from an early Hong Kong influenza virus variant A/Aichi/2/68 (X-3 1) ... the amino acid sequences of variant-A/Aichi/68 and -A/Memphis/72 haemagglutinins.
Biochem. J. (1981) 193,953-962
953
Printed in Great Britain
Amino acid sequence and oligosaccharide distribution of the haemagglutinin from an early Hong Kong influenza virus variant A/Aichi/2/68 (X-3 1) Colin W. WARD and Theo A. DOPHEIDE CSIRO, Division of Protein Chemistry, 343 Royal Parade, Parkville, Victoria 3052, Australia (Received 8 July 1980/Accepted 19 November 1980)
The amino acid sequence and oligosaccharide distribution for the haemagglutinin from the early Hong Kong influenza virus A/Aichi/2/68 (X-3 1) was investigated. The two polypeptide chains, HA 1 and HA2, were fragmented by CNBr and enzymic digestion, and the amino acid sequence of each small peptide was deduced by comparing its chromatographic behaviour, electrophoretic mobility, amino acid composition and N-terminus with that of the corresponding peptide of the haemagglutinin of known structure from the influenza-virus variant A/Memphis/102/72. Those peptides in which changes were detected were sequenced fully. The complete amino acid sequence of the haemagglutinin HA1 chain (328 residues) and 188 of the 221 residues of the HA2 chain were established by this approach, and revealed only twelve differences between the amino acid sequences of variant-A/Aichi/68 and -A/Memphis/72 haemagglutinins. These occurred at positions 2, 3, 122, 144, 155, 158, 188, 207, 242 and 275 in the HAl chain and 150 and 216 in the HA2 chain. The highly aggregated hydrophobic region (residues 180-121) near the C-terminal end of the HA2 chain was not resolved by peptide sequencing. The oligosaccharide distribution in variant-A/Aichi/68 haemagglutinin was identical with that found in that of A/Memphis/72, with sugar units attached at asparagine residues 8, 22, 38, 81, 165 and 285 in the HAl chain and 154 on the HA2 chain. The monosaccharide compositions of the individual carbohydrate units on variant-A/Aichi/68 haemagglutinin differed from those of the corresponding units in variant-A/Memphis/72 haemagglutinin, and evidence was found for heterogeneity in the oligosaccharide units attached at single glycosylation sites.
Influenza remains a poorly controlled infection that frequently affects a large proportion of the population, largely because of the ability of the virus to undergo frequent and often dramatic antigenic change. Although both viral coat proteins (haemagglutinin and neuraminidase) can change, the haemagglutinin is considered the more important antigen because of its central role in the infection process (Drzeniek, et al., 1966; Laver & Kilbourne, 1966; Klenk et al., 1975; Lazarowitz & Choppin, 1975). Two kinds of antigenic variation have been observed in influenza virus: major changes, termed 'antigenic shift', and minor changes, termed 'antigenic drift'. Since the first influenza virus was isolated from man in 1933 (Smith et al., 1933), antigenic shifts have occurred in 1957 (Asian influenza), 1968 (Hong Kong influenza) and 1977 (Russian influenza). Since the appearance of the Hong Kong sub-type of influenza type A virus in man in 1968, rapid antigenic drift has occurred, Vol. 193
resulting in the appearance of new variants each year (Schild et al., 1974; Pereira, 1976). Peptide 'maps' have shown that antigenic shift and drift are associated with major and minor differences respectively in the amino acid sequences of the haemagglutinin (Laver & Webster, 1968, 1972). Previous reports have described the amino acid sequence of the haemagglutinin from the Hong Kong variant A/Memphis/102/72 as determined by protein (Dopheide & Ward, 1978, 1979, 1980; Ward & Dopheide, 1979, 1980; Ward et al., 1980a) and nucleic acid (Sleigh et al., 1980) procedures, and these data have been used to determine the partial amino acid sequences of several natural Hong Kong field strains (Laver et al., 1980), and variants selected with monoclonal hybridoma antibodies (Laver et al., 1979). In the present paper we describe our investigations of the amino acid sequence of the haemagglutinin from X-3 1, a recombinant virus containing the haemagglutinin from the early Hong Kong strain A/Aichi/68. We have determined the 0306-3275/81/030953-10$01.50/1 © 1981 The Biochemical Society
954 structure to assist the characterization of the extent of chemical and antigenic change that has occurred in the Hong Kong sub-type during antigenic drift, and to provide the sequence data required for the interpretation of the 0.3 nm (3 A)-resolution electrondensity map derived from X-ray-crystallographic analysis of X-31 haemagglutinin crystals (Wiley & Skehel, 1977; Wilson et al., 1980).
