﻿Volume 1 1 Number 5 1983 Nucleic Acids Research
The sequence of RNA segment 1 of influenza virus A/NT/60/68 and its comparson with the
corresponding segmet of strain A/PR/8/34 and A/WSN/33
K.L.Jones, J.A.Huddleston and G.G.Brownlee
Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford OXI
3RE, UK
Received 7 January 1983; Revised and Accepted 2 February 1983
ABSTRACT
The complete nucleotide sequence of RNA segment 1 of influenza virus
A/NT/60/68, corresponding to the PB2 protein, has been determined. It is
2341 nucleotides long, encoding a predicted product of 759 amino acids with
a net charge of +271 at neutral pH. The predicted amino acid sequence has
been compared to the equivalent sequences in influenza viruses A/PR/8/34 and
A/WSN/33. Evolutionary divergence, assuming a direct lineage from A/PR/8/34
and allowing for "laboratory drift", is 0.08% per year. The alignment of RNA
segment 10 of A/NT/60/68 with segments 1 and 3 is completed, confirming that
it is a mosaic of regions from these two segments.
INTRODUCTION
Influenza A viruses have negative stranded, segmented RNA genomes
consisting of 8 essential segments (1), which code for at least 10 proteins
(2). The largest of these segments are over 2,200 nucleotides long and code
for the 3 polymerase proteins (PB2, PBl, PA)(3,4). Together with the viral
RNA and nucleocapsid protein (NP), they make up the active transcribing
structures (5,6). RNA segment 1 of influenza virus A/NT/60/68 encodes the
PB2 protein (the smaller of the 2 basic P proteins), found in other influenza
strains, to recognise host cell cap 1 structures (7-11). The capped RNA is
cleaved 10-14 nucleotides from its 5' end (12-14), preferentially after a
purine residue (15), by a cap 1-dependent virion endonuclease (15), thus pro-
ducing an RNA primer. The larger basic P protein (PBl) appears to catalyse
the addition of the first ribonucleotide (7,10), which is complementary to
the 2nd or 3rd residue of the viral RNA (14,16) to the free 3' OH group of
this primer. Additional processes such as elongation and termination of the
nascent RNA chain remain to be assigned to specific proteins, although the
acidic P protein (PA) may be involved in at least one of these functions (10).
The proposed function of the 3 polymerase proteins and cross reference to the
older nomenclature is summarized in Table 1.
© IRL Press Limited, Oxford, England. 1 555
Nucleic Acids Research
TABLE 1 Function and nomenclature of polymerase proteins
RNA Previous New Proposed
segment nomenclature nomenclature function
1 P3t or P2* PB2 Cap recognition
2 P1 PB1 Initiation of
transcription
3 P2t or P3* PA ?Elongation
t in A/PR/8/34 (38) * in A/FPV/Rostock (37)
Influenza is still a cause of mortality among the elderly. Specific
vaccines directed against the surface glycoproteins, the haemagglutinin (HA)
and neuraminidase (NA) are in use and have a protective value but the high
rate of evolution of these proteins reduces the potential lifetime of such
agents (17). The P proteins represent an alternative target for anti-viral
agents, and it is of great interest to elucidate their mode of action in an
attempt to give both a deeper understanding of the virus and to design physio-
logically safe drugs.
In the present study, we present the nucleotide sequence of RNA segment
1 of influenza virus A/NT/60/68, which codes for the PB2 protein. Its predic-
ted amino acid sequence is compared with the corresponding segments of influ-
enza viruses A/PR/8/34 (18) and A/WSN/33 (19) in order to estimate its rate
of evolution and to look for conserved regions of possible functional signifi-
cance. This sequence also completes the comparison of RNA segment 10 (20), a
small RNA isolated from a preparation of A/NT/60/68, with segments 1 and 3 of
A/NT/60/68 (21). We confirm that segment 10 is indeed a mosaic of these 2
segments, formed by some form of intragenic recombination.
