VIROLOGY
130,134-143
(1983)
Antigenic Variation in the Influenza A Virus Nonstructural LORENA
E. BROWN, V. S. HINSHAW,
Division
AND
Protein, NSl
R. G. WEBSTER’
of Virology and Molecular Biology, St. Jude Children’s Research Hospital, Memphis, Tennessee 38101
Received Apd 8, 1983; accepted July 5, 1983 The antigenic structure of the nonstructural (NSl) protein encoded by influenza type A virus was examined using monoclonal antibodies prepared against purified NSl inclusions isolated from the cytoplasm of infected cells. Topographical analysis by competitive radioimmunoassay indicated that three different overlapping antigenic regions were present on the NSl of A/WSN/33 (HlNl). Immunoprecipitation studies using infected cell lysates showed that antigenic determinants on A/WSN/33 NSl are common to NSl proteins encoded by a wide range of viruses of human, swine, equine, and avian origin. Several avian strains, however, were found to encode antigenically variant NSl proteins which had either extensive changes in one or more antigenic regions or small changes in epitopes within a region suggestive of antigenic drift. There was no correlation between surface antigen subtype and the antigenic profile of the NSl protein. The antigenic relationships of NSl proteins shown in this study are in agreement with the available sequence data. INTRODUCTION
Although the surface glycoproteins of influenza virus are known to have the capacity for extensive antigenic variation, the internal proteins of the virus are type-specific and have been considered antigenically highly conserved in nature @child and Pereira, 1969; Schild, 1972). In recent years, however, sequence data on the matrix (M) and nucleoprotein (NP) genes revealed that differences in amino acid composition, though relatively minor, occurred in different isolates apparently by the accumulation of point mutations in the genome (for review, see Webster et aC, 1982). With the aid of monoclonal antibodies it was shown that the genetic changes in the NP gene were reflected in changes in antigenicity of this protein (van Wyke et aL, 1980). In addition to virion internal antigens, the influenza nonstructural proteins (NSl and NS2), both encoded on gene 8 of the virus (Inglis et al, 1979; Lamb and Choppin, 19’79), have been considered antigenically and genetically highly conserved. The NSl 1 To whom dressed.
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for
reprints
0042-6822/83 $3.06 Copyright All rights
0 1983 by Academic Press, Inc. of reproduction in any form reserved.
should
be ad-
protein is one of the most abundant viral proteins in infected cells and yet its function remains a mystery. It is synthesized early in infection and migrates to the nucleus (Lazarowitz et al, 1971; Breidis et al, 1981) where it accumulates in the nucleolus (Dimmock, 1969; Krug and Etkind, 1973). It is also found in cell fractions containing polysomes (Shaw and Compans, 1978). Evidence suggesting that the NS proteins may be involved in regulation of viral replication comes from temperature-sensitive mutants with lesions in gene 8 that are defective in the synthesis of virion RNA and/or late proteins at the nonpermissive temperature (Wolstenholme et aL, 1980; Koennecke et ah, 1981). The NSl protein has also been shown to have an affinity for RNA (Yoshida et aL, 1981) and, in some strains combines with cellular RNA to form electron-dense paracrystalline inclusion bodies in the cytoplasm of infected cells (Morrongiello and Dales, 1977; Shaw and Compans, 1978). Sequence data now available for several NSl proteins (Porter et ah, 1980; Baez et aL, 1980; Lamb and Lai, 1980; Winter et aL, 1981; Baez et aL, 1981; Krystal et aL, 1983) have revealed differences in amino acid 134
ANTIGENICITY
OF INFLUENZA