Materials and methods Virus The primary inoculum of the X-31 virus was kindly supplied by Dr. W. G. Laver, Australian National University, Canberra, Australia. Virus was grown in the allantoic sac of 11 day-old-chick embryos and purified as described by Laver (1969). A total of 4000 chick embryos yielded 600mg of virus.
Haemagglutinin isolation and peptide fragmentation The procedures for haemagglutinin isolation by electrophoresis on cellulose acetate blocks, the separation of the heavy (HAl) and light (HA2) chains by centrifugation on guanidine hydrochloride density gradients, reduction and S-carboxymethylation with iodo[2-"4C]acetate, CNBr cleavage
and the separation of the CNBr peptides have been fully described, as have the procedures for pyroglutamyl aminopeptidase (EC 3.4.11.8), trypsin (EC 3.4.21.4), thermolysin (EC 3.4.24.4), pepsin (EC 3.4.23.1) and Staphylococcus aureus proteinase (EC 3.4.21.19) digestion (Dopheide & Ward, 1978, 1979, 1980; Ward & Dopheide, 1979, 1980; Ward et al., 1980a). The digestion of peptide HA2 CN1 (for nomenclature, see below) with trypsin (1% w/w) was extended for 48h at 370C in 0.1M-N-ethylmorpholine/acetate, pH8.0, with stirring; a further addition of enzyme was made after 24h. Soluble tryptic peptides were intially fractionated on two coupled Sephadex G-50 (Fine grade) columns (150cmxO.9cm each, in series) in 0.O1MNH4HCO3/10% (v/v) propan-2-ol. Pooled fractions were further purified by high-voltage electrophoresis and, where necessary, chromatography in butanol/ acetic acid/water/pyridine (15 :3 :12: 10, by vol.) on Whatman 3 MM paper.
Analytical Amino acid analyses were performed after hydrolysis in 5.7 M-HCl/0.004 M-thioglycollic acid at 1080 C in vacuo for 24h. Tryptophan-containing peptides were identified by staining side strips with Erhlich reagent (Easley, 1965), and were hydrolysed in 4M-methanesulphonic acid containing 0.2% tryptamine at 1150C for 24h (Simpson et al., 1976).
C. W. Ward and T. A. Dopheide
C-Terminal amino acids were detected by selective 3H-labelling (Holcomb et al., 1968). Automated sequence degradation of the deblocked HA1 chain was performed with a protein program (Inglis et al., 1979), and manual dansyl (5-dimethylaminonaphthalene-l-sulphonyl)-Edman degradations were performed as described previously (Ward & Dopheide, 1979). Glucosamine was determined on an amino acid analyser after hydrolysis in 3M-p-toluenesulphonic acid at 1000C for 24h (Allen & Neuberger, 1975). Neutral sugars were determined as alditol acetates by g.l.c. (Albersheim et al., 1967). All samples (12-70nmol) were hydrolysed in 2ml of 2.5 M-trifluoroacetic acid at 1000C for 2h in a sealed tube under N2. After hydrolysis, the acid was removed by rotary evaporation and the hydrolyses reduced with NaBH4 and acetylated (Albersheim et al., 1967). The resulting alditol acetate derivatives were separated by g.l.c. on a column (1.5 m x 2mm) of 3% SDP 2340 on Supelcoport 100/120.
Peptide nomenclature Peptides have been assigned a prefix according to the digest from which they were recovered (CN, CNBr; T, tryptic; C, chymotryptic; Th, thermolytic; P, peptic; S.a., Staphylococcus aureus proteinase) and a number (1, 2, 3) according to the order in which they occur in the final sequence. Peptides resulting from partial cleavages, sharing common regions of sequence, are denoted la, lb, Ic etc.