MATERIALS AND METHODS
Cloning of full length segment 1 RNA of A/NT/60/68 Double stranded DNA was
synthesized from a mixture of full length cDNA of segments 1, 2 and 3 of A/NT/
60/68 virion RNA (kindly provided by Dr B M Moss) and cloned in the PvuII site
of pBR322 as previously described (22,23). This entailed the use of 2 oligo-
nucleotide primers such that the first 12 and last 13 nucleotide residues of
the cloned RNA segments were derived from these primers and could differ
slightly from the RNA sequence. 240 potential recombinants were first
screened (24) with [32P] labelled short-copy cDNA obtained from A/NT/60/68
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virion RNA allowing us to isolate full length segment 2 and segment 3 clones
as previously described (22). Surprisingly, segment 1 clones were absent in
the initial screening but were subsequently detected by screening (24) with
[32p] labelled short-copy cDNA derived from the heterologous virion RNA, A/
USSR/90/77, also provided by Dr B M Moss. Two independent segment 1 clones,
referred to as A/NT/60/68/1 and 2, gave a distinctive HinfI restriction enzyme
profile when analysed by 5% polyacrylamide gel electrophoresis versus marker
segment 2 and segment 3 clones (results not shown). A/NT/60/68/1 and 2 dif-
fered from one another in a total of 7 of their HinfI bands (results not
shown), suggesting they were cloned in opposite orientations in the PvuII site
of pBR322 and that, in addition, clone 2 lacked an internal HinfI site. Sequ-
encing of residues 1174 to 1428 confirmed this hypothesis (36). Subsequent
cloning of material from the same full length double-stranded DNA preparation
into a modified pBR322 vector gave 2 clones with an internal HinfI restriction
pattern identical to that of clone 1. Thus 3 out of 4 RNA segment 1 clones
are taken to be identical to that sequenced.
Subcloning of sonicated fragments in M13mp9 and sequencing 15 Fgs of plasmid
DNA (25) derived from clone A/NT/60/68/1 was sonicated (26) to an average size
of 400 base pairs and the ends repaired by incubation with the Klenow subfrag-
ment of E.coli DNA polymerase I in the presence of all 4 deoxynucleoside tri-
phosphates. After heating the reaction mixture at 70 for 10 mins, a one-
tenth aliquot was blunt-end ligated (27,9) using T4 DNA ligase (from N. Gas-
coyne) to approximately 5 jg of SmaI cut and calf intestinal phosphatase
treated (23) M13mp9 (28). The ligation mixture (10l1) was used to transform
competent E.coli JM103 (29) and recombinants selected as clear plaques by
insertional inactivation of 6-galactosidase (29). 200 recombinants were
transferred as an ordered array onto a lawn of E.coli JM103 on a fresh plate
and phage was transferred by blotting in duplicate onto nitrocellulose filters
for screening (30) for specific influenza segment 1 containing sequences. Two
probes were used: firstly a [32p] labelled cDNA prepared against an A/NT/60/
68 vRNA template (23) in the presence of all 4 deoxynucleoside triphosphates,
and secondly llkg of A/NT/60/68 vRNA was partially degraded by boiling for 15
mins in 2 Ll of deionised formamide in lmM MgCl2 and labelled with [ _32p]
ATP using T4 phosphokinase (31). Free deoxynucleoside triphosphates were
separated on a lml Sephadex G100 column. Single stranded DNA from the 60
specific influenzal clones was prepared by standard procedures (32). The
phage inserts were sequenced using the Sanger dideoxy sequencing technique
(33) in conjunction with a 'universal' 17 nucleotide long primer (34). 95%
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Nucleic Acids Research
of the sequence was obtained in this manner. A consensus of the sequence data
was prepared using the Staden programs (35). Two contigs were constructed,
with an intervening unknown region, estimated to be 10-20 nucleotides (corres-
ponding to nucleotides 689-703) in length by comparison with the known sequ-
ence of segment 1 of A/PR/8/34 (18). To complete this unknown region, a Bst
Ni restriction fragment was labelled and sequenced by standard Maxam and
Gilbert sequencing procedures (36). Further ambiguous regions were sequenced
in this manner. Nucleotides 2165-2314, 490-658 and 863-921 were obtained by
a common DdeI digestion followed by digestion of the specific bands with AccI,
RsaI and RsaI respectively. The sequence shown in figure 1 represents a con-
sensus of 75% of viral (negative) strand and 93% of the positive strand with
3% of this sequence, (nucleotides 1323-1333, 1371-1378 and 1851-1878 inclu-
sive), present in a single copy. A consensus of the above information is
available upon request.
RESULTS
Figure 1 shows the sequence of RNA segment 1 of influenza virus A/NT/60/
68 as deduced from clone A/NT/60/68/1, written in the mRNA (positive) sense.