composition. Considerable variability has also been found in charge and phosphorylation (Petri et al, 1982). Antigenic studies to date, however, have failed to detect any significant degree of heterogeneity among NSl proteins from different strains of virus. Using antisera prepared to purified inclusions from A/WSN/33-infected cells (Shaw et aL, 1982) and to A/Fowl Plague/ Restock/34 NSl isolated from polyacrylamide gels (Petri et aL, 1982), extensive cross-reactivity was demonstrated in NSl proteins from type A strains of human, avian, porcine, and equine origin. To examine the antigenic structure of NSl with more sensitive probes, we have prepared monoclonal antibodies to A/WSN/33 NSl for topographical analysis of the molecule and investigated variation of different antigenic areas in a number of influenza A virus serotypes. MATERIALS
AND
METHODS
Viruses. Influenza virus A/WSN/33 (HlNl) and representative strains (see Fig. 4 and Table 2) from each of the 13 hemagglutinin (HA) subtypes were used. Viruses were grown in lo-day-old embryonated chicken eggs and the allantoic fluid used as a source of virus for infection of tissue culture cells. Influenza virus (A/ WSN/33), used in enzyme-linked immunosorbent assays (ELISA), was concentrated from allantoic fluid by adsorption to and elution from chicken erythrocytes and purified by banding in sucrose gradients (Laver and Webster, 1968). Sources of NSl. Cytoplasmic inclusions containing NSl were isolated from A/ WSN/33-infected BHKZl-F (baby hamster kidney) cells by the method of Shaw and Compans (1978). This method involves homogenization of infected cells, removal of nuclei by centrifugation, fluorocarbon extraction of the cytoplasmic extract, and isolation of inclusions from the aqueous phase by discontinuous sucrose density gradient centrifugation. Gradient fractions were examined by polyacrylamide gel electrophoresis (PAGE) and ELISA. Those containing NSl inclusions were dialyzed
NSl
PROTEIN
135
against 0.01 M Tris-HCl pH 8.5, 0.02 M EDTA, and stored at 4’. Cytoplasmic extracts of Madin-Darby canine kidney (MDCK) cells, infected in the presence of [?S]methionine, were used as a source of NSl for radioimmunoprecipitation (RIP) tests. Infected cells were harvested after 24 hr, pelleted, and lysed in 0.05 M Tris pH 7.2 containing 0.6 M KC1 and 0.5% Triton X-100. Nuclei were removed from the cell extract by low-speed centrifugation. For viruses that produced very little cytoplasmic NSl, the pelleted nuclei were resuspended in 0.05 M Tris pH 7.2, containing 0.6 M KC1 and 0.5% Triton X-100, and sonicated. Nuclear extracts were spun at 9000 g for 5 min and the supernatant used in RIP tests. Production of mono&ma1 antibodies. BALB/c mice were infected intranasally with 50 ~1 of diluted A/WSN/33 allantoic fluid (a6000 HAU). Serum was taken at 4 weeks postinfection from the survivors and examined by hemagglutination-inhibition. Mice with titers against A/WSN/33 virus of a1600 were boosted, at least 2 months after infection, with 300 pg of purified NSl inclusions intraperitoneally and 200 pg of inclusions subcutaneously in the hind leg. Four days later the spleen cells from these mice were fused with the 8-azaguanineresistant clone of MOPC-21 myeloma cells (P3/X-63-Ag8) at a ratio of 2:l (spleen cells:myeloma cells) using polyethylene glycol (Kiihler and Milstein, 1976). The resulting hybridoma cells were screened for the production of antibodies to NSl by radioimmunoprecipitation (RIP) and positive cultures were cloned in soft agar. Cloned cell lines were inoculated into the peritoneal cavity of BALB/c mice, together with 0.5 ml Pristane (Aldrich Chemical Co.) and the resulting ascitic fluid used as a source of antibody. The previously characterized monoclonal antibody (174/l) to influenza virus matrix protein (Hackett et al, 1980) was also used in this study. Pur$kation and radioiodination of mono&ma1 antibodies. Monoclonal antibodies were purified from ascitic fluid by affinity chromatography using protein ASepharose 4B (Pharmacia). Immunoglobulin G (IgG) was eluted from the adsorbent
136
BROWN,
HINSHAW,
