J. gen. Virol. (1982). 58, 1-12.
Printed in Great Britain
Key words: ND V/non-structural proteins/glycoproteins/strain variation
Non-structural Proteins in Newcastle Disease Virus-infected Cells
By P. C H A M B E R S *
AND A. C. R. S A M S O N
Department of Genetics, The University, Newcastle upon Tyne NE1 7R U, U.K.
(Accepted 18 August 1981)
SUMMARY
Examination of pulse-labeUed Newcastle disease virus (NDV)-infected chick
embryo fibroblasts (CEF) by two-dimensional polyacrylamide gel electrophoresis
revealed the presence of two virus-coded non-structural polypeptides ofmol, wt. 36K
and 33K. Longer pulses and pulse-chase incubations revealed the production of an
additional, glycosylated, non-structural polypeptide of mol. wt. 40K (gp40). Kinetic
arguments suggest that 36K and 33K are primary translation products but that gp40
is not. 36K was stable in chase incubations, but 33K was not. Partial digest peptide
analysis showed that gp40 and an additional glycosylated polypeptide gp62, which is
sometimes present (Chambers & Samson, 1980), are related to the HN polypeptide.
Partial digest peptide analysis of the 36K polypeptide generated only a few peptides,
which were not sufficient to conclude whether 36K was related to the major virus
polypeptides, and since polypeptide 33K was metabolically unstable, insufficient
radioactivity was incorporated for peptide studies. Extensive strain-dependent
variation in the isoelectric points and mol. wt. of all the NDV polypeptides which are
soluble in the isoelectric focusing gels, including 36K and 33K, is reported. This
variation, and the insensitivity of the synthesis of 36K and 33K to actinomycin D,
show that both non-structural polypeptides are virus-coded.
INTRODUCTION
Paramyxoviruses, of which SV5, Sendai and Newcastle disease virus (NDV) are the
best-studied examples, contain six virus coded polypeptides (Kingsbury, 1977) together with
some actin derived from the cells in which they were grown. The virus envelope contains three
of the virus-coded polypeptides: HN, F and M. Nucleocapsids contain the other three
polypeptides: L, NP and P. The mol. wt. of the corresponding polypeptides are similar for
Sendal, SV5 and NDV, with the exception of P. The mol. wt. of HN is approx. 75K, F 67K,
M 39K, L 200K and NP 55K. The mol. wt. of the P polypeptide is 79K in Sendai virus (Lamb
et al., 1976), 53K to 56K in NDV (Chambers & Samson, 1980; Smith & Hightower, 1981 a),
and 46K in SV5 (Buetti & Choppin, 1977). The analogy of the 'P' polypeptides is weakened
by this variation in mol. wt., and the 'P' polypeptide of NDV was termed NAP
(nucleocapsid-associated protein) in an earlier publication (Chambers & Samson, 1980). This
terminology is also used in this communication.
The synthesis of all these structural polypeptides is readily detectable in virus-infected cells,
and several studies have suggested that non-structural polypeptides are also synthesized. In
the case of Sendai virus, it has been shown that the peptide fingerprint of the non-structural
polypeptide C (22K) is different from those of the structural polypeptides (Lamb & Choppin,
1978). A polypeptide of similar mol. wt. (24K) has been detected in SV5-infected cells (Peluso
et al., 1977). In NDV-infected cells, a non-structural polypeptide, 36K, has been described
(Alexander & Reeve, 1972; Hight0wer & Bratt, 1974; Chambers & Samson, 1980) but not
characterized.
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P. CHAMBERS
AND
A. C. R. SAMSON
Glycosylated non-structural polypeptides are also known. The active F protein is made up
of two disulphide-linked polypeptides, F~ and F 2, derived from a glycosylated precursor F 0 by
proteolytic cleavage. HN0, a precursor to the HN glycoprotein, has been detected for two
extremely avirulent NDV strains (Nagai et al., 1976a; Nagai & Klenk, 1977). F 0 and HN o
may be either structural or non-structural, depending on the virus-cell system (Nagai et al.,
1976a). Two glycosylated polypeptides, mol. wt. 62K and 40K (gp62, gp40), have been
detected in NDV-infected cells (Chambers & Samson, 1980) but no previous studies have
detected similar material in virions.
