Journal of General Virology (1994), 75, 3742. Printed in Great Brita#l
37
A small percentage of influenza virus M1 protein contains zinc but zinc
does not influence in vitro M 1 - R N A interaction
Christine Elster, ~ Eric Fourest, 2 Florence Baudin, ~ Kjeld Larsen, ~ Stephen Cusack 1
and Rob W. H. Ruigrok 1.
1 E M B L Grenoble Outstation, c / o I L L , B P 156, 38042 Grenoble Cedex 9 and 2 C E N - G / D B M S / T S V ,
38041 Grenoble Cedex, France
B P 85X,
A peptide containing the C C H H motif, the putative
zinc-binding sequence o f influenza virus M1 protein,
was found to bind zinc in a one-to-one complex with the
characteristics of a typical zinc-binding peptide. Intact
influenza virus also contained zinc and we show that this
zinc is bound to the M1 protein in the virus. However,
only a small proportion of M1 contained zinc: 4 % in
virus and 6 to 9 % in isolated protein. One strain,
B/Yamagata/16/88, consistently contained more zinc:
15 to 2 0 % both in virus and in isolated protein. We
also determined the RNA binding and transcription inhibition activities of various M1 proteins and found that
the zinc content of M1 had no influence on either
activity. We suggest that the zinc in M1 has a structural
role in the virion other than nucleic acid binding.
Introduction
C y s X a a ~ - C y s X a a 7 His-Xaa~ His ( C C H H motif)
(residues 148 to 162 of A / P R / 8 / 3 4 M1 protein; Allen et
al., 1980), first remarked upon by Wakefield & Brownlee
(1989), which could be involved in R N A binding. This
C C H H motif is situated in a part of the M1 sequence
that may be involved in transcription inhibition (Ye et
al., 1989). Zinc-binding sequences have been implicated
in specific protein-nucleic acid interactions and protein
dimerization domains (see references in Discussion). In
this paper we describe the zinc-binding activity of a 27
amino acid peptide containing the C C H H motif and
have determined the zinc content of intact influenza virus
and purified M 1. We further show that the zinc content
of M1 does not influence the in vitro R N A binding and
transcription inhibition activities of M1.
Influenza viruses are enveloped viruses with a segmented
negative strand R N A genome. Each v R N A segment is
complexed with the major RNA-binding protein nucleoprotein (NP) and carries a copy of the polymerase
complex. These structures, called ribonucleoprotein
particles (RNPs), are independent active transcription
units. The total genome consists of eight RNPs which are
contained within a shell of M1 protein which lines the
inside of the viral lipid bilayer. Embedded in this lipid
bilayer are the viral glycoproteins which are essential for
entry into and release from the host cell.
The structure of the virion suggests an important role
for the M 1 protein. M 1 may interact with the cytoplasmic
tails of the glycoproteins, the lipid membrane and the
RNPs and so form the 'glue' between them. Interaction
of M 1 with lipid has been shown in intact virus using
light-activated cross-linking (Gregoriades & Frangione,
1981) and also in vitro using isolated M1 and liposomes
(Bucher et al., 1980). Contact between M1 and the
glycoproteins has not yet been shown but interaction of
M1 with RNP is suggested by the fact that purified M1,
when added to transcribing RNPs, inhibits transcription
(Zvonarjev & Ghendon, 1980; Ye et al., 1989). hi vitro
interaction of M 1 with R N A has been shown with filterbinding assays and a blotting technique (Wakefield &
Brownlee, 1989; Ye et al., 1989).
The primary sequence o f all influenza A and B M1
proteins contains the putative zinc-binding sequence
0001-1904 © 1994 SGM
Methods
Virus and M1 protein. InfluenzavirusesA/PR/8/34, B/Beijing/1/87
and B/Yamagata/16/88 were grown in embryonated hen's eggs and
obtained in purified form from Pasteur-Mrrieux, Marcy L'Etoile,
France. M1 protein was isolated from spikeless virus. Spikes were
removed by bromelain digestion (Brand & Skehel, 1972) which was
stopped by addition of 100 mM-iodoacetamide. Spikeless virus was
then purified by pelleting through 14% sucrosein PBS. The virus was
then disrupted with 1% Triton X-I00 in PBS and centrifugedon a 10
to 30 % continuousglycerolgradientin PBS (SW41rotor, 36000 r.p.m.,
4 °C, 16 h). M1 was collectedfrom the upper fractions of the gradient
and stored immediatelyat -20 °C. The purity was judged to be higher
than 90% by PAGE (Fig. 1).