with 0.5 M glycine in 0.14 M NaCl (pH 3.1) and the pH immediately readjusted to 7.4. Protein concentrations were determined both by the method of Lowry (1951) and by absorbance at 280 nm, assuming an adsorption coefficient of 1.34 for a 1 mg/ml solution. For iodination, 100 pg of purified IgG was reacted with 400 &i Na ‘%I (Amersham) and 10 pg chloramine T. After 3 min, the reaction was terminated by the addition of 30 pg metabisulfite and 10 ~1 of 6 X 10e2 M KI. Unincorporated ‘%I was removed by passage through Sephadex G-25 (Pharmacia) equilibrated with phosphate buffered saline (pH 7.4) containing 5 mg/ ml bovine serum albumin and 0.05% Tween 20 (BSA5PBST). Serological assags. Hemagglutination and hemagglutination-inhibition tests were carried out as previously described (Laver and Webster, 1968). The method for indirect ELISAs was that used by Kida et al. (1982). Antigen solutions for coating plates were either 200 HAU per/well of disrupted purified A/WSN/33 virus or purified NSl inclusions at 80 ng protein/well. Radioimmunoprecipitation. Radioimmunoprecipitation (RIP) tests were carried out as described by Kida et al. (1982). Briefly, protein A-Sepharose 4B beads (Pharmacia) (50 ~1 of 30% suspension), coated with saturating amounts of rabbit anti-(mouse immunoglobulin) serum, were reacted with 5 ~1 undilute ascitic fluid for 1 hr at 25”. After washing, labeled antigen was added and incubated for a further 1 hr. The beads were again washed, resuspended in Laemmli sample buffer (Laemmli, 1970), boiled for 3 min, pelleted, and the supernatant applied to either a 12 or a lo-20% gradient polyacrylamide sodium dodecyl sulfate gel as described by Laemmli (1970). After electrophoresis for 18 hr at 70 V, the gels were processed for fluorography by immersion in a 1 M sodium salicylate solution for 30 min (Chamberlain, 1979).
Estimation
of antibody in a&tic fluid.
Each ascitic fluid preparation and purified IgG preparation was titrated by ELISA for binding to purified NSl inclusions. The amount of IgG present in an ascitic fluid
AND
WEBSTER
was then calculated from the difference in its binding titer compared to the corresponding Ig preparation of known concentration. This estimation is valid only if the binding capacity of the IgG has not been diminished on low pH treatment during purification. Since in each case the slope of the curve for purified IgG was the same as that of the corresponding ascitic fluid, indicating similar affinity of interaction, it is unlikely that acid treatment has affected these antibodies. Competitive radioimmunoassag. Flexible 96-well plates,(Dynatech) were coated with purified NSl inclusions in PBS (at 30 ng/ 50 pi/well) for 2 hr. The coating solution was then removed and replaced with 100 ~1 of 10 mg/ml BSA in PBS (BSA,,,PBS). After 1 hr the plates were washed with PBS containing 0.05% Tween 20 (PBST). Dilutions of ascitic fluid in BSA,PBST (25 ~1) were then added, immediately followed by a constant amount of iodinated purified IgG in 25 ~1 BSA,PBST. The plates were incubated overnight and then washed six times with PBST. The wells were separated and radioactivity determined in a gamma counter. A suitable amount of radioactive IgG was determined by titration in the absence of unlabeled competitor. Slightly less than that required to completely saturate the available binding sites was used. Unless otherwise specified this was 675 ng [1251]IgG/well. The results of the competition experiments are expressed as percentage inhibition of binding: 100 X (cpm bound in absence of competitor - cpm bound in the presence of competitor) + cpm bound in absence of competitor. Anti-matrix ascitic fluid was included as competitor in each experiment as a negative control. RESULTS
Characterization Antibodies
of Anti-NSl
Mono&ma1
Eight monoclonal antibodies were prepared that failed to react in ELISA with purified disrupted A/WSN/33 (exemplified by 9/l and 19/l in Fig. 1A) but bound to purified A/WSN/33 inclusions (Fig. lB), suggesting specificity for the NSl protein.