Experiments were devised to determine whether the NDV-induced non-structural
polypeptides are primary gene products or are related to the structural polypeptides.
METHODS
Staphylococcus aureus V8 protease was obtained from Miles Laboratories, Slough, U.K.;
40% ampholines pH range 5 to 7 were from LKB, South Croydon, U.K.; Nonidet P40
(NP40) was from BDH. Falcon micro-test II plates, 96 flat-bottomed well (96-well plates)
were from Becton-Dickinson, Runcorn, U.K. All other materials were as described previously
(Chambers & Samson, 1980).
Virus and cells. NDV strains AV, HP16, N, LK and Beaudette C, were obtained from
Professor C. F. Fox, University of California, Los Angeles. NDV strains B1, F and Texas
were obtained from Dr R. Avery, Warwick University. Large scale preparation of purified
NDV was as described previously (Chambers & Samson, 1980). Alternatively, for use in
labelling experiments, a smaller scale preparation was used. Approximately 104 p.f.u, w a s
injected into each 10-day-old embryonated egg. After 48 h at 37 °C, allantoic fluid was
harvested and clarified by centrifugation at 10000 g for 10 min. The supernatant was divided
into 1 ml amounts and stored frozen at - 7 0 °C. Virus was used without further purification
for labelling experiments on monolayers. Secondary monolayers of chick embryo fibroblasts
(CEF) were grown in 96-well plates in medium 199 supplemented with 5 % calf serum. BHK
cells were grown in medium 199 supplemented with 10 % calf serum.
Radiolabelled virus. Allantoic membranes from l 1-day-old embryonated eggs were
collected and transferred to allantoic fluid from NDV-infected eggs (previous section) for 30
rain at 37 °C. Membranes were then rinsed three times in Hanks' salts and transferred to
medium 199 containing 0.1 /~g/ml leucine. After 6 h incubation at 37 °C, membranes were
transferred to medium 199 containing 5 ~tCi/ml [14C]leucine and incubated overnight at
37 °C. Medium was pipetted off, mixed with purified non-radioactive NDV, and [14C]leucinelabelled NDV prepared by centrifugation on sucrose/tartrate gradients (Chambers & Samson,
1980).
[3H]glucosamine-labelled NDV was prepared by a similar procedure, except that media
contained 10 ~tg/ml leucine throughout, and overnight incubation was in medium
supplemented with 50 aCi/ml [3H]glucosamine rather than [laC]leucine.
R adioIabelled monolayers
[14C]leucine-labelled monolayers. Confluent monolayers of CEF or BHK cells in 96-well
plates were incubated overnight in medium 199 containing 0.4 /tg/ml leucine. Monolayers
were rinsed with Hanks' salts, then incubated at 37 °C for 30 rain under 30 ~1 clarified
NDV-infected allantoic fluid, ensuring an m.o.i, greater than 100. After infection, cells were
rinsed with Hanks' salts, then incubated with medium 199 containing 0.1 /zg/ml leucine until
the labelling period commenced. The monolayers were labelled with 20/~1 of medium 199
lacking non-radioactive leucine, supplemented either with 17 pCi/ml [14C]leucine for labellings
of duration greater than 1 h, or with 33 pCi/ml [14C]leucine for labellings of duration 1 h or
less. After labelling, radioactive medium was removed and the monolayer was taken up into
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N D V non-structural proteins
30/A of sample buffer [8 % (w/v) NP40, 9-5 M-urea, 5 % (v/v) 2-mercaptoethanol, 2 % (w/v)
ampholines pH range 3.5 to 10]. The whole extract was then loaded on to either an isoelectric
focusing (IEF) gel or a non-equilibrium pH gradient electrophoresis (NEPHGE) gel
(O'Farrell et al., 1977).