It is 2341 nucleotides in length with a viral RNA base composition of 23% A,
24.6% C, 17.9% G and 34.5% U. There is one long open reading frame from
nucleotide numbers 28-2304 coding for a predicted product of 759 amino acids.
No other open reading frames longer than 250 nucleotides exist in the mRNA
sense. Since the longest two other open reading frames contain neither a
methionine residue nor a splicing acceptor site close to the 5' ends, and
alternative forms of segment 1 mRNAs have not been demonstrated, it is unlike-
ly they are utilized. The second clone, A/NT/60/68/2, with the altered HinfI
profile (see Methods) has a G residue at nucleotide 1312, causing a change in
the predicted amino acid sequence from Asn -O Asp at residue 429.
The predicted protein of 759 amino acids (Fig. 1) is basic, with a net
charge of +271 at pH 7.0, assuming glutamic and aspartic acid take the value
-1, arginine and lysine the value +1, and histidine the value +j and that no
post-translational modification has occurred. The calculated molecular weight
of 85,947 daltons, is consistent with the predicted weight of 80-100 daltons
for the protein from SDS gels.
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E1 R I K E L R M L H 13359 TSR ZIPT K T T V ONH N A I I
ROM UAUMMUAAAOAAOAUCOUUCCWUCCCCOCOACAAUCMACCOUOCSACSA
10 20 30 40 50 s0 70 30 so 100 110 120
130 140 150 1g0 170 ISO 150 200 210 220 220 240
ED8 0 I T L M So? N lO0AD 3
S0 R V H V S P L A V T N NM105 N 0 P H T I T V H Y
250 230 270 230
P1R V YK T Y F 1 K V ESPL
ccAAAUUCAAUAUUAAAUOAOUA
370 330 330 400
AO0L S AK E Al10D V I M E
OCAGACCUCAOUO3CCAAOOAOOCACAAOAUAOA
490 500 510 520
E LI D CIKI 95200L M V AY
OAACUCCAAOAUUDCAAAAUUUCUCCUUOUOUCUC
310 320 630 340
240
L M L TUO
GT C ME 3 VYT
mAAcououumAoAm
730 740 750 750
V S AD0 P L A SYTL E N C H
850 3o0 370 330
I A A MD0L R I
1 F S F
370 3o0 330 1000
LIRI R V M D 0 390 E P T N
-----
OGNCUAUmOUC0OAOAALRCACAAUD
1060 1100 1110 1120
IS6V A E AZ I¶40 A M V F
c
1210 1220 1230 1240
440
NMIL L RMHPSIK OAK V L
CPAI
1330 1340 1350 1330
EM M
9NR DI 51048 K M 0 V
36in36C- AOCAA-AAAWOOGCGUa
1450 1460 1470 1430
L LU8 P1E E V Bar7 I 0 T E
1570 1530 1530 1500
M III M-NM E T 90K I 3 U I
1630 1700 1710 1720
V SO VSY T L 5105 S M 5 0
1310 1320 1330 1S40
1330 1340 1350 1330
AD 7 LII E3 P
ao
0 T 3 I
20530 2030 2070 2060
ELI M
NL AK 7
K A N V L
2170 2130 2130 2200
ISPISP M A I 1
230
AWACAOOM
410
530
650
CADDUODA
770
173
OCACACAG
390
1010
1130
1220
1370
0 EY
GAGAAC
1430
K L T
WACUOACA
1730
VLOZU
WIAAUACUL
20300
2210
300
420
=CAAUGAAOUUX
540
MAOMGACUUOUJC
VMO
730
300
1020
1140
1230
13300
1UCAGCACAOSO
I T YS
1740
T FD0T
IwUUoAU~
133
0
2100
aODV V
2220
310 320
430 440
D A RI L T
550 560
670 630
MUGOM35LAUMEAMM
730 300
RSMV D I L
MW8AVU003ACALMiCtU
310 3208
1030 1040
I LRIK A T
1150 IISO
3CAOLUUMOOLIGUALCUM
1270 12300
HNI DMNVMN
CAUAUCOACAAVUAAUOMJ
1330 1400
1510 1520
1330 1640
1750 1760
ACCCMBALNUMUMMCUA
1370 1330
1330 2000
OPFL I LO0
2110 2120
2230 2240
330
V I2oI S
WCrIomAAUA-OO
450
.106A a
M£ODMUCACA
L 2YOI A
310A-N
SY3rN P
WOAGAA=~A
330
1050
R 310L V
1170
N¶O V N
340 350 3300
460 470 430
L T I TICKl-R-R-E
O TSI8V Y IE V
700 710 720
320 330 540
340 3M0 330
1030 1070 1030
I L I VIO
0RD0E
1130 1130 1200
A N 3R L NPM
1230 1300 1310 1320
O
I0V L P N T PI8T
1410 1420 1430 1440
0510F L S V 5 0
1530 1540
1 5P P E 5 V L
1770 17300
LYOPF A A A P P
%=CCWUVOCAOCCACCCCACC
1330 1300
NOrT T I S L T
2010 2020
S7 S V 0 P
2130 2140
RYS0 3 I *L T D
2250, 2230
1550 1530
A IRaDaI
1730 1500
WOMWAOAUAGOASA
1310 1320
MAWUPMCOOAMAAOAV
203 2040
2150 2130
2270 2230
Figure 1. Nucleotide sequence of segment 1 of A/NT/60/68 and the predicted
amino acid sequence of the PB2 protein.