Virus protein concentrationswere determined by the Lowry method
(Lowry et al., 1951) using BSA as a standard. The concentration of
isolated M 1 protein was determinedwith the Lowry and the Bradford
C. Elster and others
38
1
2
3
4
5
6
HA
NP
MI
Fig. 1. Polyacrylamide gel of intact influenza viruses (lanes 1 to 3) and
isolated M1 proteins (lanes 4 to 6) used in this study, non-reducing
conditions. Lanes 1 and 4: A/PR/8/34; lanes 2 and 5 B/Beijing/1/87 ;
lanes 3 and 6: B/Yamagata/16/88. The gel was stained with Coomassie
blue G250. HA, Haemagglutinin.
(1976) assays, both using BSA as standard, and by absorbance
measurements using the known amino acid composition of A/PR/8
M 1 and the molar extinction coefficients of Tyr and Phe. These three
methods yielded values within 20 % of each other and any one of these
methods was used throughout this work depending on concentration
and volume of the protein solutions.
Peptide studies. A 27-residue peptide with the A/PR/8/34 M1
sequence EVAFGLVCATCEQIADSQHRSHRQMVT (MP3) was
synthesized using the solid phase technique at EMBL, Heidelberg,
Germany. Peptide concentrations were determined by weighing out
powder: the factor between powder weight and peptide concentration
was determined by amino acid analysis. All cysteines in a fresh peptide
solution were reduced but the solutions had to be used immediately
after preparation in order to avoid oxidation since oxidized peptide did
not bind metals. Metal binding studies were performed in 20 mMHEPES, 50 mM-NaC1 pH 7.0. Absorption spectra between 700 and
250 nm were measured in a Kontron Uvikon 941 spectrophotometer.
Cobalt titration was performed by adding 1 gl of a 10 mM-CoCI 2
solution to 1 ml peptide solution (60 gM) from 0 to 120 gM-Co~+ in
10 gM steps. The dissociation constant for the peptide/Co -"+ complex
was determined using a least-square model fitting approach.
Atomic absorption spectroscopy. The amount of zinc complexed with
virus and purified proteins was determined after extensive dialysis
against PBS by atomic absorption spectroscopy using a Perkin Elmer
model 2380 single-beam atomic absorption spectrophotometer. An
ai~acetylene flame was used and the absorption was measured at
213-9 nm. The measurements were calibrated against a standard zinc
solution in PBS. PBS without zinc was used for zero point calibration.
Measurement in PBS diluted 20-fold, in order to try to reduce the
noise level, gave very similar results. Typical concentrations used were
I0 mg/ml for virus and 1 mg/ml for M1 and RNP. The detection limit
for zinc was about 10 ng/ml. Quantification of other metal ions was
carried out at the following wavelengths : 248.3 nm for Fe z+, 232 nm for
Ni 2+ and 324.8 nm for Cu 2+.
RNA binding assays. Segment 8 viral RNA was produced by in vitro
run-off synthesis using T7 RNA polymerase. Prior to transcription, the
plasmid DNA (pHgaNS, a gift from Mark Krystal, Mount Sinai
School of Medicine, New York, N.Y., U.S.A.) was digested with the
restriction enzyme HgaI, which defines the 3' terminus of the transcripts
as nucleotide + 890 of the gene. Digested DNA was used as template
to produce NS vRNA (Davanloo et al., 1984). The binding constant of
NS vRNA to M 1 was determined by a nitrocellulose binding assay. The
standard reaction buffer for the binding assay was 10 mM-HEPES
pH 8.0, 25 mM-NaC1, 5 mM-MgCI 2 and 2-5 mM-DTT. The reaction
volume was 45 gl. The M 1 protein was incubated with the a"-P-labelled
vRNA (30 pmol) for 30 min at 20 °C and filtered through nitrocellulose
filters (Whatman, 0.45 pm pore size). The RNA complexed with M1
bound to the filters and could be detected by scintillation counting.
Since the amount of RNA used was extremely small, the M1
concentration at 50% RNA retention is taken to be the dissociation
constant K~j.
