_Archives ...
Vfrology
Arch Virol (1992) 123:29-45
© Springer-Verlag 1992 Printed in Austria
Molecular characterization and heterogeneity of feline immunodeficiency virus isolates N. Maki 1, T. Miyazawa 2, M. Fukasawa ~, A. Hasegawa 1, M. Hayami 3, K. Miki 1, and T. Mikami 2
1Fundamental Research Laboratory, Tonen Corporation, Saitama, 2Department of Veterinary Microbiology, Faculty of Agriculture, The University of Tokyo, Tokyo, and 3Research Center for ImmunodeficiencyVirus, Institute for Virus Research, Kyoto University, Kyoto, Japan Accepted August 15, 1991
Summary. We have molecularly cloned the complete genomic DNA of TM2 strain of feline immunodeficiency virus (FIV) isolated in Japan and compared its nucleotide and the deduced amino acid sequence with those of previously described U.S. isolates, FIV Petaluma and FIV PPR. The infectious molecular clone of FIV TM2 is different from FIV Petaluma in host cell range; the clone can not infect Crandell feline kidney cells which were permissive for FIV Petaluma. The amino acid sequence homologies, in gag, pol, and env genes between FIV TM2 and Petaluma were 90%, 87%, and 81%, respectively. On the other hand, comparative analysis of each gene between FIV Petaluma and PPR showed 96, 95, and 85%, respectively. These results suggested that the genomic diversity was present among FIV strains isolated from geographically distant areas. Interestingly, tat- and rev-like short open reading frames contained inframe stop codons in the FIV Petaluma but not in the FIV TM2.
Introduction Feline immunodeficiency virus (FIV) is a T-lymphotropic lentivirus isolated from a domestic cat with an acquired immunodeficiency syndrome (AIDS)-like disease in the United States in 1986 [45]. Additional isolates have been reported in the United Kingdom [22], the United States [47], Japan [29, 37], and other countries [13, 39]. Seroepidemiological surveys revealed that this virus had spread all over the world [17, 20, 26, 28, 29, 49, 55]. FIV infection in cats is associated with immunodeficiency-like diseases such as lymphoadenopathy, opportunistic infection, stomatitis, anemia, and so on [27-29, 46, 60, 61]. Pathogenic lentiviral infections are well known in other mammalian species
30
N. Maki et al.
(sheep, goat, horse, cattle, monkey, and h u m a n ) and the viruses share similarities in their genomic organization [40]. The nucleotide sequence analyses of h u m a n immunodeficiency virus type I and type 2 (HIV-1, HIV-2) showed genetic variation a m o n g several isolates [2, 4, 7, 8, 12, 14, 23, 62]. These genetic divergences were also d o c u m e n t a t e d in simian immunodeficiency virus (SIV) [30, 31, 33]. In the case of FIV, the total nucleotide sequence has been reported only for two strains t b u n d in the United States (Petaluma and P P R strains) [43, 44, 47, 56], but the extent of genetic variation of F I V is n o t yet clear. Recently, we reported isolation of two Japanese strains (TM t and TM2) of F I V [37]. The F I V T M strains differed biologically from F I V Petaluma. F I V T M strains could infect feline T-lymphocytes, but n o t Crandell feline kidney ( C R F K ) cells that were permissive for replication of F I V Petaluma [61]. In the present study, we have determined the complete nucleotide sequence of F I V T M 2 a n d partial sequence o f F I V T M 1, and c o m p a r e d t h e m with F I V P e t a l u m a and F I V PPR. Materials and methods
Virus isolation and cloning
Fresh peripheral blood mononuclear cells from two FIV-seropositive cats which inhabited Tokyo area were cocultivated with Con-A stimulated peripheral blood mononuclear cells from a specific-pathogen-free cat, and two strains (TM 1 and TM2) of FIV were isolated as described previously [-37]. At the peak of reverse transcriptase (RT) activity, culture supernatants were clarified and stored in aliquots at - 7 0 °C as the primary virus stock. For molecular cloning of FIV TM 2, the primary stock virus was transmitted to a feline CD4-positive lymphoblastoid cell line, MYA-1 [38]. Fourteen days after infection, the FIV-infected MYA-1 cells were cocultured with uninfected MYA-1 cells at the ratio of 1 : 10, and two days after cocultivation, the extrachromosomal DNA was prepared by the Hirt method [-25]. The extrachromosomal DNA was cleaved with Sad restriction endonuclease and selected by size on an agarose gel electrophoresis. The DNA fraction containing 8--10 kilobase (kb) fragments was purified as described by Maniatis et al. [35]. The DNA was ligated to the SacI site of kZAP-II vector (Stratagene), and the recombinant phages (1 x 106) were plated and then screened by the mixture of 32P-labelled synthetic oligonucleotide probes. The sequences of synthetic probes were 5'-AGGAGTCTCTTTGTTGAGGACTTTTGAGTT and 5'-AGTATCTGTGTAATCTTTTTTTACCTGTGAG, which respectively corresponded to the R and U5 regions of the long terminal repeat (LTR) sequence of FIV Petaluma [56]. The probes were synthesized by a phosphoamidite chemistry on an ABI DNA synthesizer. Positive clones obtained were identified by use of the FIV Petaluma probe (p2-FIV, a 2.6 kb Sad fragment that covers the gag and Y-end of the pol regions of FIV Petaluma), kindly supplied by Dr. R. A. Olmsted (Georgetown University, Rockville, MD). The molecular cloning of FIV TM1 was previously reported [36]. DNA sequencing
Fragments of viral DNA from the phage library were subcloned into pUC118 and pUC119 (Takara, Japan), or M13mpl8 and M13mpl9. The DNA fragments were sequenced bidirectionally by dideoxy-chain termination method [50] using either Escherichia coli DNA
Genomic diversity of FIV
31
polymerase Klenow Fragment or T7 DNA potymerase (Sequenase, United States Biochemical). The nucleotide and the deduced amino acid sequences were compared with those of the three other reported sequences of strain TM1 [37], Petaluma [56] and PPR [-47] by the Interigenetics Bion software. The complete FIV TM2 sequence (Fig. 2) has been deposited in the GenBank data base (accession no. M59418).
DNA transfection The full length plasmid clone (pFIVTM2) was reconstituted to produce proviral form (one LTR on both sides of its genome) by the same strategy as described previously [36], and designated as pTM229. 5 gg of the pTM229 was transfected into CRFK cells [9] by the calcium phosphate coprecipitation method [ 19, 59] and the virus production was monitored by Mg2+-dependent RT activity [41] and indirect immunofluorescence assay [37]. Results
Molecular cloning of FIV TM2 A r e c o m b i n a n t )~ZAP-II phage library constructed f r o m e x t r a c h r o m o s o m a l D N A isolated from the F I V TM2-infected MYA-1 cells was screened with synthetic oligonucleotides of the L T R region as probes. Five positive clones were identified and analyzed by restriction endonuclease digestion and Southern blot hybridization with the p 2 - F I V as a probe (data n o t shown). The clone p F I V T M 2 appeared to contain the full length genome of F I V T M 2 and was selected for further characterization.
Nucleotide sequence and genomic organization The restriction endonuclease patterns of the F I V T M strains were quite different f r o m that of F I V Petaluma. Figure 1 shows the restriction m a p of F I V T M 2 in c o m p a r i s o n with those of F I V TM1 [36] and Petaluma [56]. Of the 25 0
1
2
3
4
5
6
7
8
1
I
t
I
t
1
1
I
l
BHHS E
A'V~,I
H
t
tA I'
NP
NP H S
H
I
lJi, H
PXX X E B
NP PP P LTR g EEl I, a g j I
E
I- LI i
PXX X
BHHS E
B~I
Bg
P P Bg
E H
11
F
P B NX pol
H
BgEV
L BHH
~ IF
I'~
X P BgEV
J I/ × BH Bg X vif A t L,,,J I I
9 Kb
XN
Fig. 1. Comparison of restriction map of
BHH
FIV TM2 (A) with those of TM1 [36] (B) and Petaluma (C). Restriction map of FIV Petaluma was generated from the published nucleotide sequence [-56]. B BamHI; Bg BglII; E EcoRI; EV EcoRV; H HindIII; N NheI; P PstI; S SacI; X XbaI
XN H
env
N LTR r'-I ~B
Fig. 2. Complete nucleotide sequence of FIV TM2 viral DNA genome. The sequence is numbered from the beginning of the U3 region. Underlined is the primer binding site (PBS) complementary to the Y-end of tRNA Ly~and polypurine tract (PPT). Two putative spliceacceptor (SA) sites for orf B are indicated by vertical lines
27 54 T G G G A A C~%T T A T T G G G A T C C T G A A G A ~ A T A G A G A A A A T G C T G A T G G A C T G A G G G U3 81 108 CC-C A C A T A A A C A AGT G A C A G A T G A A A A A C A G C T G A A T A T G A G T C A G A G T T A A A T 135 G C T A G C A G C T G C T T A ACC G C A A A A C C A C A T C C T A T G