1559
c
h
u
m
a
A
9
c
4
a
K
0
c
a
c
a
w
c
c
x
a
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9
if
j
m
a
10
m
Nucleic Acids Research
DISCUSSION
1. Comparison of the predicted amino acid sequence of the PB2 protein among
differing influenza A virus strains
Figure 2 shows the differences in the amino acid sequence between that
reported here and the corresponding segments of A/PR/8/34 (18) and A/WSN/33
(38). Of the 30 amino acid differences (Fig. 2) between A/NT/60/68 and A/PR/
8/34, 22 are due to single nucleotide mutations. The remainder show 2 sub-
stitutions within the codon, but, in each case, one of these could be a silent
change. A similar comparison between the amino acid sequences of A/NT/60/68
and A/WSN/33 indicates 35 differences, 32 of which are single site mutations.
2 changes are due to 2 substitutions within a single codon and one nucleotide
change is probably silent. In the single case of amino acid residue 453, all
3 nucleotides differ. Most amino acid changes between the three strains are
conservative in nature (e.g. R -L) or semi-conservative (S -* A, N *S).
But 2 of the differences between A/PR/8/34 and A/NT/60/68 are non-conserved,
at residues 309 (G --I-D) and 453 (P -* H). Between A/WSN/33 and A/NT/60/68,
5 non-conserved changes occur at residues 195 (G -D), 296 (N -D), 630
(G R), 740 (N -* D), and 453 (S -, H). These alterations give a slightly
increased overall charge of +28 for the PB2 protein of A/PR/8/34 and +29 for
A/WSN/33. We consider it unlikely that these non-conserved residues play an
important functional role in Cap recognition because of their charge varia-
bility between different strains of influenza. The marked change in second-
Influenza
43 67 82 103 105 107 114 121 185 195 250 251 271 292 298
strain rr
A/NT/60/68 S V 8 G K S V / I D V R A T D
A/PR/8/34 A I N A/ N/ I R - - -
Influenza
299 309 338 344 358 368 382 394 444 451 453 463 471 478 480
A/WSN/33 V\ V\ K\ R I I\ A
A/NS/60/68 |I
LX
D K
V
V |IE H V V
A/PR/8/34 R jG . I
R I I V I
Influenza
491 493 494 588 613 630 636 638 655 661 667 674 676 684 740
strain AI i11IV I T
A/WSN/33 A AK
I G I I V p T N\
A/NT60/_8 TI R V I T R L IV A T i T S D
A/PR/8/34 IA F' V A/ /
T A
Figure 2. Amino acid differences between the PB2 proteins of A/NT/60/68,
A/WSN/33 and A/PR/8/34.
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Nucleic Acids Research
ary structure which can occur upon substitution of a proline residue by any
other amino acid suggests that residue 453 has limited structural significance.