Transcription inhibition assay. For transcription assays, glycerol
gradient-purified RNPs (Honda et al., 1988) were used. bl vitro
transcription was carried out at 30 °C for 2 h. Standard reaction
mixture (Honda et al., 1986; Ishihama et al., 1986) contained 50 mMTris-HC1 pH 7"8, 30 mM-NaC1, 5 mM-MgCI~, 1 mM-DTT, 200 gMNTPs, 10 ~tCi [c~-3~P]UTP, 200 gM-ApG as primer and 0.015 gM-RNP
(based on NP concentration and assuming 75 copies of NP per RNP on
average). The reaction was stopped by addition of 50 gl 8 % sodium
pyrophosphate and RNA precipitation was carried out by addition of
100 gl of 10% TCA. Insoluble material was collected on Whatman
G F / C filters, extensively washed with 20ml 10% TCA and the
radioactivity was measured by scintillation counting.
Results
Z i n c binding by C C H H - b o x peptide
The absorption spectrum of the Co~+/peptide complex
w i t h v a r i o u s a m o u n t s o f C o ~'+ is s h o w n in F i g . 2(a). T h e
s p e c t r u m is c h a r a c t e r i s t i c f o r t e t r a h e d r a l l y c o o r d i n a t e d
C o ~'+ w i t h p e a k a b s o r b a n c e s at 284 n m (e = 3680 M-1.
c m 1) a n d 6 2 0 n m
(e=570M
1.cm-1). W h e n
the
a b s o r b a n c e at 2 8 4 n m is p l o t t e d a g a i n s t the C o .-'+
c o n c e n t r a t i o n (Fig. 2b), a l i n e a r i n c r e a s e in signal is
o b s e r v e d until e q u i m o l a r a m o u n t s o f C o .'.+ a n d p e p t i d e
are r e a c h e d , s u g g e s t i n g a 1:1 C o 2 + / p e p t i d e c o m p l e x .
L e a s t - s q u a r e c u r v e - f i t t i n g suggests a K d o f 0.2 gM at
p H 7.0. A d d i t i o n o f Z n 2+ to t h e C o 2 + / p e p t i d e c o m p l e x
leads to a d e c r e a s e in the a b s o r b a n c e a n d since e q u i m o l a r
a m o u n t s o f Z n 2+ c o m p l e t e l y d i s p l a c e t h e C C +, the K d f o r
Z n 2+ m u s t be c o n s i d e r a b l y l o w e r t h a n t h a t f o r C o 2+. T h e
s h a p e o f the a b s o r p t i o n s p e c t r u m , t h e m o l a r a b s o r b a n c e s
a n d t h e K d are v e r y s i m i l a r to t h o s e o f o t h e r Z n 2+b i n d i n g p e p t i d e s , s u c h as t h o s e d e r i v e d f r o m r e t r o v i r a l
nucleic acid-binding proteins, alcohol dehydrogenase
a n d a l a n y l - t R N A s y n t h e t a s e ( G r e e n & Berg, 1989; Berg,
1993; B e r g m a n et al., 1992; M i l l e r et al., 1991).
Z h w content in virus and isolated M 1
T h e z i n c c o n t e n t o f p u r i f i e d i n t a c t virus, spikeless virus,
i s o l a t e d M 1 a n d R N P w a s d e t e r m i n e d by a t o m i c
a b s o r p t i o n s p e c t r o s c o p y . T h e results ( T a b l e 1) c l e a r l y
s h o w t h a t t h e r e is zinc in i n f l u e n z a virus. T h e zinc is n o t
a s s o c i a t e d w i t h p u r i f i e d R N P o r w i t h t h e spikes b u t w a s
found associated with purified M1 protein. When
e x p r e s s e d as m o l e s o f z i n c p e r m o l e o f M 1 it b e c o m e s
c l e a r t h a t o n l y 4 to 6 % o f the M 1 m o l e c u l e s c o n t a i n a
Zinc in it~uenza virus M1 protein
39
Table l. Zinc content of influenza virus and isolated
protein components
0.25
0.2
0-15
e~
©
e~
0.1
<
0.05
-0.05
i i !i
i
300
200
0.3
i
(b)
I
400
500
600
Wavelength (nm)
i
i
i
i
i
700
i
i
800
] - -
0-2
0.1
0.0
20~
i
401
i
60L
~
810
L
i
i
i
100
Co 2+ (BM)
+ i 0-3
i
i
i
i
i
i
O
rl
Sample*
Intact virus
A/PR/8/34
B/Beijing/1/87
B/Yamagata/16/88
Batch 1
Batch 2
Spikeless virus:~
A/PR/8/34
B/Beijing/1/87
B/Yamagata/16/88
Isolated M1 protein
A/PR/8/34
B/Beijing/1/87
B/Yamagata/16/88
Isolated RNP
A/PR/8/34
Zinc
(rig per mg sample)~
Mole zinc per
mole MI$
13"1 ± 1"0 (n = 7)
13"8±0"7 (n = 3)
0'042±0"003
0"044±0"002
46.4_+ l'3 (n = 3)
63-5 ( n = 1)
0"148±0'004
0"203
70-5
69
239
221 _+40 (n = 2)
149_+11 (n = 2)
438+101 (n = 2)
0-059
0.058
0"198
0"092
0.062
0'182
10±18 (n = 5)§
*The seven determinations of intact A/PR/8 virus and three of
intact B/Beijing virus were independent experiments using virus from
three and two different batches respectively. The measurements on
B/Yamagata virus were also done on virus from two separate batches,
as indicated.