[3H]glucosamine-labelled monolayers. The general procedure was as described above,
but all media contained 10/lg/ml leucine and labelling was with medium 199 supplemented
with 100 ~tCi/ml [3H]glucosamine. Monolayers were taken up in final sample buffer
(Laemmli, 1970).
Actinomycin D, when used, was present in post-infection incubation and labelling media at
a final concentration of 2/~g/ml.
Two-dimensional gel electrophoresis. NP40 was used in all IEF procedures, although the
virus nucleocapsid protein is less soluble than in gels containing Tween 80 (Chambers &
Samson, 1980). The overall results were more reproducible, and the anomalous, slow
migration of F in IEF gels containing Tween 80 was avoided (Samson et al., 1981). The
general procedure of O'Farrell (1975) was followed, with slight modifications, depending on
the isoelectric point (pI) of the polypeptides under study. (i) To resolve basic polypeptides,
such as gp40 and 33K, N E P H G E gels contained 2% ampholines pH range 3.5 to 10. These
were loaded at the anode (acid) and electrofocused for 2500 V.h. (ii) To resolve neutral and
acidic polypeptides, such as HN, F 0, F l, NAP and 36K, IEF gels contained 1% ampholines
pH range 3.5 to 10 and 1% ampholines pH range 5 to 7. These were loaded at the anode and
electrofocused for 4000 V.h (Samson et al., 1981). Measurement of pH gradients,
equilibration of the IEF gels and running the second dimension were as described by O'Farrell
(1975). Second dimensions were run on 10% polyacrylamide gels as described previously
(Chambers & Samson, 1980) then either prepared for fluorography (Bonner & Laskey, 1974)
or fixed in 10 % (v/v) acetic acid and dried down for autoradiography.
SDS-polyacrylamide gel electrophoresis (SDS-PAGE). This was performed on 10%
polyacrylamide slab gels (Laemmli, 1970).
Partial digest peptide analysis of NDV polypeptides. NDV-induced polypeptides were
located in two-dimensional autoradiographs, cut out, and the gel slices swollen for 15 rain in
water followed by 30 min in equilibration buffer [0.5% (w/v) SDS, 0.1% (v/v)
2-mercaptoethanol, 0.00I % (w/v) bromophenol blue, 0.063 M-tris-HCt pH 6.8]. Slices were
rinsed in equilibration buffer lacking 2-mercaptoethanol before loading into stacking gel
sample wells which already contained 3 /~g V8 protease set in 0.3 % (w/v) agarose, 0.1%
(w/v) SDS, 0.001% bromophenol blue, 0.063 t~-tris-HC1 pH 6-8. Peptides were then
generated and analysed by one of two alternative procedures. Firstly, the procedure of
Cleveland et al. (1977) was used. Electrophoresis was at 40 mA/gel until the dye front
reached the interface of stacking gel and resolving gel (17% polyacryl~mide). The gel
apparatus was then filled with water prewarmed to 37 °C and incubated at 37 °C for 30 min.
After incubation was complete, the current through the apparatus was restarted and the gel
was run with an ample supply of cooling water until the dye front was within 1 cm of the gel
end. Secondly, the electrophoresis was at 15 W/gel constant power until the dye front was 5
mm from the interface of stacking gel and resolving gel [10 % polyacrylamide containing 50 %
(w/v) sucrosel. The current was then stopped, the apparatus was filled with prewarmed water
and incubated as above. After incubation, electrophoresis was restarted and continued until
the dye front was within 1 cm of the gel end. In either case, the gel was stained with
Coomassie Brilliant Blue, then processed for fluorography.
The 10% polyacrylamide:sucrose gel system allows resolution of a wide range of
polypeptide tool. wt., from above 95K to below 6K with less risk of gels cracking during
drying than when using high polyacrylamide concentrations (above 10%). Normal Laemmli
buffers were used throughout, and sucrose was dissolved in the separating gel mix before
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4
P. CHAMBERS
A N D A . C. R . S A M S O N
polymerization. One gl of N,N,N',N'-tetramethylethylenediamine (TEMED) was used to
catalyse polymerization of 25 ml of gel mix, rather than the normal 10 gl used for 25 ml of
SDS-PAGE gel mix. Digestion within the stacking gel rather than at the stacking gel
resolving gel interface gave more extensive digestion of the substrate polypeptide by V8
protease under the conditions described.