ANTIGENICITY
2
B
32
128
512
2
ANTIBODY DILUTION
8
32
OF INFLUENZA
128
512
X IO-’
FIG. 1. Specificity of monoclonal antibodies. Ascitic fluid containing antibodies raised against matrix protein (17411, 0) and purified NSl inclusions (9/l, 0 and 19/l, n ) were titrated by ELISA against purified A/WSN/33 influenza virus (A) and purified NSl inclusions (B).
Alternatively anti-matrix ascitic fluid bound to high titers to disrupted virus but only very weakly to the purified inclusion preparation (Figs. 1A and B). Furthermore, each of the eight monoclonal antibodies precipitated proteins of molecular weight corresponding to NSl from cytoplasmic extracts of A/WSN/33 infected cells (Fig. 2). The precipitated proteins were resolved into two bands as have also been observed
NSl
PROTEIN
137
by Petri et al. (1982) and Shaw et al. (1982). Two proteins indistinguishable from those precipitated by anti-NSl monoclones were precipitated by anti-matrix monoclonal antibody if A/WSN/33-infected cell extracts were used in RIP tests. However, when extracts from cells infected with other viruses, such as A/seal/Massachusetts/l/80 (H7N7), were used a difference in the specificity of the anti-NSl and antimatrix antibodies was clearly observed (Fig. 2). The existence of two different molecular weight forms of matrix protein has also been shown by Petri et al. (1982) and it is conceivable that these would migrate at the same positions on a gel as the two forms of NSl. However, to firmly establish that the two bands were not a result of coprecipitation of matrix and NSl, radiolabeled A/WSN/33 cytoplasmic extracts were adsorbed prior to use in RIP tests, with anti-matrix-coated protein A-sepharose 4B beads. No proteins were precipitated with anti-matrix antibody from the adsorbed cytoplasmic extracts, indicating that adsorption had successfully resulted in significant depletion of matrix protein (results not shown). Precipitation from the matrix-depleted extracts with antibodies to NSl still resulted in the appearance of two bands on the gel, indicating that these were both NSl and not a result of coprecipitation of NSl and matrix.
Antigenic Analysis of NSl by Competitive Binding Studies
9/f
Wf
atd
9/t
19x1
OM
FIG. 2. Radioimmunoprecipitation of NSl and M from infected cell lysates. Cytoplasmic extracts (left lane in both panels) from cells infected with A/WSN/ 33 (HlNl) or A/seal/Massachusetts/l/80 (H7N7) were precipitated with antibodies to NSl (9/l, 19/l) or to matrix protein (cuM). Precipitates were run on a 12% polyacrylamide gel.
Competitive RIA was used to determine whether the anti-NSl monoclonal antibodies possess different fine specificities. These studies also provide information on the relative affinities of the antibodies and the topographical relationships of antigenie regions on the NSl surface. Each of the anti-NSl ascitic fluid preparations was tested for their ability to compete with iodinated anti-NSl IgG for binding to purified NSl inclusions. One example of the inhibition curves obtained is shown with ‘251-31/2 in Fig. 3. The amount of competing IgG necessary to inhibit the binding of iodinated monoclonal antibody by 50% was calculated from each curve (Table 1). Three
138
BROWN,
HINSHAW,
00
5,000
1,250
312
70
COMPETING ANTIBODY (ng)
FIG. 3. Inhibition of binding of anti-NSl by ascitic fluid preparations in competitive radioimmunoassays. Iodinated 31/2 anti-NSl IgG (625 rig/well) was added to purified NSl inclusions in the presence of serial dilutions of 20/l (0). 3112 (m), 51/l (0), 3012 (O), 91/l (A), 9/l (A), 19/l (A), 18/l (A) and anti-matrix (A) ascitic fluids. The results are expressed as the percentage inhibition of binding of 31/2 IgG in the presence of competitor relative to its binding in the absence of competing antibody.