?Amounts of virus and spikeless virus are given as mg of protein.
:~Assuming that all zinc is associated with M 1 and that 13 % of viral
protein is M1. This amount of M1 is based on physical measurements
on monodisperse, egg-grown virus (Ruigrok et al., 1984; Cusack et al.,
1985). Virus contains 50% spikes, 25% lipid and 25% internal
proteins (NP, M1 and polymerases) and spikeless-virus protein is
assumed to be 50% M1.
§This value is ng zinc per mg NP; this corresponds to 0-002 ions of
zinc per molecule NP and to 0.15 zinc ions per RNP (assuming on
average 75 NP per RNP) and consequently to 0.15 zinc ions per
polymerase complex. The determined amount of Zn is at the limit of
detection, hence the large relative standard deviation.
0-2
0-1
0-00
i
210
i
i
i
i
40
60
Zn 2+ (BM)
C
m
i
;
8
m
L
100
Fig. 2. Metal binding by peptide MP3. (a) Absorption scans of MP3
with various amounts of CoCI~. The curves correspond to 60 BM-MP3
plus 0, 10, 20, 30, 40, 50, 60, 70, 80 and 90 gM-CoCI~. (b) Absorbance
at 284 nm of 60 gM-MP3 as a function of CoCI~ concentration. (c)
Absorbance of a 60 BM-Co2+/MP3 complex as a function of added
concentration of ZnCla. The residual density after complete displacement of the cobalt by the zinc is probably due to light-scattering. The
spectra showed a sloping, f?atureless line with none of the characteristic
absorption peaks of the cobalt complex.
zinc ion in intact and spikeless virus of A / P R / 8 and
B/Beijing compared with 15 to 20% in B/Yamagata.
The fact that similar amounts of zinc per M 1 molecule
are found in virus and in purified M 1, and the covariation
of zinc content in intact virus and purified M1 of
A / P R / 8 and B/Beijing virus on the one hand and
B/Yamagata on the other, are strong arguments that
zinc is indeed associated only with M 1. We were surprised
to find that B/Yamagata contained more zinc than
B/Beijing and A / P R / 8 (of which three different batches
were tested always with the same result). We therefore
tested a second batch of B/Yamagata that was produced
a year later and again found a significantly higher
zinc content, suggesting that this difference between
B/Yamagata and the other two viruses is real.
Oxford & Perrin (1975) also detected zinc in a purified
influenza virus preparation (3 ng zinc per mg viral
protein) and suggested that it could be associated with
the viral polymerases. However, the amount of zinc in
purified R N P determined here is at or just below the
reliable detection level and suggests that there cannot be
more than 0-15 ions of zinc per polymerase complex
(Table 1).
We tested for the presence of other divalent metal ions
and found no significant amounts of copper, nickel or
iron. The zinc in isolated M1 could not be removed by
C. Elster and others
40
0.8
I
I fill
I
I
I
I I Illl
I
I
I
I I Illl
I
I
I
I
.......
f I l[ll
0.6
o
0.4
""
O.2
,
0"0
O,
,~,l
1
P,
1
•i
.....
I
........
10
M1 (nM)
100
1000
Fig. 3. RNA-binding activity of purified M1 proteins. MI was mixed
with segment 8 vRNA and filtered over a nitrocellulose membrane. The
fraction of retained RNA is shown. The experiments were performed
on the same day with fresh M I and a single RNA preparation.
Experiments showed more variation when different RNA preparations
were used and varied with the age of the MI preparation. (1)
M 1/A/PR/8/34; (C)) M 1/B/Beijing/1/87; ( x ) M 1/B/Yamagata/88.