Results In the present study we have fragmented the two chains (HA 1 and HA2) of variant-X-31 haemagglutinin by CNBr cleavage and enzymic digestion and deduced the amino acid sequence of each peptide by comparing its chromatographic behaviour, electrophoretic mobility, amino acid composition and N-terminus with that of the corresponding peptide from the haemagglutinin of variant A/Memphis/102/72 (A/Mem/72), the structure of which is already known (see the introduction for references). For those peptides in which changes were detected, the amino acid sequence was determined directly. Structure of the HAI chain CNBr cleavage of S-carboxymethylated HA 1 (700nmol) gave five peptides, CN1-CN5, which could be readily separated by gel filtration (Fig. 1). The large fragments CN1 (residues 1-168), CN2 (residues 169-260), and CN3 (residues 269-320) were digested with trypsin and the digests fractionated as shown in Figs. 2(a), 2(b) and 2(c). The large soluble tryptic peptides, CN1.Tlb, CN1.Tlc, CN1.T3 and CN3.T2, as well as the large insoluble 1981
955
Primary structure of X-31 influenza haemagglutinin 2.5
-
1400
6000
1200 E E
CU 5000 0
2.0
_
cc
1000 - 4000
1.5
800
0 -
2000~
600
ci
~~~~~~-1000~
400
*>
I~ ~ ~~~~-3000. 1.0
0.5
I/
E
200
0
C.g o CU
X4
S4[2-14C Icarboxymethylated variant-X-31 haemagglutinin chain HA I The digest was fractionated on a column (150cm x 1.Ocm) of Sephadex G-100 in 50% (v/v) formic ) and acid. Peptides were identified by A280 ( radioactivity in lOl samples (----). The fraction size was 2.1 ml, and the flow rate 4.2 ml/h. '-7~ 0.5 _ 40
tryptic peptides, CN1.T5, CN2.T2a and CN2.T10, were further digested with either thermolysin, S. aureus proteinase or pepsin. The complete sequence of X-31 HA 1 is shown in Fig. 3, along with the known sequence of A/ Mem/72 HA1 and the location of the peptides used to deduce the sequence. Only ten sequence changes were found between X-31 and A/Mem/72 HA 1 polypeptides. These occurred at positions 2, 3, 122, 144, 155 and 158 in peptide CN1, 188, 207 and 242 in peptide CN2 and 275 in peptide CN3. The amino acid compositions, electrophoretic mobilities and amino acid sequences for the peptides containing these changes are shown in Table 1. Direct sequenator analysis (40 cycles) of carboxymethylated HA 1 (150 nmol), after the N-terminal pyroglutamic acid blocking group has been removed with calf-liver pyroglutamyl aminopeptidase, confirmed the suspected sequence changes at residues 2 and 3 in the blocked peptide CN1.Tlb.Thla (see Fig. 3). The nucleic acid sequence for the haemagglutinin gene from A/Mem/72 (Sleigh et al., 1980) showed that the 'Asx' residues at positions 137 and 250 in HA1 were asparagine not aspartic acid as found in the protein sequence (Ward & Dopheide, 1980; Ward et al., 1980a) and that the sequence at residues. 290-293 was Asn-Asp-Lys-Pro not Lys-Pro-AspAsp (Dopheide & Ward, 1978). As shown in Fig. 3, the X-31 sequence agrees with the A/Mem/72 nucleic-acid-sequence data. Peptides CNI.T5.Th5, CN2.TIO.P3 and CN3.T2.Th4 were each recovered as both neutral and acidic (deamidated) versions, supporting the assignment of residues 137, 250 and 290 or 291 as asparagine. Manual sequence analysis Vol. 193
I ' ' . " .' " .' '- '.'7' 50 60 70 80 90 100 110
l.
12Q 2000
0.4
150 0 -1
0.3
41000
0.2~~~~~~~~~~~~~~~~~0 0.1 CU - A T5 T3c 0 oftytcdgss 0i.2.Crmtgah T4~~~~~~~~~~~f CNBr
40
50
60
70
80
90
100 110 120
Fraction no.
Fig. 2. Chromatography of tryptic digests of CNBr peptides (0.6,umol, 18h digestion) from variant-X-31 haemagglutinin chain HAI Two columns (each 150cm x 0.9 cm) of Sephadex G-50 (Fine grade) connected in series were used, and the columns eluted with 0.01 M-NH4HCO3/10% (v/v) propan-2-ol at a flow rate 4.4 ml/h and ) and radioactivity in 10 or monitored by A230 ( 20,ul samples (----). Fractions (2.2 ml) were pooled as shown, and the peptides present at each fraction are indicated. (a) Tryptic peptides from S[2-'4C]-carboxymethylated peptide CN1; (b) tryptic peptides from peptide CN2; (c) tryptic peptides from S-[2-'4C]carboxymethylated peptide CN3.
of peptide CN3.T2.Th4 (100mol) confirmed the sequence of residues 290-293 as Asn-Asp-Lys-Pro. All other peptides isolated from X-31 HA 1 and indicated in Fig. 2 were identical with the corresponding peptides from A/Mem/72 in end group, composition and chromatographic behaviour (see Dopheide & Ward, 1978; Ward & Dopheide, 1980; Ward et al., 1980a). Oligosaccharide units were
C. W. Ward and T. A. Dopheide
956 .4em/72 X-31
Gix ASP Phe Pro Gly Asn Asp Asn Ser Thr Ala Thr Leu Cvs Leu (ly His His Ala Val Pro Asn Gly Thr Leu Val Lys Thr lie Thr
Tyr.