different patterns of reactivity were evident and the antibodies were divided into three groups on this basis. Antibodies 9/l, 19/l, and 91/l form group I whose TABLE
COMPETITIVE ACTIVITY OF ANTI-NSl
1 MONOCLONAL ANTIBODIES
Competing [=I]IgG
9/l
19/l
91/l
I
9/l 19/l 91/l
215” 896 564
91 478 3640
790 2086 2520
II
18/l
>b
>
>
III
20/l 31/2 51/l 30/2
> > > >
> > > >
Group
1.2 2.5 1.2 2.0
x X x x
WEBSTER
binding is efficiently inhibited by all of the antibody preparations; group II is formed by 18/l which is unique in that its binding is only inhibited by itself and is not inhibited by any of the other antibodies; and the remaining antibodies (20/l, 31/2, 51/ 1, and 30/2) form group III in which the binding of each member can only be significantly competed by other members of the group. It is evident that the competition observed is not always reciprocal. While iodinated group I antibodies can be blocked by group I, II, and III antibodies, they can only block the binding of members of group I. A possible explanation for this is that the binding affinity of group I antibodies is significantly less than those in groups II and III. Only relatively small amounts of 18/l were required to inhibit the binding of antibodies from group I suggesting that this antibody may be of higher affinity than other members of the panel. The group II antibody generally achieved 50% inhibition of group I binding at concentrations less than that of the group I antibodies themselves but this was not always the case. To determine whether the failure of some antibodies to inhibit iodinated group II and III antibodies was due to affinity differences, competition experiments were performed with increased ratios of competitor
90
20,000
AND
monoclonal
antibody
18/l
20/l
9.2 4.6 74 193
10’ 10’ 10’ 104
> > > 1.2 x 10”
52 69 1502
31/2
51/l
3012
77 77 527
76 44 112
1’76 221 140
>
>
>
>
839 179 210 4750
3239 3&1 573 2100
992 175 266 1780
222 146 280 883
a Results are expressed as the amount of competitor antibody (ng) required to inhibit binding of iodinated IgG by 50%. b >, 50% inhibition was not achieved at a dilution of ascitic fluid of l/50 representing >lO’ ng.
ANTIGENICITY
OF INFLUENZA
to labeled antibody; the amount of iodinated IgG was reduced to 125 rig/well and competing as&tic fluid was tested undilute. Under these conditions some inhibition of iodinated group II and III monoclonal antibodies by group I was observed (results not shown). Anti-matrix ascitic fluid gave no inhibition at equivalent concentrations. Even at high levels of competitor, 18/l did not compete with members of group III indicating that group II and III are truly distinct.
Antigenic Variation in NSl Determinants The NSl proteins encoded by different strains of influenza virus were examined
NSl
PROTEIN
139
for their antigenic relationship to A/WSN/ 33 NSl. Human, avian, and mammalian strains representative of each HA subtype were grown in MDCK cells. Infected cell lysates were examined by RIP with a group I (9/l), group II (18/l), and group III (20/l) monoclonal antibody (Figs. 4a and b). Examination of the human isolates (panels A, C, and E) showed that A/PR/ S/34 (HlNl) encodes an NSl protein with a relatively weak interaction with group I, II, and III antibodies despite the close surface antigen relationship to A/WSN/ 33 virus. In each of the human strains, A/ PR/8/34 (HlNl), A/Japan/305/57 (H2N2), and A/Udorn/307/‘72 (H3N2), the NSl mi-
FIG. 4. (a and b) Radioimmunoprecipitation of extracts from cells infected with influenza viruses representative of each HA subtype. Monoclonal antibody 9/l (group I), 18/l (group II), and 20/l (group III) were reacted with cytoplasmic extracts (or a nuclear extract in panel N) of infected MDCK cells in RIP tests. The first lane of each panel contained labeled cell extract (half the amount used for precipitation). The group of the antibody used in the test is shown together with the hemagglutinin (H) and neuraminidase (N) surface antigen subtype for each strain. Immunoprecipitates were run on 10-20’S gradient polyacrylamide gel. A, A/PR/8/3Q; B, A/swine/Wisconsin/ l/67; C, A/Japan/305/57; D, A/duck/Germany/l215/73; E, A/Udorn/307/72; F, A/duck/Ukraine/ l/63; G, A/equine/Miami/i/6$ H, A/pintail/Alberta/ll9/79; I, A/tern/South Africa/Gl; J, A/ turkey/Massachusetts/3740/6% K, A/turkey/Oregon/71; L, A/equine/Prague/i/56; M, A/turkey/ Ontario/6118/68, N, A/turkey/Wisconsin/i/66; 0, A/turkey/Minnesota/5/79; P, A/duck/England/ 56; Q, A/duck/Alberta/60/76; R, A/gull/Maryland/704/77.