If a similar comparison is carried out between the influenza viruses
A/PR/8/34 and A/WSN/33, 27 differences are seen (Fig. 2). Since the initial
isolates were obtained just a year apart, few changes might be expected bet-
ween them. However, both viruses have been passaged within the laboratory,
and in particular, A/WSN/33 has undergone repeated passage to select a neuro-
tropic variant. We know from a comparison of the nucleotide sequences
encoding the mature HAl polypeptide (39,40), and the MS2 (41,32) and NS (42,
27) proteins, that the predicted amino acid sequences can differ between
different laboratory isolates of A/PR/8/34. The variation is 7 out of 325,
2 out of 96, 1 out of 230, and 2 out of 121 residues in the polypeptides HAl,
KS2, NS1 and NS2, respectively, or a mean average of 1.5% per protein. Hence,
we might expect that "laboratory drift" accounts for some of the observed
differences between the three strains, particularly in relation to A/WSN/33.
A/NT/60/68 has been passaged infrequently in the laboratory; hence we think
it reasonable to assume that little laboratory drift has occurred since its
isolation in 1968.
In order to attempt to correct for the laboratory drift factor when
estimating the rate of evolution of segment 1 from A/PR/8/34 or A/WSN/33 to
A/NT/60/68, we have set up the hypothesis that the two earlier strains were
identical when isolated. Alterations which occurred in the field are taken
to be those where there is a common amino acid for the earlier strains, which
differs from that found in A/NT/60/68, e.g. residues 43, 67, 82, etc. (a total
of 20). All other alterations, e.g. 103, 105, 107, bar residues 453 and 655,
are accredited to laboratory drift alone of either A/PR/8/34 or A/WSN/33.
Positions 453 and 655, at which all 3 strains differ, are classed as field
mutations for A/NT/60/68 and laboratory drift for A/WSN/33, since, if the
A/PR/8/34 amino acid is taken as the ancestral residue, just a single amino
acid change gives both the A/WSN/33 and A/NT/60/68 residues. There are 30
amino acid changes between A/PR/8/34 and A/NT/60/68 giving a value of 30/759
or 3.9% observed evolutionary drift. Assuming that 20 residues are actual
field changes and the remainder laboratory drift we calculate a corrected
evolutionary drift of 20/30 x 3.9% = 2.6%, or 0.08% per year. Furthermore,
we calculate the laboratory drift of A/PR/8/34 to be 1.4% and that for A/WSN/
33 to be 2.1%. These values are similar to the known laboratory drift of
various proteins of A/PR/8/34 (see above).
Selection for the PB2 protein occurs regardless of whether drift
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happens in the laboratory or the field. Thus comparisons of the amino acid
sequence may still give an insight into functionally important regions.
Amino acid alterations (Fig. 2) are randomly spaced throughout the molecule
but tend to conserve its basic nature. This is compatible with its proposed
function of recognising the negatively charged host cell cap 1 structures
(7-11). The specific residues which bind the "cap" have not been identified
to date. There are no obvious regions of high basicity in the primary sequ-
ence (Fig. 1) which may carry out this function, but this does not exclude
their possible existence in the 3 dimensional structure. It would be of
interest to determine the residues involved and to see if they are conserved
between strains.
2. "Evolution" of influenza proteins
Possible amino acid sequence divergence from initial isolates during
laboratory passage may prevent us determining the absolute rates of evolution
of A/NT/60/68 proteins. Nevertheless, if the rate estimations are restricted
to those between genes of two strains, the comparative rates of evolution of
the genes can be studied. It is assumed that there is equivalent drift of
the genes, that direct lineage exists and that no major reassortment occurred
between the strains. Figure 3 shows the evolution of the proteins encoded
by segments 1 (the sequence determined in this study), 2, 3, 4, 5 and 6 of
A/NT/60/68 (21-23, 52, 53) and segments 7 (2) and 8 (54) of A/Udorn/72 with
respect to the prototype virus A/PR/8/34 (18,27,32,39,55,56,51). Segments
7 and 8 of A/NT/60/68 have not yet been sequenced. Thus those of A/Udorn/72
were chosen to complete the comparison. The resulting percentages fall into
two main groups, those less than 0.3% amino acid changes per year and those
greater than 0.9%. All internal proteins are found in the first class of
highly conserved molecules. The surface glycoproteins, the haemagglutinin
(HAl subunit) and neuraminidase (NA) show a marked percentage increase,
indicating a larger variability. It should be noted that the figure of 1%
for the HAl domain and NA represents point alterations and does not include
spaces or deletions included to allow a colinear comparison of the two sequ-
ences. It is therefore an artificially low value. Thus internal proteins
are conserved because of selection to perform specific functions within the
virion and the external proteins are variable because of antigenic selection
by the host immune systems.