TAA AGC
189 TGT A T C T T G C T C C A T T G T A A G A G T A T A T A A C C A G T G T T T
AGT CTC TCT GTT GAG C~
243 TTT CGA GTT CTC CCT
TGA GG~
162 TTG C C G A T G A C G
218 TTT AAA GCT TCG AGG 270 TCC CAC AGA TAC AAT
287 324 A A K A A A T T G A G T TTT G A G ATT G A A C C C T G T C G T G T A T C T G T G T A A TTT C T C T T A
U5 381 378 C C T GCG A A T CCC TGG AG'~ CCG G G C C A G G G A C C T CTX~ A G T TC~G CGC CCC. AAC A G G PBS 405 432 G A C T T G A A A A A G A G T G A T T A G G G A A G T G A A C ~ T A G A C C A A T A G A A A G C T G T C.%A 459 GAA CTC CTG CAG ACC TTG TGT GGG GAG CAG TTG CAG
406 ACG CTG CTG GCA G~G
513 AGT ATC TCT A G T G G A G C A G A C C T G AGC T C T G G A T T A A G T
840 C A C TAT TCG C A G GCC
C~
567 594 TAG A T A A A G A T T ATC T G O T G A C T C TTC G C G G A T C G T C A A G C C A G G A G A TTC G T C 621 GC~ GGA CAG CCA ACA AGG TAG GAG AGA TTC TAC A~C AAC
648 ATG ~ AAT GOA CAG M e t G l y ASh G l y G i n gag 675 702 G G G C G A G A C TGG A A A A T G G C C ATT A A G A G A TGT A G T A A T G T T GCT G T A G G A G T A G l y A r ~ A s p T r p Lys M e t Ala Ile Ly8 A r g Cys S e t A S h Val Ala Val G I ~ Val 729 756 AGT AAG AGT AAA AGA TTC GGA GAA GGA ~AC TTT AGA TGG GCC ATA AGG ATG G l y S e r L y s S e t Lys A r g Phe G l y G Z U G l y A s h P h e A r g T r p Ala Ile A r q Met
GCT Ala
783 AAT GTA ACT ACA GGA CGA GAA CCA GGT GAT ATA CCA GAG Ash V a l T h r Thr Gly Arg Glu Pro G l y A s p Zle P r o G l u
T T A A G A T C A A T T ATT TGT GAT Leu A r g Ser Ile Ile Cys Asp
810 ACT T T A G A A C A G Thr Leu $1U Gln
837 864 TTA C A A G A C A G A A G A G A A C A C TAT G G A TCT AGT Leu G l n A s p A r g A r g G l u His Tyr Gly Set Set
891 918 A A G G A A A T T G A T A T G G C A ATT A C C A C T T T A A A A G T T T T T G C A G T G G C A G G A A T T Lys G l u Ile A s p M e t Ala Ile T h r T h r L e u Lys V a l P h e Ala V a l Ala G l y Ile 945 992 C T A A A T A T G A C T G T A TCT A C ~ C'CC A C A G C A C ~ T G ~ A A T A T @ T A T C~T C A G A T G LeU A S h M e t Thr Val Set T h r AI8 T h r A l a Ala GIU A S h M e t T y r A l a G l n M e t 999 i026 G G A T T A G A C ACC A G A C C A T C T G T A A A A G A A A G T G G G G G A A A A G A A G A A G G A C C T G l y L e u A ~ p Thr A r g P r o Set V a l L y s G l u S e t ~ i ~ G l y L y s G l u G i u G l y P r o 1053 C C A CAG G C T ~ A T C C T ATT C A A A C A G T A A A T G G A G C A C C A C A G P r o G l n A l a T y r ~ r o lle G l n T h r V a l ASh G 1 x Ala ~ r o G l n
1000 T~T GTA GCC CTT Tyr Val Ala Leu
1187 G A Y C C A A A A A T G G T G TCC A T T T T T A T G G A G A A G G(IA A G A G A G A = p P r o Lys Met Val S e t ~le Phe M e t G l u Ly8 A I a A r g G l e
1134 GGG CTA GGA GOT G l y Leu G l y G l y
1188 1161 G A G G A G G T C Co~A C T G ~ TTC ACA GCC TTT TCA GCT AAT TTA A~% TCA AC~ GAT G l u G I ~ V a l G l n Leu T r p P h e T h r A l e P h e 8 e r A l a A S h L e u T h r S e t T h r A s p 1213 ATG GCT ACA TTA ATT ATG TCC GCA CCT GGC TGT GCA GCA GAT Me% A l a T h r Leu Ile M e t S e t Ala P r o G l y Cys A l a A l a A s p
1242 AAA GAA ATC CTA Lys G l u Ile L e u
1296 1269 GAT G A A A C A C T G A A A C A G A T G A C A ~CT GAG TAT G A ~ C G T A C C C A T C C T C C T G A T A s p Glu Thr Leu Lys G l n M e t Thr A l a G l u T y r A S p A r g T h r H i s P r o P r o A s p 1323 1350 C~ C C T A G A C C G C T G C C C T A T T T C A C T C~CC C-CA G A G A T T A T G G G G A T A G G A T T A G l y P r o A r g Pro Leu Pro T y r P h e T h r Ala A l a G l u X l e Met G l y Ile G l y L e u 1377 A C T C A A G A A C A A C A A G C A G A G C C C A G G TTT G C A C C A G C C A G A Thr G l n Glu Gln Gln Ala G I u Pro A r g Phe A l a Pmo A l a A r g
ATG Met
1404 C A G TGT A G A Gln Cys A r g
1431 1458 G C A TGG T A C C T T G ~ G C A T T A G G A ~ A G TTG GCG C~C ATA AAA C,-CC A.a2.. TCT CCC A l a T r p T y r l a u Glu AI~ L e u G l y L y s Leu A l a A l a lle L y s A l a Lys S e t P r o 1485 1512 GAC TAT TCCTCATTT ATA GAT CGA GCA GTA CAA TTG AAG CAG GGA GCT AAAGAG A r g A l a Val Gln Leu LyS G l n G l y Ala Ly~ G I u A s p T y r S e t S e r Phe Ile A s p 1539 1566 A G A C T A TTT G C T C A A A T A G A T C A A G A G C A G A A C A C A G C T G A A G T A A A G C T G T A T A r g Leu Phe Ala Gln Ile A s p Gln Glu G l n A s h Thr Ala G l u V a l Lys Le~ T y r 1593 1820 T T A ZukA C A ~ T C T T T A AGT A T A G C C ~ A T G C T A A T C C A G A T T G T ~ AGA GCA ATG LeU LyS G l n Set L~U Set Ile A l a A S h Ala A s h P r o A S p C y s L y s A r g Ala M e t 1887 J634 AGT CAT C T T AAA CCA GAA A G T ACT TTA G A G G A A A A A C T G A G G G C C T G C C A A G A A Set His Leu Lys Pro Glu S e t Thr Leu GIu GIu LyS Leu A r g A l a Cys Gln G l u
1701 G T A G G A T C A C C A G G A T A T A A A A T G C A A T T G CTG G C G G A A G C T C T C V a l G l y S e z P r o G 1 y T y r Lys Met G l n Leu Leu A l a G l u A l a Leu
1728 ACA AGG GTT Thr Arq Val
1753 !782 C A A A C A C~TT C A A A C A A A A G G A C C A A G G C T A G T G T G T T T C A A T T G T A A A A A A C C A G l n T h r V a l G i n T h r Lys G l y P r o A r g Leu Val C N S P h e A S h C y s Lys Lys Pro 1809 C A C C T G G C T A G A C A A TGT A A A G A ~ G C G A A G A G A TGT A A T A A C G l y His L e u A l a A r g G i n Cys Lys G l u A l a Lys A r g Cy8 A S h A S h
1836 TGT ~A AAA Cys G l y Lys
1863 1890 TTA GCT GCT AAT TGC TGG CAA GGA GGT AGG A~A ACC TCG GGA AAC L ~ u Ala A l a A e n C y s T r p G l n G l y G l y A r g Lys T h r S e t G l y A s h G l u A S h L e ~ G l y Lys A r g pol 1917 1944 GAG AAG GTG GGG CGA GCT GCA GCC CCA GTA AAC CAA GTG CAG CAA ATA STA CCA G I u Lys V a l G l y A r g A l a Ala Ala P r o Val A S h G l n V a l G l n G l n ~le V a l Pro G l u G l y G l y A l a S e t Cys Set P r o Ser Lys Pro Ser A l a A l a A S h Set Thr Ile CCT GGT CAC Pro Gly Mis
1971 1998 TCT GCA CCT CCA ATG GAG GAG AAA CTA TTA GAT TTA TAA ACT ATA ATA CAA TAG S e t A l a P r ~ P r o M e t G l u G l u Lys L e U Leu A s p I~u C y s T h r S e t A S h G l y G l y G l u T h r Ile A r q P h e 31e A S h T y r A S h Th= [le G l y 2025 2052 G T A C T A C C A C A A C T T TGG A A A G A A G A C C T G A A A T A C A A A T T T T T G T A A A T G G A C T h ~ T h r T h r T h r Leu G I U A r g A r g Pro G l u Ile G l n Ile P h e v a l A s h G l y His 2079 2106 A C C C T A T A A A A T T T T TAT TAG A T A C A G G A G C A G A T A T A A C A A T T T T A A A T A G A A P r o lle Ly$ P h e L e u L e u A s p T h r G l y A l a A s p lle T b r Ile L e u A s n A r q Lys 2133 2160 ~%G ATT TTC ~m~ TAG GAA ATT CTA TAG AAA ATG GGA AAC AGA ATA TGA TTG GAG A S p P h e G l n Ile G l y A s h Ser Ile G l u A s h G l y Lys G I D A S h M e t lle G i y Val 2187 2214 T A G G A G G A G G A A A A A G A G GAA C T A ATT A T A TCA A T G T A C A T C T A G A A A T T A G A G G l y G l y G l y Lys A r g Gly Thr A s n T y r Ile ASP V a l His Leu G l u Ile A ~ g A s p 2241 2268 ATG AAA ATT ATA GGA TGC AAT GTA TAT TTG GAA ATG TGT GTG TTC TAG AAG ATA G I U A s h T y r A r g M e t G i n Cys Ile Phe G l y A S h V a l C y s V a l L e u G i U A s p A s h 2295 2322 A T T C C T T A A T C C A A C C A T TAT TAG G A A G A G A T A A C A T G A T T A A G T T T A ATA T A A S e t Leu Ile G l n P r o LeU Leu G l y A r g A s p ASh Met Ile Lys P h e ASh Ile Arg 2349 2376 G A T T G G T A A T G G C T C A A A TTT CAG A A A AGA TTC C A A TAG T A A A A G T G A G Q A T G A Leu V a l M e t A l a Gln Ile Set G l u Lys Ile P r o Ile Val Lys V a l A r g Met Lys 2403 2430 A G G A C C C T A C T C ~uAG G G C C T C AGG T A A A A C A G T G G C C A T T A T C~-A A T G A~& A A A A s p P r o T h r G l n G I y Pro G l n V a l Lys G l n T r p Pro L e u S e r Asm Glu Lys Ile 2457 2484 T T G A A G C T T T A A C A G A C A TAG T A G A A A G A T TAG A A T C A G A A G G ~ A ~ K G TGA A A A GIU A l a Leu Thr A s p Ile V a l G I u A r g Leu G I u S e t G I U G l y Lys Val Lys A r g 2511 2538 GGG CTG ACC CGA ATA ATC CTT GGA ATA CCC CAG TAT TTG CTA TTA AGA AGA AGA A l a A s p P r o A S h A S h P r o ~ r p A s h T h r P r o V a l P h e A l a lle L y s Lys Ly~ S e t 2505 2592 GTG GTA AAT GGA GGA TGC TCA TAG ATT TTA GGG TCC TAA ATA AAT TAA CAG ACA G1y L y s T r p A r g M e ~ Leu lle A s p P h e A r g Val L e u A S h L y s L e u T h r A s p Lys 2619 2646 AAG ~AG CAG AAG TCC AGT TAG GAC TTC CTC ATC CTG CTG GAT TAC AAA TGA AAA G l y A l a G l u V a l G l n Leu G l y L e u P r o His P r o Ala G l y L e u G l n Met Lys Lys 2673 2700 A A C A A G T A A C G G T G T T G G A T A T A G C~=G A T G C A T A T T T T A C C A T T C C T C T A G A T C G l n V a l T h r V a l L~U A s p Ile O l y A s p A l ~ T y r P h e T h r I l e