi-
PROTEINS
g 300
110
i L 0 0
100
a00
0 f 100
SO
10 FRACTION
30
30
233
OF NDV
Oroszlan, et al., 1971). This gel eluate of VP 9 protein did not show any detectable hemagglutinin or neuraminidase activities, nor did it react with anti-S (nucleocapsid protein) antibody in CF-tests or antibodies active in hemagglutination inhibition (HI) and neuraminidase inhibition (EI). Hyperimmune serum prepared against VP 9 (Ab-9) in rabbits lacked the capacity to neutralize hemagglutinin or neuraminidase activity of intact NDV particles and could not react with viral S-antigen in CF tests (Table 1). Complement-fixation tests were also performed to determine whether the Ab-9 serum could react with NDV
NUMBER
FIG. 2. Coelectrophoresis of NDV virion polypeptides labeled with “C-labeled amino acids and polypeptide, VP 9 labeled with [3H]leucine (isolated from PAGE gels). Purified CEF-NDV labeled with [*H]leutine was dissociated with SDS and mercaptoethanol, and subjected to PAGE. The fastest migrating protein (VP 9) was eluted from 10 PAGE gels as described in Materials and Methods. VP 9 thus obtained was mixed with HeLa-NDV proteins labeled with “Clabeled amino acids, dissociated as described above and coelectrophoresed on the same gel. Radioactivities of gel slices and the molecular weight of VP 9 were determined as described in Fig. 1. (Abbreviations: see footnote in Fig. 1.)
TABLE
Hemagglutination inhibition: Neuraminidase inhibition: Anti-S titration:”
nary results suggested that polypeptide VP 9 might be a viral-specific protein hithertofore not reported possibly because of difficulties in demonstrating it with other NDV strains. Antigenicity of VP 9 eluted from polyacrylamide gels. The possibility of whether the VP 9 protein eluted from PAGE gels is antigenic was tested by immunizing rabbits with this material (Materials and Methods). Pooled gel slices derived from multiple gels used for electrophoretic separation of VP 9 protein were eluted in water (Materials and Methods) and finally suspended in PBS to provide material for rabbit immunization and various in vitro tests. It was of interest to determine whether the VP 9 protein eluted from polyacrylamide gels had biological activity since other workers have recovered enzymatically or serologically reactive proteins by this technique (e.g., Anfinsen, 1962;
D NDV (Miyadera strain) grown in eggs was dissociated and run in polyacrylamide gels (Materials and Methods) to separate the minor polypeptide VP 9. Gel cuts of VP 9 protein from multiple gels were eluted to provide the purified material for preparing the rabbit antiserum (Ab-9). Anti-NDV serum was prepared by immunization of rabbits with whole virus particles (Materials and Methods). The anti-S serum was prepared by rabbit immunization with nucleocapsids of NDV obtained from virus disrupted with DOC and centrifuged through a 5% to 20% sucrose gradient as described previously (Iinuma et al., 1971b). The anti-S titer of the serum after absorption with virus particles to remove antibodies against envelope antigens was l/128. Complement-fixation tests were done according to a modification of Kolmer’s method. b All titers are expressed as the reciprocal of the highest dilution of serum showing positive activity. ‘The S-antigen was obtained from HeLa cells infected with NDV. Extracts of HeLa cells were subjected to sedimentation by CsCl gradient centrifugation according to Compans and Choppin (1967). The nucleocapsid band was harvested and dialyzed against PBS.