140
BROWN,
HINSHAW,
grated at a position of higher apparent molecular weight than that of the nonhuman strains. In the latter two strains, two bands were present as had been observed with A/WSN/33 (HlNl) (Fig. 2). This was also noted with several avian strains (panels J, 0, and R). Most immunoprecipitates contained only proteins migrating in the region of NSl. However, with some samples, coprecipitation of higher molecular weight proteins was evident. An extreme example is A/ equine/Miami/l/63 (H3N8) in panel G (Fig. 4a) where the NP protein is precipitated in large amounts. This was also observed by Petri et al. (1982) and Shaw et aL (1982) and would suggest an association of these two proteins within the infected cell. Variation in the antigenicity of the NSl molecule was observed in 5 of the 18 strains. This took the form of either decreased activity with one antibody relative to the others (panel K) or complete absence of binding by a particular monoclonal antibody (panels Q and R). Two viruses, A/ duck/Alberta/ll9/79 (H4N6) in panel H and A/turkey/Ontario/608/68 (H8N4) in panel M, encoded NSl proteins that failed to react with any of the three antibodies. The strains showing variation were examined further in RIP tests with the complete panel of antibodies to determine whether the differences were confined to the epitopes examined in Figs. 4a and b or were representative of the group. The results are summarized in Table 2. Another member of each subtype was also examined to see if the variations in NSl were subtype-specific (Table 2). The antigenic analysis revealed that A/duck/Alberta/ll9/79 (H4N6) and A/turkey/Ontario/6118/68 (H8N4) NSl proteins were lacking not only in the epitopes examined in Fig. 4b but also in those defined by the rest of the antibodies in the panel. The A/duck/Alberta/ 827178 (H8N4) and A/duck/Alberta/60/76 (H12N5) NSl proteins retained reactivity with a single antigenic group while other strains showed a loss or decrease in activity with some but not all antibodies comprising each group. Although the NSl proteins from both H13N6 isolates had the same
AND
WEBSTER
antigenic profile, there was no overall correlation between the antigenic reactivity of the NSl protein and that of the hemagglutinin and neuraminidase surface antigens. DISCUSSION
In this study, monoclonal antibodies prepared to the NSl protein encoded by A/WSN/33 influenza A virus have allowed a topographical analysis of the antigenic structure of the molecule to be made and have detected different antigenic forms of this protein that had previously been considered antigenically highly conserved. Competitive radioimmunoassay data revealed three different patterns of inhibition suggestive of three different antigenic regions on the surface of the NSl protein. Antibodies from each of the three groups can exert some effect on the binding of members of the other groups, indicating that the antigenic regions defined here are not topographically remote or nonoverlapping. Members of group I could be competed for by all of the antibodies in the panel while group II (defined by only a single monoclonal antibody) and group III were only competed for by members of their own groups. Differences in affinity between the three groups of antibodies did not appear to have caused artificial division of the panel yet the definition of each group became less clear if very high competitor to labeled antibody ratios were used. Groups II and III remained distinct, however, in that the group II antibody could never compete with members of group III even when present at 1006 times more than the labeled group III IgG. The simplest topographical model that can be proposed for the antigenic profile of the NSl molecule from these results involves the site to which group I antibodies bind being central to the sites defined by groups II and III, and possibly within a pocket or groove on the surface. In this way, group I antibodies would not block the binding to sites II and III at the edges of the pocket but may themselves be blocked from entering the groove by group II and III antibodies binding at the edges.