P r o Leu A s p P r o 2727 2754 CAG ATT ATG CTC CTT ATA CTG CAT TTA CAT TAG CTA GGA AAA ATA ATG CAG GGC A s p T y r A l a Pro Tyr T h r A l a Phe T h r L e u Pro A r g Lys A s n A S h Ala G l y P r o 2781 2808 C A G G G A G G A G A T A T G T A T G G T G C A G T T TAC C A C A G G G G T G G G T T T T A A GTC C A T Gly A r g A r g Tyr V a l Trp Cys S e r Leu P r o G l n G l y T r p V a l L e u Ser P r o Leu 2835 2882 T A A ~ A T A T C A G A G T A C C T T ~ % A C A A T A T A T T A C AC~C C A T T T A T C A A A C A A ~ A T T Ile T y r G l n S e r T h r L e u A S h A S h Ile Leu G l n P r o P h e I l e Ly$ Gln A S h S e t 2889 8916 C T G AGT T A G A T A TTT A C C AAT A T A T G G A T G A T A TAT A T A T A G G A T C A A ATT T A A G I U Leu A s p Ile Tyr Gln T y r M e t A S p Asp Ile T y r Ile G l y $er Ash ~ e u A S h 2943 2970 ATA AAA AAG AAC aTa AAC AAA AAG TAG AAG AAT TAA GAA AAT TGT TAT TAT C~T Lys L y s G I u His Dys G l n Lys V a l G l u G l u L e u A r g Ly8 L e u L e u L e u T x p T r p 2997 3024 GGG GAT TTG AAA CCC CGG AAG ACA ~AT TAC AAG AAG AGC CCC CAT ATA AGT GGA G l y Phe G l u T h r Pro G l u A s p Lys Leu Gln GIU G l u Pro Pro T y r Lys T r p M e t 3051 3078 T G G GCT A T G A A T TAC ATC C A T T A A C A T G G T C A A TAC A G C A A A A A C A A T TAG A A A Gly T y r G i u Leu His Pro Leu Thr T r p Set Ile G l n G l n Lys G l n Leu G l u Ile 3105 3132 T T C C A G ~ A A G A C C C A C A T T A A A T G A2AC T A C AGA A A T T A G C A O G T A A A A T ~ % A C T P=o G l u A r q P r o T h r Leu A s h G l u L~u G I D LyS i e u Ale G l y Ly~ Ile A s P T~I 3159 8186 GGG C C A G T C A G A C T A TCC C A G A C T T A A GTA T A A AAG A A C T A A C T A ACA TGA T G A Ala Set GIn Thr Ile Pro A S p Leu Ser Ile LyS Glu Leu T h r A S h Met Met A~g 3213 32q0 G A G G A G A T C A G A A A T T A G A C T C A A T A A G A G AAT G G A C T G T A G A A G C C A A G A G A G G l y A S p O l n Lys LeU A S p Set Ile A r g GIu T r p Thr V a l G l u A l ~ LyS Arg Glu
3267 3294 RAG TAC AAA RAG CTA AGG AGG CTA TTG AGA CGC AAG CAC XGC TRA ATT ATT ATG Val Gln Ly~ Ala Lys G~U Ala Ile Glu Thr GI. Ala Gln Leu ASh Tyr THr Asp
4833 4860 TGG GAA TAR AAC ATA AAT TGG GTA TAC CAG GTA ACC CAC AAT CAC AAG CAT TGG Gly Ile Lys His Lys Leu Gly lie Pro Gly ASh Pro Gin Set Gln Ala Leu Val
3321 3348 A T C C C A ATC GAG CAT TAT A T G C A A AKT T G A GTT T A G T G G G A C C A C ATC AAA TAT Pro AS. Arg Gly Leu Tyr Ala Lys Leu S~r Leu Val G l y Pro HIS Gln Ile C~8
4007 4914 TGG A A A ATG C T A ATA ACA CAT TGA AA¢ TTT G G A TTC AGA AAT TTC TAC C A G A S A GIu ASh AI. Ash Ash Th~ LeU Lys Val Trp Ile Gln Lys ~he Leu Pro GIu Thr
3375 3402 GTT ATC AAG TGT ATC AAA AGA ACC CAG ARC ACA TTT TAT GGT A T G GTA AGA TRA Ty~ Gln Val Tyr Gln Ly~ Asn Pro Glu His ~le Leu Trp THr Gly Lys lle ASn
C T A CTT CTC TGG ATA ATG CTT TGG CCC TAG CCC TGC ATT GTC TCA ATT TTA AKC
3429
3450
A T A GAC AAA AGA AAA AAG CAG AAA A T A C C T ~ T ~ ATA TAG C T C T A A GG~ CAT G T ~
A r E Gln Lys Lys Lys Ala Glu ASh Thr Cys A s p Ile ~ l a Leu Arg AI~ Cys Tyr 3463
3510
4941
4968
Thr S~r Leu Asp ASh Ala Leu Ala Leu Ala Leu His Cys Leu ASh Phe Lys Gln 4995 5022 GGG GTA GAC TAG G G A GGA TGG CCC CTT ATG AAT TAT ACA TAC ~KC AAG AAT A r g G l y A r g Leu Gly Arg Met AI~ Pro Tyr Glu Leu Tyr Ile GI~ Gln Glu Set
A T A A A A TAA GAG AAG AGT CTA TTA T A A G A A TAG C~%A A A G RAC CAG TAT ATG A A ~ Lys Ile Arg Glu Glu Set Ile Ile Arg 21e GIH Lys G l u Pro Val Tyr Glu Ile
5049 5076 CAT TAA GAK TAC AKG ACT ATT TTT CCG CGA TTC CAC AAA AGT TAA TGA TGC RAT Leu A r g Ile Gln Asp Tyr Phe Set Ala Ile P~O Gln 5ys Leu Met Met Gln Trp
3537 3564 TAC CTG C A T CCA GAG AGG CCT ~GG RAT C A K ATC TAA T T A G A T C T C CAT ACC T T A P r o Ala Ser Arg Glu Ala Trp Glu Set A s h I~u Xle A r g S e t Pro T~r Leu ~ys
5103 5130 GGT TGT ATT ACA RAG ATC AAA AAG ATA AAA AAT C ~ A AGG GAC CAA TGA GAG TGG Leu Tyr Tyr LyE A~p Gln LyH Asp Lys LyS Trp Lys Gly Pro Met A r g Val Glu
3591 3618 AGG ~ CAC CAC CTG AC~ TAG RAT TTA TAC A T G C A G CCT T A A G T A TRA A~A GGG Ala Pro Pro Pro Glu Val Glu Phe ~le His Ala Ala Leu Set Ile Lys Arg Ala
5157 5184 AAT ATT C4~G GAC AAG GAT CAG TAT TAT T A A AGG ATG AAG AGA AGG GAA TAT TTC Tyr Trp G1 H Gln GIy Set Val Leu Leu Lys ASp Glu Glu Lys Gly Ile Phe Leu
3645 3672 CTC TAA GCA TGA TAC AAG ATG CCC CTA TRA TAG GAG C A G A K A CAT C4~T A T A TAG Leu Set Met Ile Gln Asp Ala Pro Ile lle Gly Ala Glu T h r Trp Tyr Ile ASp
5211 5238 TTG TAC CTA C4~A GAC ACA TAA GAA GAG TCC CAG AAC OCT GCA CTC TTC CTG AAG Val Pro Arg Arg H£s Ile Arg Arg Val Pro Glu Pro CHS Thr Leu Pro GIU GIy
3699 3726 A T G C ~ A GTA ~AA AAC ~A~ GAA AAG C A G CAA G A G C A G CCT ATT G~A CAA A T A CAG GIM Set Arg Lys Gln Gly Lys Ala Ala Arg Ala Ala THr Trp Thr ASh Thr Gly 3753 3780 RAT GGC AGA TAA T ~ #~3L%TAG RAG GAA GTA ATC AAA RAG C A G # ~ TAC AGG Ly$ Trp Gln Ile Met GIu Ile Glu 01 y Set ASh Gln Lys Ala Glu Wal ~ln Ala 3007 3834 CTT TAT TAT TGG CCT TAA AAG CAG OAT CAG AAG ~ T G A ATA TCA TAA CAG ATT Leu Leu Leu Ala Le~ Lys Ala Gly Ser GIu Glu Met A S h Ile lle Thr Asp Set 3861 3805 CAC RAT ATA TTT TGA ATA TTC TCA ACC AAC AAC CAG ATT TGA TGG AAG GAC TAT Gln Tyr Ile Leo ASh Ile Leu Ash Gln Gln Pro Asp Leu Met Glu G1 H Leu Trp
5265
5292
A T G A G T G A C GAA GAT TC~ CAG GTA AGT A G A AGA CTC TTT C4~A GTT CTC CAR
Met Set Asp Glu Asp Trp Gln Val Set Arg Arg Leu Phe Ala Val LeU Gln ASp GIU wlf 5319 5346 GGA GGA G T A TAC AGT GCC ATG TTG TAC ATA TCG AGT CTA CCT GAA ATG GAA CAG G l y 01 H Val Tyr Set Ala MET ~eu Tyr Ile Set Set Leu Pro Glu M~t Glu Gln 5373
5400
GAT RAA ~ T ARA AGG AGC TTT ~AG ~AA AGG CTT TCG GAa AAG GAA ACa GGA TTC ASp LHS C~S Lys Azg Set Phe LHs LHS Acg Leu Set GIu LHS GI. Thr G1 H Phe 5427
5454
A T & TTT AGA TTA AGA AAA GCT GAA GGA ATA AGG TGG AGC TTC CAT ACC AGa GAT
Ile Phe Arg Leu Arg Lys Ala Glu Gly IJe Arg TZp Set Phe His Thr Arg Asp 3915 3942 G G C A~G AC~ TCT TAG RAC AA~ TC-G AAA ~ G A AAA TAG C A A T T T T T A TAG ACT ( ~ Gln Glu v~l Le~ GlU Gln Met Glu by~ Lys Ile AI~ Ile Phe ~le Asp Trp V~l
5481
5506
T R T TAT A T A GGA TAT GTA AGA GAG ATG GTG GCC GGA TCT AGC C T A CCA GAT RGT
Tyr THr Ile Gly Tyr Val Arq Glu Met Val Ala Gly Set Ser Leu Pro Asp Set 3969 3996 TTC CTG GGC ATA AAG GTA TAC CAG G A K ATG AAG RAG T A G A T A AAC TTT GCC A ~ A P~O Gly His LHS Gly Ile Pro Gly ASh GI~ Glu Val A s p Lys LeU CHS Gln Thr
5535
5562
T T A AGA TTG TAT GTT TAT ATA AGC RAT CCA TTG TGG CAT CAA TCA TAC CGT CCT
Le. Arg Leu Tyr Val Tyr Ile ~er ASh Pro Leu Trp His Gln Set THZ Arg Pro 4023 4050 C A A T G A TGA TTA TAG RAG GTG AGG GGA TAT TAG AG~ AAA GAT CAG ARG ATG CAG M e t Met Ile Ile GIu Gly ~lu G l y Ile Leu Glu Lys A r g 5er GIU Asp Ala Gly 4077 4104 GAT A T ~ ACT TAT TAG CTG CAG CAC ~-KG A~A CAC ATT TCT T~C CT~ GAG AGG TA/% Ty~ ASp Leu Leu Ala Ala Ala Gln Glu Thr His Phe Leu Pro Gly Glu Val Arg 4131 4158 G A A T A G TAC CAA CAK AAA CKA GAA TAA TGC TAC CAA AAG GAC ACT GGG GAC TAA Ile Val Pro T~r Lys Thr Arg Xle Me~ L~u Pro L~S G l y His Trp G I ~ Leu Ile 41~5 4212 TAA TGG C~%A AAA GCT CAR TAG GAR GTA AAG GGG TGG AT~ TAT TAG GAG GAG T T A Me~ Gly Lys 5er Set lle Gly Set LHs Gly Val Asp Val Leu GI~ Gly Val Ile 4239 4266 TAG A T G AAG CAT ATA GAG GAG AAT TAG GAG TGA TTA TGA TTA ATT TAA CAA AKA A S p GIU Gly Tyr Arg Gly G l u L~u GI~ val lle Met Ile ASh Leu Thr Lys Lys 4293 4320 AAT C A K TAA CTA TAT TAG A A A AGC A~A AAA TAG CAC A A T TGA T R A TAT TC42 C T T Set Ile Thr Ile Leu GIU Lys Gln Lys ~le Ala Gln Leu Ile ~le L~u Pro Cys 4347 4374 GTA GAC ACG AAG GCC TAC AAC RAG GAG AAA TAC AAA TGA ATT CAG AAA GAG GAG Arg His Glu Gly Leu Gln Gln G1y Glu Ile Gln Met ASh Set Glu Arg G1 y Glu 4401 442~ A A A RAG GAT TC~ GAT CAG CAG GA~ TT~ T~T CAT CAT G G G TAG A T A ~ A A TTG RAG Lys Gly Ph~ Gly S~r Ala G1y V~l Phe Set S~r Trp V a l Asp Arg lle Glu Glu
3589 5616 GGC CTG A C A AAT TTT AAT ACA ~ TGG CCT TTT GTA AAT ATG TGG ATA AAG A C A G l y Leu T h r Ash Phe Ash Thr Glu Trp Pro Phe Val ASh Met Txp Ile Lys ThE 5643 5670 GGA TTT ATG TGG CAT CAT ATT GAA AGC CAG AAT ATT TGC AAA GGA GGA G A G ATT G l y Phe Met Trp ASp ASp Zle Glu Set Gln ASh Ile Cys Lys Giy Gly Glu Ile 5697 5724 T C A CAT G G A TGG GGA CCT G G A ATG GTG C~SA ATT GTG A T A A A A G C A TTT AGT TGT Set His Gly Trp Gly Pro Gly Met Val G1y Ile Val Ile Lys Ala Phe Set Cys 5751 5770 G G A G A A AGG A A G ATA A/~ ATT ACT CCT GTT ATG ATT ATA A G A GGT GAA ATA GAC Gly Glu Arg LHs Ile LHS Ile Thr Pro Val Met Ile Xle Arg Gly GIu Ile ASp 5805 5832 CCC ACA G A A TGG TGT GGA GAT TGT TGG AAT TTG ATG TGT CTT ~.% TAT TCA CCT Pro Thr G I g Trp Cys Gly Asp Cys Trp ASh Leu Met Cy~ Leu Lye Tyr Set Pro 5059 5806 C C A A~T ACA TTG CAG AGG CTT C ~ ATG CTG GCG TGT G~C AAA GAG GCT AAA GAA Pro ASh Thr LeU Gln Arg LeU Ala Met Leu Ala Cys Gly Ly3 Glu Ala Ly~ Glu 5913 5948 TGG AGA GC4~ TGT TGT AAT CAG CGT TTT GTT TCT CCT TTC AGA ACA CCA TGT G A T Trp A r g G I M Cys Cys ASh Gin Arg Phe Val Set Pro Phe Arg Thr Pro CyS A s p 5967