INHIBITORY
1
ACTIVITY OF ANTISERUM (Ab-9) PURIFIED VP 9 PROTEIN=
Rabbit Test
Preimmune serum
AGAINST
antiserum”
Ab-9
AntiNDV
AntiS
<5
<5
5120
<5
<5
<5
480
<5
<4
<4
<4
128
234
IINUMA
AND
structural proteins. As shown in Table 2, a high CF titer was obtained in the reaction only with detergent-solubilized NDV but not with intact NDV virions before or after digestion with either chymotrypsin or trypsin. In addition, this antibody could react neither with the DOC-solubilized heterologous paramyxovirus, Sendai, nor with the extracts of uninfected allantoic membranes. These results suggested that the VP 9 protein is a viral structural protein internally located in NDV virions. To confirm the observations described above using other approaches, immunofluorescent monitoring of NDV-infected cells with FITC-labeled Ab-9 was employed. HeLa cells grown on coverslips were infected with NDV at an input multiplicity of approximately 8 PFU/cell and stained with FITC-labeled Ab-9 at the 9th hr of infection with or without prior fixation. Large amounts of intracellular virus precursor proteins were previously found to accumulate at this time of infection (Iinuma et al., 1971a, 1973). The micrographs presented in Fig. 3 illustrate that when infected cells were stained after fixation, a TABLE COMPLEMENT-FIXATION
TESTS WITH VARIOUS
Antigens
NDV
SIMPSON
cytoplasmic fluorescence marked by strong staining in the perinuclear area was observed (Fig. 3 A). Conversely, staining without prior fixation resulted in no fluorescence (Fig. 3 B). Attempts to block virus-specific fluorescence by either unlabeled anti-S or anti-NDV globulin resulted in no significant difference in the extent or intensity of cytoplasmic fluorescence (Fig. 3 C and D). It is important to note that no fluorescence could be detected in noninfected cells by staining with FITC-labeled Ab-9 after fixation. These results supported the observations described above and suggested that the protein VP 9 was indeed incorporated into virions during maturation but not in the modified host cell membrane itself. Amount of VP 9 protein detected in NDV particles grown in various cell types. The results of experiments described in the preceding sections suggested that the protein VP 9 was a virus-specific protein situated within virions and internal to the envelope surface components. Further experiments were carried out to determine whether the proportion of VP 9 antigen in 2
PREPARATIONS PROTEIN“
Hemagglutination activity (HAW0.25 ml)
AND ANTISERUM
Neuraminidase activity
(OD,,,/O.l
ml,
AGAINST
PURIFIED
CF titer
of serum:
Preimmune
VP 9
Ab-9
1:600)
Purified NDV DOC-dissociated NDV SDS-dissociated NDV Chymotrypsinized NDV’ Trypsinized NDV’ Purified Sendai DOC-dissociated Sendai Allantoic membrane extract
4000 NTb NT 4000 4000 4000 NT <2
0.492 NT NT 0.283 0.508 NT NT 0
<4 <4 <4 <4 <4 <4 <4 <4
<4 256 256 <4 <4 <4 <4 <4
a The same rabbit Ab-9 antiserum used in the tests described for Table 1 was employed. Antigens were prepared as described in Materials and Methods. As controls, extracts of uninfected allantoic membranes or the heterologous paramyxovirus, Sendai, were used. Allantoic membranes were harvested from uninfected ll-dayembryonated eggs and washed throughly with PBS. The homogenized materials were subjected to three cycles of freezing and thawing and centrifugation at low speed. The resulting supernatant fluid was used as an extract of allantoic membranes. The CF titer was expressed as the reciprocal of the highest dilution of antigen showing more than 75% fixation of complement per 0.2ml volume. b NT = not tested. L Plaque-forming capacity of chymotrypsin-treated NDV was unaffected, even though the treatment reduced viral neuraminidase activity. Trypsin was without effect on the viral activities measured.
PROTEINS
OF NDV
235
Fro. 3. Fluorescent-antibody staining of NDV-infected cells, using antibody against purified VP 9 protein. HeLa cells grown on coverslips were infected with NDV at an input multiplicity of 8 PFU/cell and incubated at 37” for 9 hr. After washing with PBS, they were allowed to react with FITC-labeled Ab-9 with (A, C, D, E) or without (B) prior fixation with cold acetone. Cytoplasmic fluorescence observed after fixation (A) or without prior fixation (B). Cells pretreated with either FITC-unlabeled anti-S serum (C) or anti-NDV serum (D) before staining. The titer of unlabeled anti-S serum (1:20) or anti-NDV serum (1: 40) was sufficient to block the cytoplasmic fluorescence obtained by either FITC-labeled anti-S or anti-NDV, respectively. Specific fluorescence was completely blocked by pretreatment with FITC-unlabeled Ab-9 (E). Noninfected HeLa cells with or without prior fixation showed no fluorescence by staining with FITC-labeled Ab-9 (not shown).