Clx
eu Pro
Cly Asn
Asn
A
G;y.TAy.Y .Ly8.Tt.Hlv.Tbtr
30 ------------------Thla----------------
Mem/72 X-31
--Thlb-4 l--Th2--1
1-----.Th3 CNI
---Th4---------
---
,---Th5----
~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
I
Asn Asp Gln Ile Glu Val Thr Asn Ala Thr Glu Leu Val Gln Ser Ser Ser Thr Cly Lys Ile Cys Asn Asn Pro His Arg Ile Leu Asp Asn.AVp.1Cji .(4V. .Yqj Thr As*n Ala Thr Clu Leu Val Gln Ser Ser Ser Thr (fly Lys Ile Cys Asn Asn Pro His Arg Ile Leu Asp 60 6 11 1 T2 Tlc. . . 4 ---------------------Thl Sa3 -----------------Sal ----------4 ---------Sa2.---------
CNM
CN1 Mem/72 X-31
3---------
Gly Ile Asp Cys Thr Leu Ile Asp Ala Leu Leu Gly Asp Pro ais Cys Asp Gly Phe Gln Ain Glu Thr Trp Asp Leu Phe Val Glu Arg Gly Ile Asp Cys Thr Leu Ile Asp Ala Leu Leu Gly Asp Pro His Cys Asp Gly Phe Gln Asn Glu Thr Trp Asp Leu Phe Val Glu Arg 90 -T3
--
14-----Th2a---- A -----Th3a--- +- Th-4 4---------Th4d ---------------- 4 ---------Th5a------------ A i--Th6-1 ---Th7----
X-31
CN1
4b
CN1
Mem/72
Ser Lys Ala Phe Ser Asn Cys Tyr Pro Tyr Asp Val Pro Asp Tyr Ala Ser Leu Arg Ser Leu Val Ala Ser Ser Gly Thr Leu Glu Phe Ser Lys Ala Phe Ser Asn Cys Tyr Pro Tyr Asp Val Pro Asp Tyr Ala Ser Leu Arg Ser Leu Val Ala Ser Ser Gly Thr Leu Glu Phe
120
4T4b--1
Mem/72 X-31
T4c
1
i--Th 1- i----------------- Th2-------------i CN1I CN1 Asn Lys Arg Gly Pro s Ser Gly Phe Phe Ser Arg Asn Ala Cys Gly Gly Ser Val Thr Asn Gln Ile Glu Gly Phe Thr Trp Thr Gly Ile Glu.Gly Phe Thr Trp Thr Gly Val Thr Gln Asn Gly Gly Ser Asn Ala Cys Lys Arg Gly Pro ASer GlyPhePe r g IT 1050
T-i-
i5
Mem/72 X-31
Leu Asn Trp Leu Leu Asn Trp Leu
............
Lys SerG
Ty Lys Ser Gly
CNI
Ser Thr Tyr Pro Val Leu Asn Val Thr Met Pro Asn Asn Asp Asn Phe Asp Lys Leu Tyr Ile Trp Ser Thr Tyr Pro Val Leu Asn Val Thr Met Pro Asn Asn Asp Asn Phe Asp Lys Leu Tyr Ile Trp .........
1 T7T
--6b
b
6a
,-----Th3- -4A---------Th4- --4-------------------Th5--------------------CN1
....
^.......
......
...,---.18
T1 l
-
4
1
T2
-
-
--Th1-4 4----Th2 4---P2-----Mem/72
X-31
CN2 CN 1 Gly Val His His Pro Ser Thr Asp Gln Glu Gln Thr Ser Leu Tyr Val Gln Ala Ser Gly Arg Val Thr Val Ser Thr Lys Arg Ser Gln Gly Val His His Pro Ser Thr AsGln Glu Gln Thr Ser Leu Tyr Val Gln Ala Ser Gly Arg Val Thr Val Ser.Thr Arg Ar Ser Gln . 210 T 5a @ir 1 ~~~~~~~~~ ~~~~T2 ~ c ---44 -------------Th3 ------------------------- 4 1--Th4--A 4-----T5-$
-
P2 ------------------------------
-
-
X-31
Mem/72 X-31
__
_ Mem/72
X-31
-
*
* .. ss
........................................ .