5994
C T A G A G G T C G T C CAG AAC RAG CCT AAA AGG ~AT TTA TTG TOG ACG C.~A G A A T T A
LeU Glu Wal Val Gln ASh Lys Pro LHs Arg Asn Leu Leu Trp Thr. 01 H GIu Leu 4455 4482 AAG CAG AGT TGA ATC ATG ARK AAT TTC ATT CAG ACC CAC AAT ATT TAK GGA CGG Ala 01~ Leu ASh His GIu Lys Phe His Set ASp Pro GIn Tyr Leu Arg Thr Glu 4509 4536 AGT TCA ATC TAC CTA GAA TAG TAG CAG ~2~G AAA TAA A2~% GAA A A T GTC CCC TGT Phe ASh Leu Pro Arg ~le Val Ala GI~ Glu ~le Ly~ ~r~ Lys Cys Pro Leu Cys 4563 4590 GCA GAA TTA GAG GCG AAC RAG TAG GGG CAC AAT TAA AGA TTC GGC CTG GAA TAT At0' Ile Arg Gly 01~ Gin Val Gly Gly Gln Leu Lys Ile Arg Pro Gly Ile Trp
6021 6048 TGA AT G G A A G A A ATA ATC CCA CTG TTT RAT AAG GTT ACA GAA AAG TTA GAT AGA Met G l u Glu Ile Ile Pro Leu Phe ASh Lys Val Thr Glu Lys Leu Asp A r g ORF -A 6073 6102 G A K C C A G C T A T T AC.A TTA TTT ATC TTG C~CT TAT CAG GT~ GAC ACA TAC A G A T T T G l u Pro Ala Ile Arg Leu Phe Ile ieu ala Tyr Gln Val ASp Arg TH~ Arg Phe 6129 Ile Arg Ii~ Leu Gln Leu Leu L~U Trp Arg Asp A ~
4617 4644 C~oC A A A TGG ATT GCA CAC ATT TTA ATG G~A AAA TAA T C A TTG TAG C A G TGC ATG GI~ Met Asp Cy~ Tbr 31s Pbe Ash Gly Lys ~le Ile Ile Val Ala Val ~ s Val 4671 4690 TGG AAT CAG GAT TTT TAT GGG CAC AGA TAA TTC CAC AGG AGA CTG CAG ~TT G T A Glu Set Gly Phe Leu Trp Ala Gln Ile Ile Pro Gln Glu Thr Ala ASp Cys Thr
6156
A T T A G A ATT TTA CAA TTA TTA CTT TGG AGA GAT AGA TTT AAG TCA ATT AKT T C T
Phe Lys Set Ile ASh Set
6383 6210 •=92~ TAT TGT TTA TGC TGG CTG T~C,TC~ AAG TCT C~T TAT TC~ C ~ T~G C39A TCT L ~ Tyr Cys Le~ Cys Trp Le~ Cys Cys Lys Set AI~ Ty~ Trp Arq Leu Gln $~r Met Leu Ala VaI Leu Gln val Cys Leu Leu Ala Leu Ala Ile T y r 6237
6264
A C A TTA TCC ATA AAT ACT GCC TAG AAA TAT TTC TTT TAA TAT TTC ATC TC~ AGA
4725 4752 CAG TCA AAG CTC TTT TAC AAC TTA TAT GTG CTC ATA ATG TTA CAG AAT TAC AAA Val Lys Ala Leu Leu Gln Leu ~le Cys Ala His ASh Val Thr Glu Leu Gln Thr 47~ 4006 CAG A T A ATG GAC CAA ATT TTA AAA ATC AGA AAA TGG R A G GAT TAT TAA ATT ATK Asp ASh Gly Pro Asn Phe LHs ASh Gln Lys M ~ Glu Gly Leu Leu Ash THr Met
Thr Leu $~r ~le A~n Thr AI~ II~ Ile Mls LHs Tyr Cys Leu GI~ Ile Phe Le~ beu ~le Phe His beu 01~ Ile 6291 6310 TAT AAA CAT GGC AGA C ~ GGG ATT TAC TCA TAA TCA ACA ATG GAT AGG ACC A G A Met Ala GI~ Gly Gly Phe Th= Bi3 ASh Gln Gln Trp Xle Gly Pro Glu
6345 6372 GC^ aOC 7GA ^(;a ^T7 GT7 C~,A TT~ TGA TAT ^~C ~O~ ^C^ AAT G^A TGA AG^ AGG Glu AI~ GIu GIu l.eu L~U Asp Phe A=p Ile Ala Val GIn Her Ash Glu Glu Gly 6399 6~26 TCC A~T AAA C C C AC~ AGT AAA CCC ATT TAG C~T ACe A C ~ C A T TAC T&~C ACA AGA ~ro I ~ u A~n Pro Gly Va). Ash Pro Fhe ArT V~I Pro G l y Ile T b r Set Gin Glu 6453 6480 AAA GGA TGA TTA T';G TAA GAT CTT ACA AAC ~ ACT A C A AGA A T T A A A G A A TGA Lys Asp A=p Tyr Cy~ Lys Ile Leu Gln Thr Lys Leu Gln G l u Leu Lys Ash Glu
fi534 6507 AGT CAA AGA GGT AAA AAT TGA AGA AGG AAA TGC AC~ T A A GTT TAG AAG GC-C AAG Val Lys GIu Val Lys Ile Glu Glu Gly Ash Ala G l y Lys Phe Arg A r g Ala Arg
7657 7884 b\A AGe TGT G ~ ATT OTA T~A TAT TGC TO0 A ~ TTG GTC ~rG CAC ATC GGA 7TT tys Ala Val Glu Leu Ty[ A~,I Ile A!a Gly A~n Trp Ser CyS Thr Ser Asp Leu 7911 7998 ACC KAA A & G ATG G G G ATA TAT G A A C T G T ~ TTG TAC AAA TGG GAC TGA TAA TAG P r o LyS G l y Trp G l y Tyr Her Ash Cys Ash Cy~ T h r Ash gly Thr A~p Ash Set 7965 7992 T G A A A C AAA AAT O G C ATG CCC TAK G A A C C A C ~ TAT TTT AAG A~, TTG GTA CAA GIU T h r Lys Met Ala Cys Pro Lya A = n Gln O l y Ile Leu ArT A~n T~p Tyr Asn 8019 8048 CCC AGT CC-C AGG GTT AAG ACA C ~ 8TT AAT A A A A T A T C A AGT A G T AAA ACA GCC Pro Val Ala Gly Leu Arg Gin Ala Leu Ile Lys Tyr Gln V~l Val Lys Gln Pro
6561 6589 ATA TTT AAG ATA TTC TGA TGA AAA TGT GCT ATC CAT A G T C T A TTT GCT AAT AC~ Tyr Leu Ar9 Tyr 8e# A~p Glu ASh Val Leu 5er ~le Val T y r Leu Leu Ile Gly
8073 8100 RGA G T A TTT GAT AGT GOC ¢~3A A G A A G T TAT GCA G T A T A A ATT C A A ACA AAA GAG Glu Tyr L~u Ile Val Pro glu Glu Val Met Gln T y r Ly~ Phe LyS Gln Lys Arg
6615 6842 A T A TCT AAG ATA TT~ AAT AGA TCA TAG G A G TTT A C ~ A T C T T T G A G A C A T G A TAT Tyr Leu Arg Tyr ~eu ~le ASp ~ s Arg Set Leu G l y Set Le~ A r g His A3p Ile
8127 8154 A G C A G C T A T TCA TAT TAT GTT A G C T C T TGC G A C AGT G T T ATC TAT C43C TGG A C ~ Ala Ala Tle His Ile Met ~eu Al~ Leu ala T h r v a l Leu Set Met Ala G l y Ala
6669 6696 A G A CAT AGA AAC ACC TCA AGA C~A ACA TTA CAA T A A T A G T G A A A A C~C~ TAC CAC Asp Ile Glu Thr Pro Gln Glu Glu His Tyr Ash ASh Set G l u Lys Gly Thr Thr
6181 8208 AGG A A C ~ TGC C A C TGC TAT TGG A K T G G T G A C T C A A T A TCA T C A AGT TTT GC-C G l y T h r G l y Ala Thr Ala Ile G l y Met Val Thr Gln T y r His G l n Val Leu Kla
6728 6750 ATT AAA TAT AAA ATA TGG GAG AAG ATG TTG TAT TAG C A C A T T TAT TAT GTA TTT Leu ASh Ile Lys Tyr Gly Arq Arg Cys Cys Ile Set Th~ Phe Ile Met Tyr Leu
8235 8262 TAC TCA TCA ACA AGC ATT G G A AAA AAT AAC T G A ~ A C T G A ~ AKT AAA TAK TTT Thr His Gln Gln Ala Leu Glu Lys Ile Thr G1u Ala Le~ Lys ~le A=n ASh Leu
6777 6604 AAT CCT TTT TGC AGG AGT AGG CAT CTG CCT CGG AC~C TAG GGC G C A AGT AGT GTG Ile Leu Phe Ala Gly Val Gly Ile Trp Leu Gly A]a A r T Ala Gin Val Val Trp
8289 8316 AAG GCT AAT TAC CTT A G A GCA CCA AGT ATT AGT GAT A G G ATT AAG AGT AGA GGC A r g Leu Ile Thr Leu Glu His Gin Val Leu Val Ile G l y Leu A r g Val G1u Ala
683~ 6958 G A G ACT TCC CCC TTT AGT AGT TCC AGT AGA TGA TAC A G A ~ T AAT ATT TTG GGA ArT ~ u Pro Pro Leu v~l Val Pro Val A~p A 3 p T h r ~ l u Ile Xle Pbe T r p A = p
8343 %370 TAT A G A A]~% ATT C T T ATA TAC AC~C TTT TGC TAT C~CA A G A ATT AC-G ATG C A A TC~ Ile Glu Ly~ Pbe Leu Tyr Thr Ala ~he Ala Met Gln Glu Leu G l y Cys A~n Gln
68~8 6912 TTG TYG CC~ ACC AGA GGA ACC G~C TTG T~A AGA TTT C T T G G G A A C AAT OAT ATA Cys Trp AI~ Pro GIU Glu Pro A]a Cys Gln Asp Phe ]e~ G l y Thr Met Iie Tyr
8397 8424 AAA T C A ATT CTT TTG TAA AAT TCC TCC CAG CCT ATG G A G TAT G T A TAA CAT G A C Ash Gln Phe Phe Cys Ly8 Ile Pro Pro Set Leu Trp Set Met Tyr ASh Met Thr
6989 6966 TCT AAA AGC AAA TGT CAA TAT AAG TAT ACA AGA AC~ ACC TAC ATT C ~ AAA TTG Leu Lys Ala Ash Val Asn Ile Set Ile Gln Glu Gly Fro Thr Leu Gly Ash Trp 6993 7020 GGC AAG AGA AAT TTG GTC CAC ATT ATT TAA AAA C~C TAC AAG GCA ATG TAG AAG Ala Arg G]~ Ile Trp Set Thr Leu ?he Lys Lys Ala Tbr Arg Gln Cy~ ArT Arg
8451 8478 TTT GAA TCA AAC AAT CTG GAA TCA TGG AAA TAT CTC ATT GGG G A A TTG GTA TAA Leu ASh Gin Thr Ile Trp ASh His Gly Asn Ile Ser Leu Gly Ash Trp Tyr ASh 8505 8532 TCA AAC AAG AGA TTT GCA AAA TAA ATT TTA TGA GAT AAT AAT CGA TAT AGA ACA Gln Thr Arg A~p Leu Gln A~n Lys Phe Tyr Glu Ile Ile Met A~p Ile Glu Gln
7047 7074 CC~ ~w%G AAT CTG GAG GAG ATG GAA TGA GAC TAT AAC A C ~ A C C A T T A G G ATG TGC Oly Arg lle Trp Arg Arg Trp ASh OIU Tbr Ile Thr G l y P r o Leu Gly Cy# Ale
8559 8886 A ~ TAA TGT ACA AC-G GAA AAC TC~ AAT A C A A C A ATT ACA GAA A T G G G A AAA TTG a~n A~n Val Gln G l y Ly~ Thr Gly Ile Gln Gln Leu Gln Lys Trp Glu Ash Trp
7101 7128 AAA CAA CAC CTG TTA TAA TAT CTC C~T AGT GGT ACC T G A T T A TCA ATG TTA TGT Ash Ash Thr Cys Tyr ASh Ile Ser Val Va] V81 Pro ASp Tyr G l n Cys Tyr Val
8640 8619 C~T C ~ G ATG GAT AC~ C A A AAT CCC TCA ATA TTT AAA AGG ACT T C T TGG TAG TGT Val Gly Trp Ile G!y Lys Ile Pro Gln Tyr Leu Lys Gly Leu Le~ Gly 5er val
7155 7182 AGA CAG AGT AGA TAC ATG GTT GCA AGG GAA AGT TAA TAT TTC A C T ATG TTT GAC A~p Arq val A~p Thr Trp Leu Gln Gly Lys Val AS~ ~le Set Leu Cys Leu Thr
8694 8667 GTT C ~ AAT AGG TCT AGG AAT CTT ACT ACT ACT TAT ATG CTT GCC TAC AT~ AGT ieu Gly lle Gly Leu Gly Ile Leu Leu Leu Leu Ile Cys Leu Pro Thr Leu Val
7209 7236 A~ AC~ AI~A GAT OCT ATA TAA TAA AGA AAC AAG ACA ATT A A G T T A TTG TAC A G A Gly Gly Lys Her Lau Tyr ASh Lys Glu Thr Arg Oln L~U Set Tyr Cys Thr A~p
8P21 8748 A G A TTG TAT AAG AAA C T G TAC G A A T A A AAT ACT GGG ATA TAC AGT TAT TGC AAT Asp Cys lle Arg Asn,Cys Thr ASh LyS lle Leu Gly Tyr Thr Val lle Ala Met