RESULTS
Unless noted otherwise in the text all experiments were performed with NDV strain
Beaudette C. The base to acid orientation is left to right in all two-dimensional fluorographs
shown. NEPHGE gels for Fig. 1 and 6 contained 2% ampholines pH range 3.5 to 10. IEF
gels for Fig. 4 and 5 contained 1% ampholines pH range 3.5 to 10, 1% ampholines pH range
5to7.
Investigation of ND V primary translation products
NDV-infected CEF were labelled, and polypeptide synthesis was investigated, to determine
which NDV-induced non-structural polypeptides were primary translation products, and
which might be secondary products produced by proteolysis or other modification of the
primary products which can change the SDS-PAGE mobility.
If the non-structural polypeptides 36K, gp62, and gp40 are primary translation products,
rather than secondary products, the quantity relative to other primary products (HN, F 0,
NAP, NP and M) should be high in a short pulse-labelling experiment. Furthermore, if all
primary translation products are metabolically stable, their relative quantities will not change
in a longer pulse labelling. If any polypeptide is generated by modification of a primary
product, it might be absent from a short pulse but appear in a longer pulse or in a pulse-chase.
For example, F 0 can be chased into F 1 in a pulse-chase (Samson & Fox, 1973; Nagai et aL,
1976 a) since F 0 is the primary product and F 1is a secondary cleavage product.
Pulse and pulse-chase experiments were performed on NDV-infected cells 8 h
post-infection, and the resulting two-dimensional fluorographs are shown in Fig. 1. A 5 min
pulse is shown in (a), a 1 h pulse in (b) and a 1 h pulse followed by a 1 h chase in (c). As
expected, F 0 was cleaved to F 1 between (b) and (c). 36K was present in equal amounts in (a)
and (b) which is the behaviour expected from a metabolically stable primary translation
product. In (c), there was an increase in the more acidic, phosphorylated form of 36K
(Chambers & Samson, 1980). On the other hand, gp40 showed labelling kinetics similar to
F1, being absent from (a), with a trace appearing in (b), and maximal in (c). Thus, it seems
likely that gp40 is a secondary product formed by some modification or cleavage of one of the
primary virus polypeptides. HN charge heterogeneity increased between (a) and (b),
indicating a slow processing and the relative amount of HN declined between (b) and (c).
These experiments indicate that 36K is a primary translation product, whereas gp40 is not.
Extremely rapid processing of a larger virus polypeptide, if complete within 5 rain, could also
account for the presence of 36K.
An unexpected finding was that a highly basic polypeptide of mol. wt. 33K was present in
(a), declined in (b), and disappeared from (c). This polypeptide was not seen in uninfected
CEF and behaved like a metabolically unstable primary virus translation product, following
the arguments above. Polypeptide 33K was not found in virions (not shown). Vertical arrows
in Fig. 1 indicate host polypeptides which disappeared during chase incubation. Only the most
acidic of these polypeptides was secreted into the culture medium, so the others may be
degraded.
Comparison of the partial digestion products of ND V polypeptides
Partial digest peptide analysis of 36K, gp40 and gp62 was performed to determine whether
their partial digestion patterns resembled those of the major virus polypeptides HN, F, NP,
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ND V non-structural proteins
pH 8
7
6
5
••'~i!,i•i~iiii~•i~iiiiiiiii•i!i~!~!?iii'~+~i~•~•~ii~•~/!•il
P, •••• ~•i~•• •••i,li
Fig. 1. Fluorographs of two-dimensional polyacrylamide gel separations of NDV-infected CEF labelled
with [14C]leucine at 8 h post-infection. (a) Pulse-labelled for 5 min; (b) pulse-labelled for 1 h; (c)
pulse-labelled for 1 h, followed by a 1 h chase in medium containing 100/lg/ml unradioactive leucine.