......
.
...
21.
CN2
---------Plb----------4 CN2 CN2 Asp Tii Leu Val Ile Asn Ser Asn Gly Asn Leu Ile Ala Pro Arg Gly Tyr Phe Lys Met Arg Thr Gly Lys Ser Ser Ile Met Arg Ser Aspl Vl! Leu Val Ile Asn Ser Asn Gly Asn Leu Ile Ala Pro Arg Gly Tyr Phe Lys Met Arg Thr Gly Lys Ser Ser Ile Met Arg Ser 270 IlFTlt1 T T1 T10lI . A ______ .__---------p4.---------------CN2
_____P5_-----
12
CN4
CN3
Glu Cys Ile Thr Pro Asn Gly Ser Ile Pro Asn Asp Lys Pro Phe Gln Asn Val Asn Lys Ile Cys Cys Ile Ser ;lu Cys lie Thr Pro Asn Gly Ser Ile Pro Asn As Lys Pro Phe Gin Asn Val Asn Lys le 300 T2 --M ----- 4 ----Th3------4 4----------------------Th4--------------------- 41-----Th5--4 ----Th6-- |-
Asp Ala Pro lie Gly Thr Asp Ala Pro Ile Asp Thr
Ile Ser
CN3
CN3 X-31
..
Gln Thr Ile Ile Pro Asn Ile Gly Ser Arg Pro Trp Val Arg Gly Leu Ser Ser Arg Ile Ser Ile Tyr Trp Thr Ile Val Lys Pro Gly Gln Thr Ile Ile Pro Asn Ile Gly Ser Arg Pro Trp Val Arg Gly Leu Ser Ser Arg lie Ser Ile Tyr TreThr l 240 TIO a-T-I T7a T6 ---Pi- -----.P2.------1 ---------P3-------
--ThIT--4
Mem/72
*
---4
CN2
Mem/72
.
Thr Tyr Gly Ala Cys Pro Lys Tyr Val Lys Gln Asn Thr Leu Lys Leu Ala Thr Gly Met Arg Asn Val Pro Glu Lys Gln Thr Thr Tyr Gly Ala Cys Pro Lys Tyr Val Lys Gln Asn Thr Leu Lys Leu Ala Thr Gly Met Arg Asn Val Pro Glu Lys Gln Thr
T3b
T3c-1 T-3aCN 3
-
1t--4
T4 ----I I-
T5
-
--I TCM5
Fig. 3. Amino acid sequence ofvariant-X-31 haemagglutinin chain HAI The alignment of the tryptic and appropriate thermolytic, peptic and S. aureus-proteinase peptides used to derive the sequences is shown and is based on comparison with the known amino acid sequence of A/Mem/102/72 haemagglutinin. The N-terminal 'Glx' residue is pyroglutamic acid; methionine residues occur in both strains at positions 168, 260, 268 and 320. Carbohydrate groups are attached in both strains at asparagine residues 8, 22, 35, 81, 165 and 285. The regions sequenced directly are underlined with dots. The residues that differ between the two haemagglutinins are enclosed in boxes.
1981
957
Primary structure of X-3 1 influenza haemagglutinin
Table 1. Amino acid composition and sequence of variant-X-31 haemagglutinin peptides that difered from the corresponding peptides in varientA/Mem/72 For the location of these peptides and their comparison with A/Mem/72, see Figs. 3 and 6. Abbreviations used: >>F-
rJn
;
m cn
,L. Lr Z4 >
>
n LnP[_E
4I ¢1 >dt ¢11 ~(4fL
¢1 < ¢-:
ri)lCtcfl > > ;
eArn
eArn H- H- HLf > >f-,
J ; ;J J;J-
.4 -A
c
w r4 rN Q4 P. 11 :C e & 44 14 E>>E-> >
HE - HE-H ce cn ur
zuru
cn ~ cn(nr
u) vf) cn
0 -4u) u)u nu) u crzuf 00~.000P 0H m eflV) < < ¢: < ¢ J 4J >4 >, >4>: ;UC DC >; PW r- P. P PL c > :> > :> :> :3: 3 m m nm r- 0 a. PW 0. r4 PL X
< >. 0L :> cz 0
HE-4 F HE-H un c).