Table 2. Transcription inhibition activity of MI and
peptide MP3
Sample
M1, A/PR/8
M1, B/Beijing
MI, B/Yamagata
MP3
MP3/ZnC1z
ZnCI~
ZnC12
Concentration
(pM)
1
10
1
10
1
10
65
65/33
22
44
Transcription
(%)*
70+ 14 (n = 4)
17_+5 (n = 3)
69_+19 (n = 5)
22_+14 (n = 5)
69_+16 (n = 5)
22_+13 (n = 3)
108_+12 (n = 3)
127_+17 (n = 2)
43 (n = 1)
23 _+5 (n = 6)
*The 100% valuewas independentlydetermined for eachexperiment
in the absence of M1 or peptide.
extensive dialysis against 10 mM-EDTA or against a
100-fotd dilution o f a saturated solution of 1,10phenanthroline, suggesting that the zinc is tightly bound
and possibly not accessible to solvent.
RNA-binding activity of M1
The vRNA-binding activity of M 1 was determined using
a filter binding assay. We found that v R N A binding did
not vary with the M1 zinc content (Fig. 3). R N A binding
by B / Y a m a g a t a M1 was as strong as that by A / P R / 8
and B/Beijing M1, although it contained four times
more zinc. The average K d values for R N A binding by
M 1 are: 21 ± 6 nM (n ----7) for A / P R / 8 , 24 ___5 nM (n = 4)
for B/Beijing and 17±6rim (n = 3) for B/Yamagata.
Wakefield & Brownlee (1989) found 50% retention of
R N A at a concentration of 30 nM M1 from A / P R / 8 .
Dialysis of M 1 against E D T A did not remove zinc (see
above) and did not lead to a change in R N A binding. We
also tested the RNA-binding activity of B/Beijing M 1
that had been isolated and purified in the presence of
2mM-DTT and 100pM-ZnCl~. In this type of M1
preparation it was found that after extensive dialysis
against PBS between 40 and 100 % of M1 molecules
contained zinc (three experiments). Using this zincloaded M1 we still found a K d of 25 riM.
When R N A was incubated with the zinc-binding
peptide MP3, in the presence or absence of zinc, the
R N A was not retained on the filter. Gel retardation
experiments also indicated no binding of this peptide to
R N A (data not shown).
In vitro transcription inhibition
The transcription inhibition activities of purified M 1 and
peptide MP3 are shown in Table 2. As with the RNAbinding, the transcription inhibition activity of all three
M1 preparations was very similar and apparently
independent of the zinc content. MP3 did not inhibit
transcription, either in the presence or absence of zinc.
Interestingly, zinc by itself did inhibit transcription
probably by poisoning the polymerase. This inhibition
was overcome by adding the zinc-binding peptide,
effectively removing the free zinc. This finding is a further
indication that the zinc in influenza virus is associated
with M1 and not with the polymerase as suggested by
Oxford & Perrin (1975). We found that several divalent
cations could inhibit the polymerase activity to different
extents in the order ZnC12 > MnC12 > CaC12 > CoC12.
R N P that had been dialysed against E D T A lost all its
polymerase activity, which could be completely restored
by the addition of 1.5 mM-MgCI~ or to a lesser extent by
MnC12 (25 % restoration with 50 pM but inhibition at
1 mM).
Discussion
Generally, zinc-binding sequences in proteins are involved in specific nucleic acid recognition, protein
dimerization or otherwise holding together protein
loops; zinc can also have a catalytic function in enzymes
(Schwabe et al., 1990; Cunningham et al., 1991 ; Kaptein,
1991 ; Vallee & Auld, 1990). We show here that influenza
virus M1 protein does contain zinc but that its zinc
content is not stoichiometric and does not influence its
interaction with v R N A as measured by filter binding
assays and in vitro transcription inhibition. The peptide
containing the zinc-binding sequence did not bind to
R N A or inhibit transcription. Therefore, we think that
the zinc in M1 is not involved in an RNA-binding
structure and must have some other function.
Z i n c in influenza virus M 1 protein
Retrovirus nucleocapsid proteins (NC) contain two
stretches of a conserved sequence Cys-Xaa2-Cys-Xaa4His-Xaa4-Cys which have been shown to bind zinc both
in the isolated peptide and in the protein (Green & Berg,
1989, 1990). Some purified retroviruses contain zinc at a
stoichiometry of two zinc ions per copy of NC (Bess
et al., 1992). However, in one retrovirus, avian myeloblastosis virus, only 2 % of NC molecules contain zinc
(Jentoft et al., 1988), analogous to the case of the zinc in
influenza virus M 1.