NDV-induced polypeptides HN, Fo, NAP, NP, M and 36K were present in all separations. 33K was
detectable only in (a) and (b), gp40 only in (b) and (c), F~ only in (e). Three host polypeptides which
disappeared during chase incubation are indicated with vertical arrows, and the stable host polypeptide
actin is indicated as A. Spot intensities were matched by exposing (a) for 36 days; (b) and (c) for 3 days.
N A P and M. A fairly high level of radioactivity in the various polypeptides is needed for
partial digestion analysis, and this can be obtained by labelling infected cells for 3 h.
A 3 h pulse gave a pattern of spot intensities intermediate between that in Fig. 1 (b) and Fig.
1 (c), and since 3 3 K is metabolically unstable, insufficient label was incorporated for analysis
of this polypeptide. Results are shown in Fig. 2. The upper part shows peptide analysis on a
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P. C H A M B E R S A N D A. C. R. S A M S O N
80
40
L
20
10
HN
HN
Fo
HN
F0
gp62 Fl
gp40
M
NP NAP 36K
F1
NP
A
80
gp40
M
40
20
10
F2
HN
F0
gp40
M NP
NAP 36K
F2
(a)
Fig. 2
(b)
(c)
(d)
Fig. 3
Fig. 2. Fluorographs of tne partial digest peptides of [~4C]leucine-labelled polypeptides from
NDV-infected CEF. The upper part shows peptides analysed on a 17 % polyacrylamide gel. The lower
part shows peptides analysed on a 10 % polyaerylamide:sucrosegel. The peptides from HN, Fo, gp62,
F~, gp40, M, NP, NAP and 36K are indicated beneath the gels. gp62 and F~ were not analysed on the
lower gel. An indication of peptide mol. wt. (× 10-3) is given on the right of the figure.
Fig. 3. Fluorographs of SDS-PAGE separations of (a) [~4C]leucine-labelledpurified NDV; (b)
[3H]glucosamine-labeUed NDV; (c, d) [3H]glucosamine-labelled CEF infected with NDV strains
Beaudette C (c) or HP16 (d). The virion polypeptides L, HN, NP, A (actin derived from previous host
cells), M and F 2 are indicated at the left of the figure. The NP band also contained NAP and F r The
virion and intracellular glycosylated polypeptides HN, F 0, F~, gp40 and F 2 are indicated on the right of
the figure. In all tracks, F 2is the major labelled polypeptide migrating at the dye front.
17% polyacrylamide gel; the lower part shows analysis on a 10 % p o l y a c r y l a m i d e : s u c r o s e
gel. The upper part demonstrates that F 0 and F 1 generated very similar peptides, consistent
with the known p r e c u r s o r - p r o d u c t relationship between them. W h e n present in sufficient
quantity for analysis, gp62 generated similar peptides to H N . Although gp40 also generated
similar peptides to H N , the relationship derived from the upper gel alone is not conclusive. M,
NP, N A P and 3 6 K generated dissimilar peptides. The lower gel, a more extensive digestion,
now shows a clear relationship between H N and gp40, but reveals no more obvious
similarities in the other polypeptides.
Partial digest peptide analysis suggests that gp62 and gp40 are related to H N . P u l s e - c h a s e
experiments showed that H N declined and gp40 increased during a chase, and it therefore
seems likely that gp40 is a m a j o r b r e a k d o w n product o f H N o f unknown significance. While
partial digest peptide analysis does not suggest that 3 6 K is related to any o f the major virus
polypeptides (consistent with the short pulse-labelling d a t a o f Fig. 1) these results are not
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ND V non-structural proteins
Fig. 4. Segments of two-dimensional fluorographs containing [14C]leucine-labelled CEF infected with
NDV strains (a) Beaudette C, (b) LK, (c) Texas, (d) AV, (e) HP16, (f) B1, (g) N and (h) F.