NDV particles grown in various cell types shows’ host-controlled variation with the assumption that if VP 9 was an internal protein of this virus, the amount of VP 9 might not be affected by the host cell factors (Stanley and Haslam, 1971; Lazorowitz et al., 1973). Viruses grown in various cell systems (HeLa, HEp-2, BHK-21, BEK, CEF cells, or embryonated eggs) were prepared and purified as described in
Materials and Methods. For preparing CF test antigen, purified viruses were dissociated with 0.01 M SDS after diluting each virus preparation with saline to the same concentration for hemagglutinin or for neuraminidase units. As illustrated in Tables 3 and 4, the apparent amount of VP 9 protein based on CF titer was found to vary in NDV grown in various cell types when standardized against these surface
IINUMA
236
AND SIMPSON TABLE
RELATIVE TITER OF COMPLEMENT-FIXATION
3
VP 9 ANTIGEN
IN NDV GROWN IN VARIOUS CELL TYPES AS DETECTED IN TESTS USING STANDARDIZED AMOUNTS OF HEMAGGLUTININ UNITS (HAU)O
NDV grown in
Hemagglutinin activity” (HAW0.25 ml)
S-antigen (CF-titer)
VP 9 antigen (CF-titer)
HEp-2 HeLa BHK-21 BEK Egg CEF
1280 1920 1920 1280 1920 1280
512 512 256 256 256 64
512 512 256 128 128 64
Calculated VP 9 CF titer per 1000 HA units 400 (8.O)C 267 (5.3) 133 (2.7) 100 (2.0) 67 (1.3) 50 (1.0)
a Viruses grown in various cell types were prepared and purified as described Purified viruses were dissociated with SDS at a final concentration of 0.01 A4 and units of antibody (anti-S Ab-9) were used. b Hemagglutinin activity was determined by both serial 2- and 3-fold dilutions with SDS. ‘The value given in parentheses is the ratio of the CF titer for a given NDV CEF-grown virus. RELATIVE TITER OF VP COMPLEMENT-FIXATION
NDV grown in HEp-2 HeLa BHK-21 BEK Egg CEF
Neuraminidase activityb (Ol++$;,ml,
9 ANTIGEN
OF TESTS USING
STANDARDIZED
AMOUNTS
VP 9 antigen (CF titer)
512 256 256 256 128 64
256 256 128 128 64 64
a See footnote to Table 3. b Neuraminidase activity was determined c See footnote to Table 3.
1.0 1.0 1.0 0.5 0.5 1.0
in Materials and Methods. subjected to CF tests. Four of viruses before treatment preparation
to that of the
TABLE 4 NDV GROWN IN VARIOUS CELL TYPES AS DETECTED IN
S-antigen (CF titer)
1.004 1.042 1.450 1.402 0.932 1.308
CFS-titer
OF NEURAMINIDASE
UNITP
Calculated VP 9 CF titer per 1000 Enzyme units 256 245 90 93 68 44
(5.8)c (5.6) (2.0) (2.1) (1.5) (1.0)
CFS-titer 0.5 1.0 0.5 0.5 0.5 1.0
before treatment with SDS.
glycoproteins, the highest titers being found for VP 9 of virus grown in HEp-2 cells. Conversely, the amount of VP 9 antigen detected in these CF tests did not show this marked variation if the tests were standardized for the internal S-antigen of disrupted virions (Tables 3 and 4). These results suggested that VP 9 is an internal protein of NDV virions (Miyadera strain), which, unlike surface glycoproteins, does not appear to exhibit host-controlled variation. DISCUSSION
The data described in this communication suggest that the Miyadera strain of
Newcastle disease virus contains a minor internal protein, designated here as VP 9, with a molecular weight of approximately 26,500. While a similar constituent has not been reported in several earlier studies concerned with the structural proteins of various other strains of this paramyxovirus (Evans and Kingsbury, 1969; Haslam et al., 1969; Bike1 and Duesberg, 1969; Mountcastle et al., 1970, 1971; Iinuma et al., 1969; Scheid and Choppin, 1973), it is possible that strain differences per se could account for this discrepancy. An examination of published electropherograms of NDV structural proteins in some cases shows minor distributions of radioactive
PROTEINS
counts in a position near the anode approximating the location of the VP 9 protein of this study (see Bike1 and Duesberg, 1969; Haslam et al., 1969; Mountcastle et al., 1970, 1971). To resolve this question, we plan to test for the presence of VP 9 antigen in other NDV strains using the CF test with detergent-dissociated virus and antiserum monospecific for the Miyadera VP 9 protein. Yields of VP 9 eluted from PAGE gels were relatively low, an observation in agreement with the findings of other workers (Laver, 1970) who have attempted recovery of viral polypeptides from gel bands. Despite this limitation, however, VP 9 protein was extractable from PAGE gels in an antigenically active form and apparently free of other viral structural polypeptides. It is not surprising that VP 9 protein derived from these gels exhibits antigenicity since it is recognized that viral proteins extracted from virions and dissociated by a variety of methods retain their capacity to elicit formation of antibodies specific for individual polypeptide chains (see Horwitz and Scharff, 1969). The Ab-9 antiserum of this study, which our data indicate contains monospecific antibody for the VP 9 protein, could not react with viral envelope protein(s) or the internal S-antigen associated with viral nucleocapsids (Tables 1 and 2). These observations are consistent with the finding that cytoplasmic fluorescence obtained with FITC-conjugated Ab-9 could not be blocked by pretreating cells with either anti-NDV or anti-S antibody. Furthermore, immunofluorescent monitoring of NDV-infected cells with the Ab-9 antibody gave negative staining of the plasma membrane with unfixed cells and positive staining for cytoplasmic deposits of VP 9 antigen with fixed cells, indicating that this protein does not become incorporated into host cell membranes during viral budding. Collectively, these results suggest that VP 9 is an internal component of the NDV virion although not necessarily an integral part of the nucleoprotein complex per se. Further work will be required to establish the precise location of this virion constituent.