ANTIGENICITY
OF INFLUENZA TABLE
NSl
141
PROTEIN
2
ANTIGENIC VARIATION IN NSl DETERMINANTS Surface antigen subtype H4N6 H4N8 H7N3 H7N7 HSN4 H8N4 H12N5 H12N5 H13N6 H13N6
Influenza
virus strain
A/duck/Alberta/ll9/790 A/duck/Alberta/286178 A/turkey/Oregon/71 A/seal/Massachusetts/l/SO A/turkey/Ontario/6118/68 A/duck/Alberta/827178 A/duck/Alberta/60176 A/duck/Aiberta/461/81 A/gull/Maryland/704/77 A/gull/Massachusetts/50/80
III
II
I” 9/l
19/l
91/l
18/l
20/l
31/2
51/l
3012
-b
-
-
-
-
-
-
-
+ + t -
+ + t -
+ + + -
t t + -
t + t -
+ + + + t
+ + t -
+ t/+ -
+ t -
+ t/+ -
+ + t -
+ t t
t -
+ t t
t t t
a Antibody group defined in Table 1. b -, NSl precipitation was not detectable by autoradiography acrylamide gel electrophoresis; +, NSl was precipitated, +/-, + for that particular strain.
Group II antibodies would bind at a site on the edge sufficiently far from the site for group III antibodies so that their binding was not mutually exclusive. Such a situation has recently been proposed for the antigenic architecture of the neuraminidase molecule of influenza virus (Jackson and Webster, 1983). In this case, the antigenic sites are clustered around the enzyme’s active site. Similarly, the groove containing the putative cell receptor binding site on the HA molecule is flanked by antigenic sites (Wiley et al, 1981). With this precedent in mind, it is possible that antibodies such as these may interfere with the function of the NSl molecule and may prove useful as tools to elucidate the role of this protein. Similar inhibition studies with monoclonal antibodies have been used to show the importance of the influenza virus nucleoprotein in in vitro transcription (van Wyke et al., 1981). Reactivities of the monoclonal antibodies with NSl of different strains of influenza A show that antigenic variation does occur in this protein. The only other evidence suggestive of antigenic differences in the NSl molecule has been the differential ability of unlabeled cytoplasmic extracts of cells infected with various human
t t +
of immunoprecipitates reduced precipitation
analyzed by polyof NSl compared to
strains to compete with the binding of a rabbit anti-NSl serum to iodinated A/ WSN/33 NSl (Shaw et al, 1982). It was concluded that gradual antigenic variation had occurred over the 45 years separating A/WSN/33 and recent isolates. No such change was observed in the limited number of human strains examined here in an epitope from each antigenic region. A larger panel of monoclonal antibodies may be necessary to detect subtle differences between human strains if they exist. The determinants on NSl that we have examined are common to the majority of viruses tested despite their origin. However, ‘7 of the 23 viruses did encode antigenitally variant NSl proteins, showing either a complete loss of reactivity with certain antibodies or a decreased ability of the antibody to bind. This indicates that antigenic drift, which is the accumulation of point mutations that affect antigenically important areas of the molecule, is occurring in the NSl. Two strains did not react with any of the 8 antibodies in the panel although significant amounts of NSl appeared to be present in the infected-cell lysates. Reactivity with a more extensive panel of monoclonal antibodies or with specific polyclonal antisera is necessary to
142
BROWN,
HINSHAW,