NDV-induced polypeptides HN, F0, NAP, F 1 and 36K are indicated. F 0 was not detectable in (b); FI
was not detectable in (f), (g) or (h). Host cell actin (A) is indicated in all segments.
sufficient to conclude that 36K is unique. The minor L polypeptide was not analysed because
it was not resolved.
Presence of gp40 in ND V-infected cells
Experiments were performed to determine whether gp40 was present in CEF infected with
strains of NDV other than Beaudette C, and to determine whether any gp40 appears in
virions.
In Fig. 3, [t4C]leucine-labelled virion polypeptides of NDV strain Beaudette C (a) are
compared with [3H]glucosamine-labelled polypeptides from virions (b) or CEF infected with
NDV strains Beaudette C (c) or HP16 (d). The important point is that a prominent gp40 band
can be seen in Beaudette C-infected CEF (c), but not in (a), (b) or (d). Thus, gp40 is
non-structural in strain Beaudette C, and not present in CEF infected with NDV strain HP16.
Cells infected with most strains of NDV contain a small amount of gp40, more readily
detectable by two-dimensional electrophoresis of [3H]glucosamine-labelled extracts. Of strains
studied here, only HP16 and AV did not generate any gp40, and Beaudette C generated the
largest amount (results not shown); gp40 could also be detected in NDV (Beaudette
C)-infected BHK cells.
Polypeptide variation in ND V-infected cells
Strain-dependent variation in mol. wt. and pI~of NAP in NDV-infected CEF and BHK cells
was used as evidence that NAP is virus-coded (Chambers & Samson, 1980). NAP is not,
however, the only NDV polypeptide that exhibits strain-dependent variation. This
phenomenon is illustrated in Fig. 4, which shows CEF infected with several strains of NDV.
L, NP and M polypeptides were insoluble in the IEF first dimension and, therefore, do not
feature in the regions of the fluorographs shown.
HN and F 0 were identified on the basis of mol. wt. and glycosylation. NAP and 36K were
identified on the basis of mol. wt. and phosphorylation. F 1was identified either in pulse-chase
experiments or by its mol. wt. and glycosylation. HN, F 0, F~, NAP and 36K all exhibited
strain-dependent variation, and it is clear that a version of the 36K polypeptide is synthesized•
in all NDV-infected monolayers. It is possible to group certain strains together on the basis of
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P. C H A M B E R S
A N D A. C. R. S A M S O N
Fig. 5. Segments of two-dimensional fluorographs containing [14C]leucine-labelled 36K induced by
three strains of NDV. (a to d) CEF cells; (e to h) BHK cells. (a, e) Uninfected; (b to d) and ( f t o h)
infected with NDV strains Beaudette C (b,f), N (c, g) or HP16 (d, h). Actin (A) is indicated as a
reference point.
the general similarity in pI of the virus polypeptides. For example, in Fig. 4 strains Beaudette
C (a), LK (b) and Texas (c) had NAP and 36K of identical pI. F 1 of Beaudette C and LK
were identical (F 0 of LK was not detected, as it is rapidly cleaved to FI; Samson & Fox, 1973)
but were more acidic than F~ of Texas. HN of LK and Texas were identical, but were more
acidic than H N of Beaudette C. All polypeptides of the Beaudette C, LK, Texas group
differed from those of AV (d), but 36K, F 0 and Fx of AV were similar to those of HP16 (e).
Strains B 1 ( f ) , N (g) and F (h) showed no close resemblance to each other or to the strains in
(a to e). F 0 is not cleaved to F 1 in CEF infected with these (B1, N and F) avirulent strains of
NDV (Nagai et al., 1976a). Thus, it appears that NDV strains can be identified on the basis
of the pI of poly,peptides synthesized in infected CEF. 36K appears to be a useful strain
marker, as it is more soluble than L, NP or M.