7263 7290 TCC ACT ~CA AAT T~C~C ATT GAT TAA TTA TAC ATT T ~ G C C TA.A T C A ~ C TT~ TAT Pro I~u GID Ile PrQ Leu ~le A~n Tyr Thr Pbe G l y P r o A S h Gln Thr Cys Met 7317 7344 GTG GAA CAC ATC TTT GAT C2~A AGA TTC TGA GAT ACC AAA A T G TC~ A T G GTG G A A T~p A~n Thr Set Imu Ile Lys ASp Set Glu Ile PrO Lys Cys G l y Trp Trp Ash 7371 7398 CCA AGT AGC T~A TTA CSA TAC TTG TAA ATG GGA AGA A ~ TAA TGT GAC ATT TCA GIn Val Ala Tyr Tyr Asp Thr Cys Lys Trp GiU Glu Ala ASh Val Thr Phe Gin 7425 7452 GTG TCA CAG AAC ACA A,AG TCA ATC AC~ ATC A~"X~ GAT TAG AAC AAT CTC CTC ATG Cys His Arg Thr Gin Set G)n Set Gly Set T~p Ile Arg Thr Ile Ser Ser Trp
8775
8802
G C C T G A AAT AGA TC~% TGA A ~ A A G T A C A C C T A T C AGT G G A A T T GAG GAG AAA TC~ Pro GI~ Ile ASp A s p GIu GI~ V & I His Leu Set Val Glu Leu Arg ArT A~n G l y Met Ala 8829 8856 CAG C ~ A ATG TGG CAT ATC TGA A 2 ~ AGA G G A G G A ATG ATG AAG CAT CTC CGG ACT Arg Gin Cys Gly Ile Set GIu Lys Glu Glu Glu . G l y Ash Val Ala Tyr Leu Lys Dys Arg Arg Ash Asp GIU AIm Set Pro Asp Cys
8888
~&A
GTA AAA TAC AGG AGA GAT GCT GAG CTG ATT TCT TCC CTT TGA Lys Ile Gln Glu Arg Cy~ •
8937 7479 7506 OAA ACA AAG AAA TAG ATG GGA ATG GAG GCC AGA CTT T G A AAG T G A G A A AGT A A A Ly3 Gin Arg A5~ Arg Trp Glu Trp Arg ~ro Asp ?he Glu Set G!u Lys Val Lys 7833 7560 AAT ATC AT? ACA ATG TAA TAG TAC AAA AAA CTT AAC G T T TGC AAT G A G AAG TTC Ile Set I~u Gln Cys Ash Set Thr Lys Asn I~u Thr ?he Ala Met Arg Ser Set
G~GC 8964
R T A T~% ATC CAT TTC A A A T C A A A A TAA C A T TAA AAT CTG ~AT TGC AA A AAC Met Ash Pro Phe Gln Ile Ly3 lle Thr Leu Lys Ser Val Le~ Gin Gly Ly~ Arg 8981 ~018 GTA A G A GAC AAC G~A G A A G A A C ~ A A G A A G A A C ~ C C T TCA AAC ATA TGA TGG CAA Lys Arg Gin Arq Arg Arg A~g Lys Lys Ly8 Ala Phe Lys His He% Set AIa ASh
90q5 7557 7614 AAG TGA TTA T?A TGA TGT ACA AGG A&C ATG GAT AGA ATT TG~ ATG TCA TAG GAA Set ASp Tyr Tyr ASp Val Gin G]y A]8 Trp Ile Giu ~he Gly Cys His Arg Asn 764~ 7668 TAA ATC AAA AAG GCA CTC CGA GGC ~ G ATY TAG AAT A A G ATG TAA ATG GAA TGA Lys Set Lys Arg NiS Ser Glu Ala Arg Phe Arg ~le Arg Cya Lys Trp ASh Glu 7695 7722 AGG A~%A TAA TAT CTC ACT TAT TGA TAC GTG TC~G GAC A ~ TCC A A A TGT CAC ACX~ Gly A=n Ash I!e Se~ L~u lle Asp Thr Cy5 Gly Thr ASh ?to A=n Val Thr Gly 77~9 7776 ACC CAA CCC C~T AGA TTG TAC TAT GAA GCC AAA CAC TAT G T A C A A TTG CTC TTT Ala Asm ?to Val ASp CyS Thr MET Lys Ale A~n Th/ M E T Tyr Ash Cy8 Set L~u 7803 783O ACA AGA TAG TT~ TAC TAT GAA AAT AGA GGA TCT TAY TGT ACA TTT TAA TAT GAC Gln ASp 5er Phe Th~ Met Lys lie Glu As~ Leu ~le Val His Phe Ash Her Thr
8910 C~A TGT GTC Met Cys His
9072
ATC TAG AGA ACC GCT TTA AGA TGC TGT TTG GCA CAC CGT CAA CCA CAG ACG AAA Leu Glu ASh Arg Phe Lys Her Leu Phe Gly Thr Pro Set Thr Thr Asp 01u Thr 9099 9126 CA~ A~G AAA A~kA CAT CTG C ~ i ~ AAA AAA C ~ TG
Vfrology
Arch Virol (1992) 123:29-45
© Springer-Verlag 1992 Printed in Austria
Molecular characterization and heterogeneity of feline immunodeficiency virus isolates N. Maki 1, T. Miyazawa 2, M. Fukasawa ~, A. Hasegawa 1, M. Hayami 3, K. Miki 1, and T. Mikami 2
1Fundamental Research Laboratory, Tonen Corporation, Saitama, 2Department of Veterinary Microbiology, Faculty of Agriculture, The University of Tokyo, Tokyo, and 3Research Center for ImmunodeficiencyVirus, Institute for Virus Research, Kyoto University, Kyoto, Japan Accepted August 15, 1991
Summary. We have molecularly cloned the complete genomic DNA of TM2 strain of feline immunodeficiency virus (FIV) isolated in Japan and compared its nucleotide and the deduced amino acid sequence with those of previously described U.S. isolates, FIV Petaluma and FIV PPR. The infectious molecular clone of FIV TM2 is different from FIV Petaluma in host cell range; the clone can not infect Crandell feline kidney cells which were permissive for FIV Petaluma. The amino acid sequence homologies, in gag, pol, and env genes between FIV TM2 and Petaluma were 90%, 87%, and 81%, respectively. On the other hand, comparative analysis of each gene between FIV Petaluma and PPR showed 96, 95, and 85%, respectively. These results suggested that the genomic diversity was present among FIV strains isolated from geographically distant areas. Interestingly, tat- and rev-like short open reading frames contained inframe stop codons in the FIV Petaluma but not in the FIV TM2.
Introduction Feline immunodeficiency virus (FIV) is a T-lymphotropic lentivirus isolated from a domestic cat with an acquired immunodeficiency syndrome (AIDS)-like disease in the United States in 1986 [45]. Additional isolates have been reported in the United Kingdom [22], the United States [47], Japan [29, 37], and other countries [13, 39]. Seroepidemiological surveys revealed that this virus had spread all over the world [17, 20, 26, 28, 29, 49, 55]. FIV infection in cats is associated with immunodeficiency-like diseases such as lymphoadenopathy, opportunistic infection, stomatitis, anemia, and so on [27-29, 46, 60, 61]. Pathogenic lentiviral infections are well known in other mammalian species
30
N. Maki et al.
(sheep, goat, horse, cattle, monkey, and h u m a n ) and the viruses share similarities in their genomic organization [40]. The nucleotide sequence analyses of h u m a n immunodeficiency virus type I and type 2 (HIV-1, HIV-2) showed genetic variation a m o n g several isolates [2, 4, 7, 8, 12, 14, 23, 62]. These genetic divergences were also d o c u m e n t a t e d in simian immunodeficiency virus (SIV) [30, 31, 33]. In the case of FIV, the total nucleotide sequence has been reported only for two strains t b u n d in the United States (Petaluma and P P R strains) [43, 44, 47, 56], but the extent of genetic variation of F I V is n o t yet clear. Recently, we reported isolation of two Japanese strains (TM t and TM2) of F I V [37]. The F I V T M strains differed biologically from F I V Petaluma. F I V T M strains could infect feline T-lymphocytes, but n o t Crandell feline kidney ( C R F K ) cells that were permissive for replication of F I V Petaluma [61]. In the present study, we have determined the complete nucleotide sequence of F I V T M 2 a n d partial sequence o f F I V T M 1, and c o m p a r e d t h e m with F I V P e t a l u m a and F I V PPR. Materials and methods
Virus isolation and cloning
Fresh peripheral blood mononuclear cells from two FIV-seropositive cats which inhabited Tokyo area were cocultivated with Con-A stimulated peripheral blood mononuclear cells from a specific-pathogen-free cat, and two strains (TM 1 and TM2) of FIV were isolated as described previously [-37]. At the peak of reverse transcriptase (RT) activity, culture supernatants were clarified and stored in aliquots at - 7 0 °C as the primary virus stock. For molecular cloning of FIV TM 2, the primary stock virus was transmitted to a feline CD4-positive lymphoblastoid cell line, MYA-1 [38]. Fourteen days after infection, the FIV-infected MYA-1 cells were cocultured with uninfected MYA-1 cells at the ratio of 1 : 10, and two days after cocultivation, the extrachromosomal DNA was prepared by the Hirt method [-25]. The extrachromosomal DNA was cleaved with Sad restriction endonuclease and selected by size on an agarose gel electrophoresis. The DNA fraction containing 8--10 kilobase (kb) fragments was purified as described by Maniatis et al. [35]. The DNA was ligated to the SacI site of kZAP-II vector (Stratagene), and the recombinant phages (1 x 106) were plated and then screened by the mixture of 32P-labelled synthetic oligonucleotide probes. The sequences of synthetic probes were 5'-AGGAGTCTCTTTGTTGAGGACTTTTGAGTT and 5'-AGTATCTGTGTAATCTTTTTTTACCTGTGAG, which respectively corresponded to the R and U5 regions of the long terminal repeat (LTR) sequence of FIV Petaluma [56]. The probes were synthesized by a phosphoamidite chemistry on an ABI DNA synthesizer. Positive clones obtained were identified by use of the FIV Petaluma probe (p2-FIV, a 2.6 kb Sad fragment that covers the gag and Y-end of the pol regions of FIV Petaluma), kindly supplied by Dr. R. A. Olmsted (Georgetown University, Rockville, MD). The molecular cloning of FIV TM1 was previously reported [36]. DNA sequencing
Fragments of viral DNA from the phage library were subcloned into pUC118 and pUC119 (Takara, Japan), or M13mpl8 and M13mpl9. The DNA fragments were sequenced bidirectionally by dideoxy-chain termination method [50] using either Escherichia coli DNA
Genomic diversity of FIV
31
polymerase Klenow Fragment or T7 DNA potymerase (Sequenase, United States Biochemical). The nucleotide and the deduced amino acid sequences were compared with those of the three other reported sequences of strain TM1 [37], Petaluma [56] and PPR [-47] by the Interigenetics Bion software. The complete FIV TM2 sequence (Fig. 2) has been deposited in the GenBank data base (accession no. M59418).