Only a small proportion of M1 in influenza virus
contains zinc, about 4 % in A / P R / 8 and B/Beijing and
between 15 and 20 % in B/Yamagata. This could equally
mean that 4% (15%) of the virions have fully zincoccupied M1 (Zn/M1) or that every virion has 4%
(15%) of its M1 occupied with zinc. If zinc has an
influence on the conformation of M1, one could imagine
that a virus particle with 100% Zn/M1 might appear
different from a particle with no Z n / M 1. In fact, electron
microscopy of negatively stained influenza virus has
shown two separate types of virus with different
polymerization forms of M l: virus with 'fingerprints'
which are arrays of single lines of protein with units
spaced 4 nm apart, and virus with 'coils' which are
paired lines of protein with the same spacing between the
lines within a pair but with a larger spacing between
adjacent pairs (Ruigrok et al., 1989). We examined the
A / P R / 8 virus used in the present study and found that
less than 0"1% of particles had coils, suggesting that the
particles with coils are not the hypothetical virions with
100% Zn/M1. The two type B virus strains did not
contain any particles with coils. Further, we found no
morphological differences between B/Yamagata and
B/Beijing virus, both showing only one type of virus
having 'fingerprints' with similar spacings between the
lines and the subunits. Therefore, we propose that, with
respect to zinc content, two different types of virus
particles do not exist and that every virion contains M1
both with and without zinc.
We suggest that Zn/M1 may occupy a position in the
virus different from that of M1 without zinc. Electron
microscopy of the fine structure of the M1 layer in the
virus suggests that it is not homogeneous (Ruigrok et al.,
1989). An exciting hypothesis is that the molecules of M 1
that surround the few copies of M2 present in the viral
membrane (Zebedee & Lamb, 1988) are different from
the rest of the M 1. Interaction between M 1 and M2 has
been suggested by Zebedee & Lamb (1989) who found
that virus growth restriction by anti-M2 antibodies could
be overcome by mutations in M1.
At present we can only speculate why there is relatively
more of the zinc-binding form of M1 in B/Yamagata
than in A / P R / 8 and B/Beijing. The M1 amino acid
sequences of B/Yamagata and B/Texas/88, a B/Beijing-
41
like strain, differ by only one residue (Paul Rota,
personal communication) which is not near the CCHH
motif. The two type B viruses are in two separate cocirculating lineages (Rota et al., 1990) and the difference
between them lies largely in the sequence of the
haemagglutinin HA1 region and in the sequence of the
part of RNA segment 6 that codes for both the stalk of
neuraminidase and for NB, the type B virus homologue
of type A virus M2 (Burmeister et al., 1993). If Zn/M1
does surround M2 (or NB), then differences in NB could
possibly lead to a requirement for more Zn/M1. To test
this, more sequence information on segment 6 and zinc
determinations of type B strains would be needed.
The presence of two different forms of M 1 in influenza
virus would not be unique in the negative-strand RNA
virus group. Two biochemically different kinds of
vesicular stomatitis virus M protein, one membranous
and one cytoplasmic, were recently found after expression of M in HeLa cells (Chong & Rose, 1993). Two
different kinds of M in rabies virus also exist, one
palmitoylated and one unmodified (Gaudin et al., 1991).
Recently, two antigenically different types of M were
found in Sendai virus (de Melo et al., 1992) and
functional differences between M proteins were found in
subacute sclerosing panencephalitis virus and its measles
parental virus (Hirano et al., 1992).
We thank Dominique Nalis (EMBL, Heidelberg) for peptide
synthesis, Jean Gagnon (IBS, Grenoble) for amino acid analysis, Jean
Roux (CEN-Grenoble) for help with atomic absorption, Paul Rota
(CDC, Atlanta) for the M1 sequences of B/Yamagata and B/Texas,
Mark Krystal (Mount Sinai School of Medicine, New York) for the
plasmid containing the segment 8 sequence and Reuben Leberman for
advice and comments on the text.
References
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Influenza virus RNA segment 7 has the coding capacity for two
polypeptides. Virology 107, 548 551.
BERG, J. M. (1993). Zinc-finger-proteins. Current Opinion hz Structural
Biology 3, 11-16.
BERGMAN,T., JGRNVAL,H., HOLMQUIST, B. & VALLEE, B. L. (1992). A
synthetic peptide encompassing the binding site of the second zinc
atom (the structural zinc) of alcohol dehydrogenase. European
Journal of Biochemistry 205, 467470.
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(Received 14 April 1993; Accepted 1 September 19933