We reported earlier that synthesis of 36K was unaffected by 12 h incubation with
actinomycin D, sufficient to cause a considerable reduction in the rate of synthesis of all host
polypeptides. This indicated that 36K is virus-coded. A further test of the virus coding of 36K
is to determine whether the strain-dependent variation of 36K shown in CEF (Fig. 4) is
reproduced in BHK cells, as was shown for NAP. Sections of two-dimensional fluorographs
are shown in Fig. 5. (a to d) are from CEF cells; (e to h) from BHK cells. (a, e) are
uninfected; (b to d) and ( f to h) are infected with NDV strains Beaudette C (b,f), N (c, g) or
HP16 (d, h). Actin (A) is similar in CEF and BHK cells, and provides a useful reference point.
It is clear that the variation in pI of 36K (Beaudette C > N >HP16) seen in CEF is exactly
reproduced in BHK cells, confirming that 36K is virus-coded.
The synthesis of the 33K polypeptide was next investigated, and the results are shown in
Fig. 6. Since 33K had not been detected in many previous 3 h duration labellings a
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//
//
LL
9
N D V non-structural proteins
g
i:
j
.
Q
L Q
0
~
o
o
~
2
~
~'
b
•
:
g
i
0
2•,,•
l
c
h
QI
\
O
0
\
I
--~.o
•-~Q
d
lb
Q
•
II
Fig. 6. Segments of two-dimensional fluorographs containing [~4C]leucine-labelled 33K induced by
three strains of NDV. (a to e) CEF cells; ( f t o 0 BHK cells. (a,f) Uninfected; (b to e) and (g to £}
infected with NDV strains Beaudette C (b, g), N (c, h) or HPI6 (d, e, 13. (e) was treated with
actinomycin D throughout the post-infection incubation and labelling period. 33K spots are indicated
with oblique arrows. A host polypeptide is indicated with a horizontal arrow in all figures as a reference
point. Because the 33K spots are of low intensity, a tracing is included to show the spot positions more
clearly. Host polypeptides are indicated as open spots; 33K as filled spots. The same host and 33K
spots are indicated with arrows as in the photograph.
pulse-labelling protocol w a s e m p l o y e d . With the benefit o f hindsight, however, it is possible to
detect a very faint trace o f 3 3 K in our previous experiments. Relevant sections o f fluorographs
are s h o w n in Fig. 6, labelled for 10 min at 8 h post-infection. (a to e) are from C E F cells; ( f t o
i) are from B H K cells. (a,f) are uninfected; (b to e) and (g to i) are infected with N D V strains
Beaudette C (b, g), N (c, h) or HP16 (d, e, 0. At this extreme basic end o f the N E P H G E gel, it
is difficult to precisely reproduce variation from gel to gel. Fortunately, the version o f 3 3 K
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10
P. CHAMBERS AND A. C. R. SAMSON
induced by strain N (c, h) has a higher SDS mobility than those from strains Beandette C (b,
g) or HP16 (d, i). Strain variation in tool. wt. was reproducible, although the most basic form
of 33K of HP16 in BHK (i) had smeared and showed a slightly different position to that seen
in Fig. 6 (d) and (e). Fig. 6 (e) showed that 33K synthesis was unaffected by the presence of
actinomycin D, whereas the synthesis of host polypeptides was much reduced. 33K of strain
HP16 shows charge heterogeneity (it was assumed that both spots are forms of the same
polypeptide). Charge heterogeneity of 33K of strain Beaudette C was less marked, and 33K of
strain N showed no detectable charge heterogeneity.
These experiments showed that 33K is not synthesized in uninfected cells, shows
strain-dependent variation in mol. wt. and its synthesis is unaffected by actinomycin D. These
data are analogous to those presented for 36K and previously for NAP (Chambers &
Samson, 1980) and suggest that 33K is also virus-coded. Peptide fingerprint data on 33K are
not yet available for the reasons described earlier, so there is only kinetic evidence to show
that 33K is a primary translation product.
DISCUSSION
The data presented here demonstrate that gp40 and gp62 are breakdown products of the
HN polypeptide, but suggest that 36K and 33K are primary translation products. Thus, 36K
and 33K should be considered as genuine virus non-structural polypeptides. Rima et al.