DNA transfection The full length plasmid clone (pFIVTM2) was reconstituted to produce proviral form (one LTR on both sides of its genome) by the same strategy as described previously [36], and designated as pTM229. 5 gg of the pTM229 was transfected into CRFK cells [9] by the calcium phosphate coprecipitation method [ 19, 59] and the virus production was monitored by Mg2+-dependent RT activity [41] and indirect immunofluorescence assay [37]. Results
Molecular cloning of FIV TM2 A r e c o m b i n a n t )~ZAP-II phage library constructed f r o m e x t r a c h r o m o s o m a l D N A isolated from the F I V TM2-infected MYA-1 cells was screened with synthetic oligonucleotides of the L T R region as probes. Five positive clones were identified and analyzed by restriction endonuclease digestion and Southern blot hybridization with the p 2 - F I V as a probe (data n o t shown). The clone p F I V T M 2 appeared to contain the full length genome of F I V T M 2 and was selected for further characterization.
Nucleotide sequence and genomic organization The restriction endonuclease patterns of the F I V T M strains were quite different f r o m that of F I V Petaluma. Figure 1 shows the restriction m a p of F I V T M 2 in c o m p a r i s o n with those of F I V TM1 [36] and Petaluma [56]. Of the 25 0
1
2
3
4
5
6
7
8
1
I
t
I
t
1
1
I
l
BHHS E
A'V~,I
H
t
tA I'
NP
NP H S
H
I
lJi, H
PXX X E B
NP PP P LTR g EEl I, a g j I
E
I- LI i
PXX X
BHHS E
B~I
Bg
P P Bg
E H
11
F
P B NX pol
H
BgEV
L BHH
~ IF
I'~
X P BgEV
J I/ × BH Bg X vif A t L,,,J I I
9 Kb
XN
Fig. 1. Comparison of restriction map of
BHH
FIV TM2 (A) with those of TM1 [36] (B) and Petaluma (C). Restriction map of FIV Petaluma was generated from the published nucleotide sequence [-56]. B BamHI; Bg BglII; E EcoRI; EV EcoRV; H HindIII; N NheI; P PstI; S SacI; X XbaI
XN H
env
N LTR r'-I ~B
Fig. 2. Complete nucleotide sequence of FIV TM2 viral DNA genome. The sequence is numbered from the beginning of the U3 region. Underlined is the primer binding site (PBS) complementary to the Y-end of tRNA Ly~and polypurine tract (PPT). Two putative spliceacceptor (SA) sites for orf B are indicated by vertical lines
27 54 T G G G A A C~%T T A T T G G G A T C C T G A A G A ~ A T A G A G A A A A T G C T G A T G G A C T G A G G G U3 81 108 CC-C A C A T A A A C A AGT G A C A G A T G A A A A A C A G C T G A A T A T G A G T C A G A G T T A A A T 135 G C T A G C A G C T G C T T A ACC G C A A A A C C A C A T C C T A T G
TAA AGC
189 TGT A T C T T G C T C C A T T G T A A G A G T A T A T A A C C A G T G T T T
AGT CTC TCT GTT GAG C~
243 TTT CGA GTT CTC CCT
TGA GG~
162 TTG C C G A T G A C G
218 TTT AAA GCT TCG AGG 270 TCC CAC AGA TAC AAT
287 324 A A K A A A T T G A G T TTT G A G ATT G A A C C C T G T C G T G T A T C T G T G T A A TTT C T C T T A
U5 381 378 C C T GCG A A T CCC TGG AG'~ CCG G G C C A G G G A C C T CTX~ A G T TC~G CGC CCC. AAC A G G PBS 405 432 G A C T T G A A A A A G A G T G A T T A G G G A A G T G A A C ~ T A G A C C A A T A G A A A G C T G T C.%A 459 GAA CTC CTG CAG ACC TTG TGT GGG GAG CAG TTG CAG
406 ACG CTG CTG GCA G~G
513 AGT ATC TCT A G T G G A G C A G A C C T G AGC T C T G G A T T A A G T
840 C A C TAT TCG C A G GCC
C~
567 594 TAG A T A A A G A T T ATC T G O T G A C T C TTC G C G G A T C G T C A A G C C A G G A G A TTC G T C 621 GC~ GGA CAG CCA ACA AGG TAG GAG AGA TTC TAC A~C AAC
648 ATG ~ AAT GOA CAG M e t G l y ASh G l y G i n gag 675 702 G G G C G A G A C TGG A A A A T G G C C ATT A A G A G A TGT A G T A A T G T T GCT G T A G G A G T A G l y A r ~ A s p T r p Lys M e t Ala Ile Ly8 A r g Cys S e t A S h Val Ala Val G I ~ Val 729 756 AGT AAG AGT AAA AGA TTC GGA GAA GGA ~AC TTT AGA TGG GCC ATA AGG ATG G l y S e r L y s S e t Lys A r g Phe G l y G Z U G l y A s h P h e A r g T r p Ala Ile A r q Met
GCT Ala
783 AAT GTA ACT ACA GGA CGA GAA CCA GGT GAT ATA CCA GAG Ash V a l T h r Thr Gly Arg Glu Pro G l y A s p Zle P r o G l u
T T A A G A T C A A T T ATT TGT GAT Leu A r g Ser Ile Ile Cys Asp
810 ACT T T A G A A C A G Thr Leu $1U Gln
837 864 TTA C A A G A C A G A A G A G A A C A C TAT G G A TCT AGT Leu G l n A s p A r g A r g G l u His Tyr Gly Set Set
891 918 A A G G A A A T T G A T A T G G C A ATT A C C A C T T T A A A A G T T T T T G C A G T G G C A G G A A T T Lys G l u Ile A s p M e t Ala Ile T h r T h r L e u Lys V a l P h e Ala V a l Ala G l y Ile 945 992 C T A A A T A T G A C T G T A TCT A C ~ C'CC A C A G C A C ~ T G ~ A A T A T @ T A T C~T C A G A T G LeU A S h M e t Thr Val Set T h r AI8 T h r A l a Ala GIU A S h M e t T y r A l a G l n M e t 999 i026 G G A T T A G A C ACC A G A C C A T C T G T A A A A G A A A G T G G G G G A A A A G A A G A A G G A C C T G l y L e u A ~ p Thr A r g P r o Set V a l L y s G l u S e t ~ i ~ G l y L y s G l u G i u G l y P r o 1053 C C A CAG G C T ~ A T C C T ATT C A A A C A G T A A A T G G A G C A C C A C A G P r o G l n A l a T y r ~ r o lle G l n T h r V a l ASh G 1 x Ala ~ r o G l n
1000 T~T GTA GCC CTT Tyr Val Ala Leu
1187 G A Y C C A A A A A T G G T G TCC A T T T T T A T G G A G A A G G(IA A G A G A G A = p P r o Lys Met Val S e t ~le Phe M e t G l u Ly8 A I a A r g G l e
1134 GGG CTA GGA GOT G l y Leu G l y G l y
1188 1161 G A G G A G G T C Co~A C T G ~ TTC ACA GCC TTT TCA GCT AAT TTA A~% TCA AC~ GAT G l u G I ~ V a l G l n Leu T r p P h e T h r A l e P h e 8 e r A l a A S h L e u T h r S e t T h r A s p 1213 ATG GCT ACA TTA ATT ATG TCC GCA CCT GGC TGT GCA GCA GAT Me% A l a T h r Leu Ile M e t S e t Ala P r o G l y Cys A l a A l a A s p
1242 AAA GAA ATC CTA Lys G l u Ile L e u
1296 1269 GAT G A A A C A C T G A A A C A G A T G A C A ~CT GAG TAT G A ~ C G T A C C C A T C C T C C T G A T A s p Glu Thr Leu Lys G l n M e t Thr A l a G l u T y r A S p A r g T h r H i s P r o P r o A s p 1323 1350 C~ C C T A G A C C G C T G C C C T A T T T C A C T C~CC C-CA G A G A T T A T G G G G A T A G G A T T A G l y P r o A r g Pro Leu Pro T y r P h e T h r Ala A l a G l u X l e Met G l y Ile G l y L e u 1377 A C T C A A G A A C A A C A A G C A G A G C C C A G G TTT G C A C C A G C C A G A Thr G l n Glu Gln Gln Ala G I u Pro A r g Phe A l a Pmo A l a A r g
ATG Met
1404 C A G TGT A G A Gln Cys A r g
1431 1458 G C A TGG T A C C T T G ~ G C A T T A G G A ~ A G TTG GCG C~C ATA AAA C,-CC A.a2.. TCT CCC A l a T r p T y r l a u Glu AI~ L e u G l y L y s Leu A l a A l a lle L y s A l a Lys S e t P r o 1485 1512 GAC TAT TCCTCATTT ATA GAT CGA GCA GTA CAA TTG AAG CAG GGA GCT AAAGAG A r g A l a Val Gln Leu LyS G l n G l y Ala Ly~ G I u A s p T y r S e t S e r Phe Ile A s p 1539 1566 A G A C T A TTT G C T C A A A T A G A T C A A G A G C A G A A C A C A G C T G A A G T A A A G C T G T A T A r g Leu Phe Ala Gln Ile A s p Gln Glu G l n A s h Thr Ala G l u V a l Lys Le~ T y r 1593 1820 T T A ZukA C A ~ T C T T T A AGT A T A G C C ~ A T G C T A A T C C A G A T T G T ~ AGA GCA ATG LeU LyS G l n Set L~U Set Ile A l a A S h Ala A s h P r o A S p C y s L y s A r g Ala M e t 1887 J634 AGT CAT C T T AAA CCA GAA A G T ACT TTA G A G G A A A A A C T G A G G G C C T G C C A A G A A Set His Leu Lys Pro Glu S e t Thr Leu GIu GIu LyS Leu A r g A l a Cys Gln G l u
1701 G T A G G A T C A C C A G G A T A T A A A A T G C A A T T G CTG G C G G A A G C T C T C V a l G l y S e z P r o G 1 y T y r Lys Met G l n Leu Leu A l a G l u A l a Leu