H4(-4'-
--~~~~~,.~-
aC'CS
:>3~>
-
CC
--000
rf-c
< bE bo .
C
> >C40 U (H>> U 0u ;~~~~~~~~2 :Rt; zZZ; z
zz;_U)
zzz U) (4) U)
zzA.
z
(-(
zz
A. F- F-4 F- -- Faaa N ae aa L UU)z7=a E n cn M n ev:u cec) c En U)
Ls: w > >>
w w >>
J-);-
;--
3 33 k:X X
3 3 X X
A.
aaaaa ^ m Ln cn m
000 s
C: n mz
-1
¢ -1
1
Hf4 r-
;zXXXz V U > :> > :>
CNuzum m fnm m
Oz zozoHH
-
En
> H4 E- HX H E-H H H HE-H E-S d:i PL4 PL4 13v Pi g; > > :> > :> :~~~~~~~> H E- HE-H E- E :
0C000e' cotaca C aCcrO L4 PIN. > > O::::, 000 000 P. 0- PW 00
(-H
cr, Cf U --X-
0
o
£>^
SA
Fw H
0
ooo
U)U)
cn cnuU
4(4)4)4
>
;
>>>
0 z z 0z z Z 2i!;Z Z Z 'Z; Z : ZZ Z;z I.L 000000 OOOP R P m (n V0 :
>
-- - -4 > > > > > > P44) P Z HZ Z Z H Z 1111 1- i4 1--
PIL~ 11 j: 4(4 :4
m cnV) ;I:> > Er.:uzu:u V CD c: :> z ur n UP En En VI'z %zz z z 0000 O0 z z OP Cax0-0 > > > > > > > > > >-E 4 > >. > >1 >4 Vu uu CDiw w w w w - O E0 H E- H E-HE 0 0 0EA H 0 -£ 14 W : E
HE-HE-
J nu]
cn
W
"
(f U) :
Hti
-4
Z
e
bo
Z
-C-¢ 0
e~~~lm
C
*t W: cd
P-4
r zoozzo 4 >4 H 1
u C z- z EE -4
0 0 0 0 0 u 4 Y4 4 4 H H " H cl Oc Cr cr c
914
w4 V4 :z
z 4X:4nP O~~~~~~~~~~~~~~E z z z z E4 H4
0
> > > > > >
(P4 P4P4
CM
be
Q
o 0 C.
en
C
>
H H(->z EH ¢ V: Vl: a3C 111 1 HH HEcr cr C) 0R C-I Z w:Z Z u t; CL -- CDu D :XWP-e E-EHa - E E- --I
¢j j
-
tiA ~ -4
i-
4
F
EH E-H E- HEH>>>>> > > > >R > > -il;,F-1 .-I4; --I t W
a;a,,'aa
E-
p4lp4p4p 0 0 0 z :K 4 HEH "H -4 ' cn cn ur un uz mn c
U~ Ln ull Vm , l P. P, 4)4)4)4 c;C SC ; cr, ul n V). VI * Un
C
0:
rn cn
0
000000
U) cn U]
004
:, "0 C......... 1C 0 P uC-; ; P.L,P-4 C C4 Ct W.az C0O! c;; : p! < p:> >
U
V
UU j fj
U4
C9\4 PfI
V
P_LzC; '4 P4 01
>Z
H EEv H
tJ Zz
EH
EH
C:n
Z. 0ZZe H CM _000
>;
2~~~EEr,Vn E-4 EfE' UJ 3
;>
tr_. z
00
Ot
PL4^ -.;C4 r-~
U, ce0000cxt l U) CO-4 0 C 4 0C
HE!XDr
m CD
Ucm
H-'i-aI--{H
=4
X X =
I
r-
Z~ )4)4Z Z EnOv
ZZ
ZZ
t
gQH2 E CCB (JN
HH 00
ce Cy c CO"
-C-C-C
p
u
'z o-;; 'C:;:¢
W
Z4; 4)
I>>(
>>
.
r 000 .t 'cU-t'
X
00(4)4)C;4)4)4O(4 00C < ~0000 Q CD H
11 Z
oC
(-2
-
C5
-:
4
P4
>RaH
P14 0-0-ta-HOC0 r 000
0
:t
cm
E-
> >
00 HOEN (; U CS
,n :3 n3
H
CD
)