(1980) detected two non-structural polypeptides in mumps virus-infected cells, tool. wt. 23K
(C) and 17K (S). A metabolically unstable non-structural polypeptide (mol. wt. 15K to 18K)
has been detected in measles virus-infected cells and canine distemper virus-infected cells (Hall
et al., 1980; Rima & Martin, 1979; Campbell et aL, 1980). Preliminary results with Sendal
virus-infected cells (P. Chambers, unpublished results) show the presence of two nonstructural polypeptides in both CEF and BHK cells. One is the previously described C / C '
complex; the other has a mol. wt. of 56K and is unstable in chase incubations. Synthesis of
both Sendal non-structural polypeptides is unaffected by actinomycin D. C/C' shows strain
variation (Etkind et aL, 1980) but strain variation of 56K has not yet been investigated. Two
non-structural polypeptides may be a general feature of paramyxoviruses, with one
metabolically stable and the other unstable.
The role of these non-structural polypeptides is still a matter for speculation. Since
paramyxovirus virion nucleocapsids contain the various enzymes necessary for transcription
(Huang et al., 1971; Weiss & Bratt, 1976; Colonno & Stone, 1975, 1976a, b) but do not
contain either 36K or 33K, it seems that virus transcription does not require these
polypeptides. Replicative processes, such as synthesis of full length positive and negative
strands of RNA are not performed by isolated virion nucleocapsids, and perhaps require the
participation of non-structural polypeptides. Virus assembly and budding processes are other
possible sites of action. One way to determine the role of non-structural polypeptides in
replication and assembly is to identify mutants with a lesion in a particular polypeptide
showing an electrophoretic shift, and to correlate this physical variable with a biological
function (King & Newman, 1980; Samson et al., 1981). A second approach is to examine the
kinetics of nucleocapsid and virus assembly, and the polypeptides associated with
nucleocapsids and cellular fractions (Portner & Kingsbury, 1976; Nagai et aL, 1976 b; Lamb
& Choppin, 1977; Smith & Hightower, 1981 b).
Both 36K and 33K show charge heterogeneity. That of 36K is due to phosphorylation, but
the cause of that of 33K is not yet known. Rapid disappearance of 33K in pulse-chase
experiments, and charge heterogeneity, increase the possibility of metabolic control exerted by
the non-structural polypeptides.
Not all strains of NDV generate gp40, so there may not be a biological role for this
polypeptide. Pulse-chase experiments with several strains of NDV (Beaudette C, Texas, N,
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N D V non-structural proteins
11
AV, HP16) show that where HN is stable in a chase, no gp40 appears (AV, HP16), but that
where HN is unstable in a chase gp40 can de detected (Beaudette C, N, Texas). The gp40
itself disappears in a 4 h chase, and may be more stable in strain Beaudette C than in the other
strains in which it can be detected. Since half of the HN molecule is basic (gp40), the other
half must be acidic, as the H N polypeptide itself has a neutral pI. Salt-shock mapping could be
used to determine which part of the HN molecule corresponds to gp40 (Samson et al., 1980)
and since inspection of fluorographs suggests that gp40 contains a disproportionate amount
of the HN carbohydrate, a rough map of the HN molecule could be drawn up, showing
regions rich in acidic amino acids, basic amino acids and carbohydrate. Thus, continued study
of gp40 could yield interesting information about the biochemical structure of the H N
molecules of paramyxoviruses. The disulphide-linked fragments of HN do not appear in
two-dimensional gels, possibly lying outside the pI range studied (Samson et al., 1980).
Small m R N A s which may code for 36K and 33K have not yet been detected (Weiss &
Bratt, 1976; Thomas et al., 1978). The positions of 36K and 33K in the genetic map of N D V
remain to be determined, but Collins et at. (1980) found that the u.v. target size increment of
the H N gene was large enough to encode a small additional polypeptide.
The technical assistance of Mrs J. Hughes is gratefully acknowledged. Phil Chambers was supported
by a Science Research Council Studentship. A. C. R. Samson is the recipient of a research grant from
the Wellcome Trust.
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