1728 ACA AGG GTT Thr Arq Val
1753 !782 C A A A C A C~TT C A A A C A A A A G G A C C A A G G C T A G T G T G T T T C A A T T G T A A A A A A C C A G l n T h r V a l G i n T h r Lys G l y P r o A r g Leu Val C N S P h e A S h C y s Lys Lys Pro 1809 C A C C T G G C T A G A C A A TGT A A A G A ~ G C G A A G A G A TGT A A T A A C G l y His L e u A l a A r g G i n Cys Lys G l u A l a Lys A r g Cy8 A S h A S h
1836 TGT ~A AAA Cys G l y Lys
1863 1890 TTA GCT GCT AAT TGC TGG CAA GGA GGT AGG A~A ACC TCG GGA AAC L ~ u Ala A l a A e n C y s T r p G l n G l y G l y A r g Lys T h r S e t G l y A s h G l u A S h L e ~ G l y Lys A r g pol 1917 1944 GAG AAG GTG GGG CGA GCT GCA GCC CCA GTA AAC CAA GTG CAG CAA ATA STA CCA G I u Lys V a l G l y A r g A l a Ala Ala P r o Val A S h G l n V a l G l n G l n ~le V a l Pro G l u G l y G l y A l a S e t Cys Set P r o Ser Lys Pro Ser A l a A l a A S h Set Thr Ile CCT GGT CAC Pro Gly Mis
1971 1998 TCT GCA CCT CCA ATG GAG GAG AAA CTA TTA GAT TTA TAA ACT ATA ATA CAA TAG S e t A l a P r ~ P r o M e t G l u G l u Lys L e U Leu A s p I~u C y s T h r S e t A S h G l y G l y G l u T h r Ile A r q P h e 31e A S h T y r A S h Th= [le G l y 2025 2052 G T A C T A C C A C A A C T T TGG A A A G A A G A C C T G A A A T A C A A A T T T T T G T A A A T G G A C T h ~ T h r T h r T h r Leu G I U A r g A r g Pro G l u Ile G l n Ile P h e v a l A s h G l y His 2079 2106 A C C C T A T A A A A T T T T TAT TAG A T A C A G G A G C A G A T A T A A C A A T T T T A A A T A G A A P r o lle Ly$ P h e L e u L e u A s p T h r G l y A l a A s p lle T b r Ile L e u A s n A r q Lys 2133 2160 ~%G ATT TTC ~m~ TAG GAA ATT CTA TAG AAA ATG GGA AAC AGA ATA TGA TTG GAG A S p P h e G l n Ile G l y A s h Ser Ile G l u A s h G l y Lys G I D A S h M e t lle G i y Val 2187 2214 T A G G A G G A G G A A A A A G A G GAA C T A ATT A T A TCA A T G T A C A T C T A G A A A T T A G A G G l y G l y G l y Lys A r g Gly Thr A s n T y r Ile ASP V a l His Leu G l u Ile A ~ g A s p 2241 2268 ATG AAA ATT ATA GGA TGC AAT GTA TAT TTG GAA ATG TGT GTG TTC TAG AAG ATA G I U A s h T y r A r g M e t G i n Cys Ile Phe G l y A S h V a l C y s V a l L e u G i U A s p A s h 2295 2322 A T T C C T T A A T C C A A C C A T TAT TAG G A A G A G A T A A C A T G A T T A A G T T T A ATA T A A S e t Leu Ile G l n P r o LeU Leu G l y A r g A s p ASh Met Ile Lys P h e ASh Ile Arg 2349 2376 G A T T G G T A A T G G C T C A A A TTT CAG A A A AGA TTC C A A TAG T A A A A G T G A G Q A T G A Leu V a l M e t A l a Gln Ile Set G l u Lys Ile P r o Ile Val Lys V a l A r g Met Lys 2403 2430 A G G A C C C T A C T C ~uAG G G C C T C AGG T A A A A C A G T G G C C A T T A T C~-A A T G A~& A A A A s p P r o T h r G l n G I y Pro G l n V a l Lys G l n T r p Pro L e u S e r Asm Glu Lys Ile 2457 2484 T T G A A G C T T T A A C A G A C A TAG T A G A A A G A T TAG A A T C A G A A G G ~ A ~ K G TGA A A A GIU A l a Leu Thr A s p Ile V a l G I u A r g Leu G I u S e t G I U G l y Lys Val Lys A r g 2511 2538 GGG CTG ACC CGA ATA ATC CTT GGA ATA CCC CAG TAT TTG CTA TTA AGA AGA AGA A l a A s p P r o A S h A S h P r o ~ r p A s h T h r P r o V a l P h e A l a lle L y s Lys Ly~ S e t 2505 2592 GTG GTA AAT GGA GGA TGC TCA TAG ATT TTA GGG TCC TAA ATA AAT TAA CAG ACA G1y L y s T r p A r g M e ~ Leu lle A s p P h e A r g Val L e u A S h L y s L e u T h r A s p Lys 2619 2646 AAG ~AG CAG AAG TCC AGT TAG GAC TTC CTC ATC CTG CTG GAT TAC AAA TGA AAA G l y A l a G l u V a l G l n Leu G l y L e u P r o His P r o Ala G l y L e u G l n Met Lys Lys 2673 2700 A A C A A G T A A C G G T G T T G G A T A T A G C~=G A T G C A T A T T T T A C C A T T C C T C T A G A T C G l n V a l T h r V a l L~U A s p Ile O l y A s p A l ~ T y r P h e T h r I l e P r o Leu A s p P r o 2727 2754 CAG ATT ATG CTC CTT ATA CTG CAT TTA CAT TAG CTA GGA AAA ATA ATG CAG GGC A s p T y r A l a Pro Tyr T h r A l a Phe T h r L e u Pro A r g Lys A s n A S h Ala G l y P r o 2781 2808 C A G G G A G G A G A T A T G T A T G G T G C A G T T TAC C A C A G G G G T G G G T T T T A A GTC C A T Gly A r g A r g Tyr V a l Trp Cys S e r Leu P r o G l n G l y T r p V a l L e u Ser P r o Leu 2835 2882 T A A ~ A T A T C A G A G T A C C T T ~ % A C A A T A T A T T A C AC~C C A T T T A T C A A A C A A ~ A T T Ile T y r G l n S e r T h r L e u A S h A S h Ile Leu G l n P r o P h e I l e Ly$ Gln A S h S e t 2889 8916 C T G AGT T A G A T A TTT A C C AAT A T A T G G A T G A T A TAT A T A T A G G A T C A A ATT T A A G I U Leu A s p Ile Tyr Gln T y r M e t A S p Asp Ile T y r Ile G l y $er Ash ~ e u A S h 2943 2970 ATA AAA AAG AAC aTa AAC AAA AAG TAG AAG AAT TAA GAA AAT TGT TAT TAT C~T Lys L y s G I u His Dys G l n Lys V a l G l u G l u L e u A r g Ly8 L e u L e u L e u T x p T r p 2997 3024 GGG GAT TTG AAA CCC CGG AAG ACA ~AT TAC AAG AAG AGC CCC CAT ATA AGT GGA G l y Phe G l u T h r Pro G l u A s p Lys Leu Gln GIU G l u Pro Pro T y r Lys T r p M e t 3051 3078 T G G GCT A T G A A T TAC ATC C A T T A A C A T G G T C A A TAC A G C A A A A A C A A T TAG A A A Gly T y r G i u Leu His Pro Leu Thr T r p Set Ile G l n G l n Lys G l n Leu G l u Ile 3105 3132 T T C C A G ~ A A G A C C C A C A T T A A A T G A2AC T A C AGA A A T T A G C A O G T A A A A T ~ % A C T P=o G l u A r q P r o T h r Leu A s h G l u L~u G I D LyS i e u Ale G l y Ly~ Ile A s P T~I 3159 8186 GGG C C A G T C A G A C T A TCC C A G A C T T A A GTA T A A AAG A A C T A A C T A ACA TGA T G A Ala Set GIn Thr Ile Pro A S p Leu Ser Ile LyS Glu Leu T h r A S h Met Met A~g 3213 32q0 G A G G A G A T C A G A A A T T A G A C T C A A T A A G A G AAT G G A C T G T A G A A G C C A A G A G A G G l y A S p O l n Lys LeU A S p Set Ile A r g GIu T r p Thr V a l G l u A l ~ LyS Arg Glu
3267 3294 RAG TAC AAA RAG CTA AGG AGG CTA TTG AGA CGC AAG CAC XGC TRA ATT ATT ATG Val Gln Ly~ Ala Lys G~U Ala Ile Glu Thr GI. Ala Gln Leu ASh Tyr THr Asp
4833 4860 TGG GAA TAR AAC ATA AAT TGG GTA TAC CAG GTA ACC CAC AAT CAC AAG CAT TGG Gly Ile Lys His Lys Leu Gly lie Pro Gly ASh Pro Gin Set Gln Ala Leu Val
3321 3348 A T C C C A ATC GAG CAT TAT A T G C A A AKT T G A GTT T A G T G G G A C C A C ATC AAA TAT Pro AS. Arg Gly Leu Tyr Ala Lys Leu S~r Leu Val G l y Pro HIS Gln Ile C~8
4007 4914 TGG A A A ATG C T A ATA ACA CAT TGA AA¢ TTT G G A TTC AGA AAT TTC TAC C A G A S A GIu ASh AI. Ash Ash Th~ LeU Lys Val Trp Ile Gln Lys ~he Leu Pro GIu Thr
3375 3402 GTT ATC AAG TGT ATC AAA AGA ACC CAG ARC ACA TTT TAT GGT A T G GTA AGA TRA Ty~ Gln Val Tyr Gln Ly~ Asn Pro Glu His ~le Leu Trp THr Gly Lys lle ASn
C T A CTT CTC TGG ATA ATG CTT TGG CCC TAG CCC TGC ATT GTC TCA ATT TTA AKC
3429
3450
A T A GAC AAA AGA AAA AAG CAG AAA A T A C C T ~ T ~ ATA TAG C T C T A A GG~ CAT G T ~
A r E Gln Lys Lys Lys Ala Glu ASh Thr Cys A s p Ile ~ l a Leu Arg AI~ Cys Tyr 3463
3510
4941
4968
Thr S~r Leu Asp ASh Ala Leu Ala Leu Ala Leu His Cys Leu ASh Phe Lys Gln 4995 5022 GGG GTA GAC TAG G G A GGA TGG CCC CTT ATG AAT TAT ACA TAC ~KC AAG AAT A r g G l y A r g Leu Gly Arg Met AI~ Pro Tyr Glu Leu Tyr Ile GI~ Gln Glu Set
A T A A A A TAA GAG AAG AGT CTA TTA T A A G A A TAG C~%A A A G RAC CAG TAT ATG A A ~ Lys Ile Arg Glu Glu Set Ile Ile Arg 21e GIH Lys G l u Pro Val Tyr Glu Ile
5049 5076 CAT TAA GAK TAC AKG ACT ATT TTT CCG CGA TTC CAC AAA AGT TAA TGA TGC RAT Leu A r g Ile Gln Asp Tyr Phe Set Ala Ile P~O Gln 5ys Leu Met Met Gln Trp