3. The Origin of RNA segment 10
One of the aims of this study was to confirm previous work defining the
origin of a small RNA (segment 10) found in a preparation of A/NT/60/68 virus
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Nucleic Acids Research
1.0
a
0
0
C
0
a
c
0
0
C
a
0
*
A/NT/60/68 A/UDORN/72
Figure 3. Histogram shoving the different rates of evolution of viral
proteins. Rates are calculated as 5 amino acid change of A/NT/60/68 for PB2,
PBl, PA, NP, HA1, NA and for A/Udorn/72 for the NS and NS proteins with
reference to A/PR/8/34. Data are calculated from references 22, 21, 23, 52,
53, 2, 54, 18, 55, 56, 39, 51, 32, and 27.
10 20 30 40 A 0 so 70 s* 90 *00 * io
MSCAMMCA inACLM CmAROM OmAwmLC hCAMJOOWX CAAUCCWM WWXUCOAAC A50wuwo X
130 140 C 120 o o 170 10 zoo D 210 22 0 230 E 240
5050504ina UsouinmUmi UCOCMUCUC OCAUCmCOA MIUCMd&hCACWOCUAOCJ,,510
AUJ~A11UII1 OAOOMJCII
250 u 2 27 230 290 F 300 310 320 330 340 5 250
__eUCON UUOfANMM A w - OCUAU mC UUCA sOLwasCUCC maN
CacM
370 300 300 400 410 420
ALu_ CARUMOM MAUNMs CAMU.UMC AAAAAAICUAIUOUUUCUA CU
Figure 4. The nucleotide sequence of segment 10 of A/NT/60/68 and its
alignment with segments 1 and 3 from A/NT/60/68. Arrows indicate alternative
Junctions between the sections.
Region A nucleotides 1 to (75 -- 76) of segment 3
Region B " (1971 -*1972) to 2014 of segment 3
Region C " 30 to (89 9
91) of segment 1
Region D " (2015 -S 2017) to (2051 -- 2055) of segment 3
Region 1 " (86 - 89) to (126 -4 130) of segment 3
Region F " (2065 -+ 2069) to 2233 of segment 3
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Nucleic Acids Research
F
D3
E r
<
N~~~~~~~v......
I ~~~~~A
segment I segment 3
_____ Recombinatlon
AAAv Track of polymerase
Figure 5. A hypothetical arrangement for intragenic recombination between
RNA segments 1 and 3.
(20). Sequence analysis of this RNA and its comparison to segments 1 and 3 of
A/PR/8/34 indicated a mosaic structure for segment 10 (18). Intragenic
recombination had not previously been seen in influenza viruses. Figure 4
shows the alignment of segment 10 to specified regions of 1 and 3, found
using a computer program to scan for homology (43). No mismatches within the
regions were found, although ambiguities in the positioning of Junctions
remain. Figure 5 illustrates a hypothetical arrangement of segments 1 and 3
allowing such a recombination event by "polymerase jumping". The two ends of
segment 3 are exactly reproduced in band 10.
4. General discussion
Control of influenza infection by the conventional methods of vaccina-
tion has achieved little success. A cap-recognising protein, such as PB2,
presents a potential target for specific anti-influenza virus drugs. A 24K
cap binding protein (CBP), found within the host cell, has also been described
(44,45). It would be of interest to compare the amino acid sequence and
active residues of this protein to those of PB2 to study any functional simi-
larities and to determine whether agents against PB2 might not also affect
CBP. It has been shown that the RNA segment coding for the PB2 protein of
A/PR/8/34 is involved in the recognition of target cells by an anti-influenza
cytotoxic T cell line (46). This may be due to the presence of a small amount
of PB2 on the surface of an infected cell or an antigen-presenting cell or
may be an indirect effect of PB2 on some other viral product.
Studies on functional P proteins have been limited by their scarcity
within the virion (approximately 8% of total protein in the viral core (38)).
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The development of both eukaryotic (47,48) and prokaryotic expression systems
of cloned genes (49,50) should allow increased quantities of these proteins
to be made available for research.
ACKNOWLEDGEMENTS
We thank the Medical Research Council for financial support to K.L.J.
for a research training grant and to G.G.B. for an MRC programme grant.
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