OF NDV
237
The possible functional role of the VP 9 polypeptide and its relationship to other structural components of NDV Miyadera is also unknown. It is entirely conceivable that VP 9 represents a monomer of one of the larger NDV structural polypeptides. In this connection, the VP 9 protein of Miyadera virus particles dissociated with deoxycholate can be separated in the upper fractions of sucrose-DOC gradients (Iinuma et al., 1971b) which also contain the polypeptides VGP 4, VGP 5, and VP 8 (Fig. 1). The hemagglutinin but not the neuraminidase activity associated with these gradient fractions can be specifically removed by absorption with red blood cells with concomitant loss of VGP 4 protein as judged by PAGE analysis. Additionally, recent experiments (Iinuma, unpublished) have demonstrated that neuraminidaseactive fractions of enzymatically dissociated NDV Miyadera virus separated by column chromatography contain no detectable VP 9 protein but only VGP 5 and VP 8. While these findings would suggest that VGP 4 is associated with the hemagglutinin activity of this strain whereas VGP 5 and VP 8 are functionally linked with its neuraminidase activity, they do not exclude the possibility that the minor component VP 9 derives from one of these polypeptides. Such postulated structure-function relationships for the proteins of NDV Miyadera must be further substantiated in light of the recent report that the hemagglutinin and neuraminidase activities of NDV (Hickman strain) reside in the same glycoprotein (Scheid and Choppin, 1973). Work now in progress indicates that the VP 9 protein is synthesized and incorporated into mature virions late in the infectious cycle. We have also found that VP 9 is rich in arginine and that this amino acid is required for the synthesis of this polypeptide as well as viral progeny. These and other findings lend further credence to the earlier hypothesis that VP 9 may be an essential protein for the NDV assembly process (Iinuma et al., 1971a; Iinuma et al., 1973). An interesting aspect of this work was the finding that the relative amount of VP 9 protein present in NDV Miyadera grown
238
IINUMA
AND SIMPSON
in various cell types is constant when the test antigen (dissociated virus) used for its determination in CF tests was standardized against the internal S-antigen of these preparations (Tables 3 and 4). Conversely, standardization of these CF titrations using constant hemagglutinin or neuraminidase units of the test viruses gave up to an g-fold variation in the amount of VP 9 antigen detected in virus from various cell types. Such a variation most likely reflects host-controlled modifications of these glycoproteins of the type previously documented for various myxoviruses and paramyxoviruses (Ishida and Homma, 1961; Kates et al., 1961; Drake and Lay, 1962; Matsumoto and Maeno, 1962; Stenbeck and Durand, 1963; Simpson and Hauser, 1966; Klenk and Choppin, 1969; Compans et al., 1970; Homma, 1971, 1972). ACKNOWLEDGMENTS We thank Miss Janeen E. Dougherty for her excellent technical assistance throughout this investigation. The initial phase of this work was conducted at the Cancer Research Institute (Department of Virology), Nagoya University School of Medicine, Nagoya, Japan. This study was supported, in part, by funds from Research Contract NIH-NIAID-72-2504 and USPH Grant AI-09124 from the National Institute of Allergy and Infectious Diseases. Additional support was provided by funds from Research Contract NIH-NCI-E-71-2077 (The Virus Cancer Program) from the National Cancer Institute. REFERENCES C. B., SELA, M., and COOKE, J. P. (1962). The reversible reduction of disulfide bonds in polyalanyl ribonuclease. J. Biol. Chem. 237, 182551831. BAEILANIAN,R., EGGERS, H. J., and TAMM, I. (1965). Studies on the mechanism of poliovirus-induced cell damage. I. The relation between poliovirusinduced metabolic and morphological alterations in cultured cells. Virology 26, 100-113. BIKEL, I., and DUESBERG, P. H. (1969). Proteins of Newcastle disease virus and of the viral nucleocapsid. J. Virol. 4, 388-393. COMPANS, R. W., and CHOPPIN, P. W. (1967). The length of the helical nucleocapsid of Newcastle disease virus. Virology 33, 344-346. COMPANS, R. W., KLENK, H., CALXXJIRI, L. A., and CHOPPIN, P. W. (1970). Influenza virus proteins. 1. Analysis of polypeptides of the virion and identification of specific glycoproteins. Virology 42,
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