determine whether these strains encode a unique subtype of NSl or whether shared determinants are present. All of the antigenically variant NSl proteins examined here were of avian origin. There was no overall correlation between variation observed in NSl and the surface antigen subtype as was also found with the NP molecule (van Wyke et aZ., 1980). Several strains isolated from ducks in the same location over a period of 5 years encoded antigenically different NSl proteins even though some isolates shared the same hemagglutinin and neuraminidase genes. Two H13N6 viruses isolated from gulls, however, did encode the same antigenic form of NSl. Whether this particular NSl protein is characteristic of viruses that can replicate in gulls will require examination of a large number of isolates from this species. Although we cannot as yet determine whether the antigenic reactivity of NSl can be used as a marker for replication in certain avian populations, it is clear that monoclonal antibodies to NSl will be valuable in antigenic classification of this molecule. The only previous attempt at classification of the NSl protein has used estimations of base sequence homology of gene 8 derived by molecular hybridization (Scholtissek and von Hoyningen-Huene, 1980). This placed the human strains in a group separate from the avian strains. The avian strains were then further subdivided into two groups between which the homology was about 40%. These groups do not coincide with the antigenic relationships determined in this study which show that many of the avian strains are highly cross-reactive with human strains. In contrast, complete sequence data for the NSl of A/Fowl Plague/Restock/34 (Porter et al, 1980), A/Udorn/72 (Lamb and Lai, 1980), A/PR/8/34 (Baez et aL, 1980; Winter et aL, 1981), and A/duck/Alberta/60/76 (Baez et aL, 1981) revealed that the former three strains showed 89% homology whereas less conservation of sequence was observed with A/duck/Alberta/60/76 (68% homology between A/duck/Alberta/60/76 and A/PR/8/34). Our antigenic analysis indicates that the NSl proteins of A/ Udorn172 and A/PR/8/34 share epitopes
AND
WEBSTER
in each of the three antigenic areas while A/duck/Alberta/60/76 NSl has only one antigenic region in common with these proteins. Although our studies examine only a small proportion of the molecule, i.e., the antigenic determinants, the relationships we observe appear to correlate well with the known sequence information. We are now in a position to use the monoclonal antibodies for detailed classification of influenza A isolates and, perhaps of greater interest, as tools to elucidate the function of NSl. ACKNOWLEDGMENTS This work was supported by U. S. Public Health Research Grant AI08831 and AI02649 from the National Institute of Allergy and Infectious Diseases, Cancer Center Support (CORE) Grant CA21765, and AISAC. The authors wish to thank Kenneth Cox for his excellent technical assistance. REFERENCES BAEZ, M., TAUSSIC, R., ZAZRA, J. J., YOUNG, J. F., PALESE, P., REISFELD, A., and SKALKA, A. (1986). Complete nucleotide sequence of the influenza A/ PR/8/34 virus NS gene and comparison with the NS genes of the A/Udorn/72 and A/FPV/Rostock/ 34 strains. Nucleic Acids Rex 8, 5845-5858. BAEZ, M., ZAZRA, J. J., ELLIOTT, R. M., YOUNG, J. F., and PALESE, P. (1981). Nucleotide sequence of the influenza A/duck/Alberta/66/76 virus NS RNA: Conservation of the NSl/NSX overlapping gene structure in a divergent influenza virus RNA segment. VZrolosy 113, 387-402. BREIDIS, D. J., CONTI, G., MUNN, E. A., and MAHY, B. W. J. (1981). Migration of influenza virus-specific polypeptides from cytoplasm to nucleus of infected cells. lrirology 111, 154-164. CHAMBERLAIN, J. P. (1979). Fluorographic detection of radioactivity in polyacrylamide gels with the water soluble fluor, sodium salicylate. And Rio&em 98,132-135. DIMMOCK, N. J. (1969). New virus-specific antigens in cells infected with influenza virus. virdogy 39,224234. HACKER, C. J., ASKONAS, B. A., WEBSTER, R. G., and VAN WYKE, K. (1986). Monoclonal antibodies to influenza matrix protein: Detection of low levels of matrix protein on abortively infected cells. J. Gen viral 47.497-501. INGLIS, S. C., BARREN, T., BROWN, C. M., and ALMOND, J. W. (1979). The smallest genome RNA segment
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