3537 3564 TAC CTG C A T CCA GAG AGG CCT ~GG RAT C A K ATC TAA T T A G A T C T C CAT ACC T T A P r o Ala Ser Arg Glu Ala Trp Glu Set A s h I~u Xle A r g S e t Pro T~r Leu ~ys
5103 5130 GGT TGT ATT ACA RAG ATC AAA AAG ATA AAA AAT C ~ A AGG GAC CAA TGA GAG TGG Leu Tyr Tyr LyE A~p Gln LyH Asp Lys LyS Trp Lys Gly Pro Met A r g Val Glu
3591 3618 AGG ~ CAC CAC CTG AC~ TAG RAT TTA TAC A T G C A G CCT T A A G T A TRA A~A GGG Ala Pro Pro Pro Glu Val Glu Phe ~le His Ala Ala Leu Set Ile Lys Arg Ala
5157 5184 AAT ATT C4~G GAC AAG GAT CAG TAT TAT T A A AGG ATG AAG AGA AGG GAA TAT TTC Tyr Trp G1 H Gln GIy Set Val Leu Leu Lys ASp Glu Glu Lys Gly Ile Phe Leu
3645 3672 CTC TAA GCA TGA TAC AAG ATG CCC CTA TRA TAG GAG C A G A K A CAT C4~T A T A TAG Leu Set Met Ile Gln Asp Ala Pro Ile lle Gly Ala Glu T h r Trp Tyr Ile ASp
5211 5238 TTG TAC CTA C4~A GAC ACA TAA GAA GAG TCC CAG AAC OCT GCA CTC TTC CTG AAG Val Pro Arg Arg H£s Ile Arg Arg Val Pro Glu Pro CHS Thr Leu Pro GIU GIy
3699 3726 A T G C ~ A GTA ~AA AAC ~A~ GAA AAG C A G CAA G A G C A G CCT ATT G~A CAA A T A CAG GIM Set Arg Lys Gln Gly Lys Ala Ala Arg Ala Ala THr Trp Thr ASh Thr Gly 3753 3780 RAT GGC AGA TAA T ~ #~3L%TAG RAG GAA GTA ATC AAA RAG C A G # ~ TAC AGG Ly$ Trp Gln Ile Met GIu Ile Glu 01 y Set ASh Gln Lys Ala Glu Wal ~ln Ala 3007 3834 CTT TAT TAT TGG CCT TAA AAG CAG OAT CAG AAG ~ T G A ATA TCA TAA CAG ATT Leu Leu Leu Ala Le~ Lys Ala Gly Ser GIu Glu Met A S h Ile lle Thr Asp Set 3861 3805 CAC RAT ATA TTT TGA ATA TTC TCA ACC AAC AAC CAG ATT TGA TGG AAG GAC TAT Gln Tyr Ile Leo ASh Ile Leu Ash Gln Gln Pro Asp Leu Met Glu G1 H Leu Trp
5265
5292
A T G A G T G A C GAA GAT TC~ CAG GTA AGT A G A AGA CTC TTT C4~A GTT CTC CAR
Met Set Asp Glu Asp Trp Gln Val Set Arg Arg Leu Phe Ala Val LeU Gln ASp GIU wlf 5319 5346 GGA GGA G T A TAC AGT GCC ATG TTG TAC ATA TCG AGT CTA CCT GAA ATG GAA CAG G l y 01 H Val Tyr Set Ala MET ~eu Tyr Ile Set Set Leu Pro Glu M~t Glu Gln 5373
5400
GAT RAA ~ T ARA AGG AGC TTT ~AG ~AA AGG CTT TCG GAa AAG GAA ACa GGA TTC ASp LHS C~S Lys Azg Set Phe LHs LHS Acg Leu Set GIu LHS GI. Thr G1 H Phe 5427
5454
A T & TTT AGA TTA AGA AAA GCT GAA GGA ATA AGG TGG AGC TTC CAT ACC AGa GAT
Ile Phe Arg Leu Arg Lys Ala Glu Gly IJe Arg TZp Set Phe His Thr Arg Asp 3915 3942 G G C A~G AC~ TCT TAG RAC AA~ TC-G AAA ~ G A AAA TAG C A A T T T T T A TAG ACT ( ~ Gln Glu v~l Le~ GlU Gln Met Glu by~ Lys Ile AI~ Ile Phe ~le Asp Trp V~l
5481
5506
T R T TAT A T A GGA TAT GTA AGA GAG ATG GTG GCC GGA TCT AGC C T A CCA GAT RGT
Tyr THr Ile Gly Tyr Val Arq Glu Met Val Ala Gly Set Ser Leu Pro Asp Set 3969 3996 TTC CTG GGC ATA AAG GTA TAC CAG G A K ATG AAG RAG T A G A T A AAC TTT GCC A ~ A P~O Gly His LHS Gly Ile Pro Gly ASh GI~ Glu Val A s p Lys LeU CHS Gln Thr
5535
5562
T T A AGA TTG TAT GTT TAT ATA AGC RAT CCA TTG TGG CAT CAA TCA TAC CGT CCT
Le. Arg Leu Tyr Val Tyr Ile ~er ASh Pro Leu Trp His Gln Set THZ Arg Pro 4023 4050 C A A T G A TGA TTA TAG RAG GTG AGG GGA TAT TAG AG~ AAA GAT CAG ARG ATG CAG M e t Met Ile Ile GIu Gly ~lu G l y Ile Leu Glu Lys A r g 5er GIU Asp Ala Gly 4077 4104 GAT A T ~ ACT TAT TAG CTG CAG CAC ~-KG A~A CAC ATT TCT T~C CT~ GAG AGG TA/% Ty~ ASp Leu Leu Ala Ala Ala Gln Glu Thr His Phe Leu Pro Gly Glu Val Arg 4131 4158 G A A T A G TAC CAA CAK AAA CKA GAA TAA TGC TAC CAA AAG GAC ACT GGG GAC TAA Ile Val Pro T~r Lys Thr Arg Xle Me~ L~u Pro L~S G l y His Trp G I ~ Leu Ile 41~5 4212 TAA TGG C~%A AAA GCT CAR TAG GAR GTA AAG GGG TGG AT~ TAT TAG GAG GAG T T A Me~ Gly Lys 5er Set lle Gly Set LHs Gly Val Asp Val Leu GI~ Gly Val Ile 4239 4266 TAG A T G AAG CAT ATA GAG GAG AAT TAG GAG TGA TTA TGA TTA ATT TAA CAA AKA A S p GIU Gly Tyr Arg Gly G l u L~u GI~ val lle Met Ile ASh Leu Thr Lys Lys 4293 4320 AAT C A K TAA CTA TAT TAG A A A AGC A~A AAA TAG CAC A A T TGA T R A TAT TC42 C T T Set Ile Thr Ile Leu GIU Lys Gln Lys ~le Ala Gln Leu Ile ~le L~u Pro Cys 4347 4374 GTA GAC ACG AAG GCC TAC AAC RAG GAG AAA TAC AAA TGA ATT CAG AAA GAG GAG Arg His Glu Gly Leu Gln Gln G1y Glu Ile Gln Met ASh Set Glu Arg G1 y Glu 4401 442~ A A A RAG GAT TC~ GAT CAG CAG GA~ TT~ T~T CAT CAT G G G TAG A T A ~ A A TTG RAG Lys Gly Ph~ Gly S~r Ala G1y V~l Phe Set S~r Trp V a l Asp Arg lle Glu Glu
3589 5616 GGC CTG A C A AAT TTT AAT ACA ~ TGG CCT TTT GTA AAT ATG TGG ATA AAG A C A G l y Leu T h r Ash Phe Ash Thr Glu Trp Pro Phe Val ASh Met Txp Ile Lys ThE 5643 5670 GGA TTT ATG TGG CAT CAT ATT GAA AGC CAG AAT ATT TGC AAA GGA GGA G A G ATT G l y Phe Met Trp ASp ASp Zle Glu Set Gln ASh Ile Cys Lys Giy Gly Glu Ile 5697 5724 T C A CAT G G A TGG GGA CCT G G A ATG GTG C~SA ATT GTG A T A A A A G C A TTT AGT TGT Set His Gly Trp Gly Pro Gly Met Val G1y Ile Val Ile Lys Ala Phe Set Cys 5751 5770 G G A G A A AGG A A G ATA A/~ ATT ACT CCT GTT ATG ATT ATA A G A GGT GAA ATA GAC Gly Glu Arg LHs Ile LHS Ile Thr Pro Val Met Ile Xle Arg Gly GIu Ile ASp 5805 5832 CCC ACA G A A TGG TGT GGA GAT TGT TGG AAT TTG ATG TGT CTT ~.% TAT TCA CCT Pro Thr G I g Trp Cys Gly Asp Cys Trp ASh Leu Met Cy~ Leu Lye Tyr Set Pro 5059 5806 C C A A~T ACA TTG CAG AGG CTT C ~ ATG CTG GCG TGT G~C AAA GAG GCT AAA GAA Pro ASh Thr LeU Gln Arg LeU Ala Met Leu Ala Cys Gly Ly3 Glu Ala Ly~ Glu 5913 5948 TGG AGA GC4~ TGT TGT AAT CAG CGT TTT GTT TCT CCT TTC AGA ACA CCA TGT G A T Trp A r g G I M Cys Cys ASh Gin Arg Phe Val Set Pro Phe Arg Thr Pro CyS A s p 5967
5994
C T A G A G G T C G T C CAG AAC RAG CCT AAA AGG ~AT TTA TTG TOG ACG C.~A G A A T T A
LeU Glu Wal Val Gln ASh Lys Pro LHs Arg Asn Leu Leu Trp Thr. 01 H GIu Leu 4455 4482 AAG CAG AGT TGA ATC ATG ARK AAT TTC ATT CAG ACC CAC AAT ATT TAK GGA CGG Ala 01~ Leu ASh His GIu Lys Phe His Set ASp Pro GIn Tyr Leu Arg Thr Glu 4509 4536 AGT TCA ATC TAC CTA GAA TAG TAG CAG ~2~G AAA TAA A2~% GAA A A T GTC CCC TGT Phe ASh Leu Pro Arg ~le Val Ala GI~ Glu ~le Ly~ ~r~ Lys Cys Pro Leu Cys 4563 4590 GCA GAA TTA GAG GCG AAC RAG TAG GGG CAC AAT TAA AGA TTC GGC CTG GAA TAT At0' Ile Arg Gly 01~ Gin Val Gly Gly Gln Leu Lys Ile Arg Pro Gly Ile Trp
6021 6048 TGA AT G G A A G A A ATA ATC CCA CTG TTT RAT AAG GTT ACA GAA AAG TTA GAT AGA Met G l u Glu Ile Ile Pro Leu Phe ASh Lys Val Thr Glu Lys Leu Asp A r g ORF -A 6073 6102 G A K C C A G C T A T T AC.A TTA TTT ATC TTG C~CT TAT CAG GT~ GAC ACA TAC A G A T T T G l u Pro Ala Ile Arg Leu Phe Ile ieu ala Tyr Gln Val ASp Arg TH~ Arg Phe 6129 Ile Arg Ii~ Leu Gln Leu Leu L~U Trp Arg Asp A ~
4617 4644 C~oC A A A TGG ATT GCA CAC ATT TTA ATG G~A AAA TAA T C A TTG TAG C A G TGC ATG GI~ Met Asp Cy~ Tbr 31s Pbe Ash Gly Lys ~le Ile Ile Val Ala Val ~ s Val 4671 4690 TGG AAT CAG GAT TTT TAT GGG CAC AGA TAA TTC CAC AGG AGA CTG CAG ~TT G T A Glu Set Gly Phe Leu Trp Ala Gln Ile Ile Pro Gln Glu Thr Ala ASp Cys Thr
6156
A T T A G A ATT TTA CAA TTA TTA CTT TGG AGA GAT AGA TTT AAG TCA ATT AKT T C T
Phe Lys Set Ile ASh Set
6383 6210 •=92~ TAT TGT TTA TGC TGG CTG T~C,TC~ AAG TCT C~T TAT TC~ C ~ T~G C39A TCT L ~ Tyr Cys Le~ Cys Trp Le~ Cys Cys Lys Set AI~ Ty~ Trp Arq Leu Gln $~r Met Leu Ala VaI Leu Gln val Cys Leu Leu Ala Leu Ala Ile T y r 6237
6264
A C A TTA TCC ATA AAT ACT GCC TAG AAA TAT TTC TTT TAA TAT TTC ATC TC~ AGA
4725 4752 CAG TCA AAG CTC TTT TAC AAC TTA TAT GTG CTC ATA ATG TTA CAG AAT TAC AAA Val Lys Ala Leu Leu Gln Leu ~le Cys Ala His ASh Val Thr Glu Leu Gln Thr 47~ 4006 CAG A T A ATG GAC CAA ATT TTA AAA ATC AGA AAA TGG R A G GAT TAT TAA ATT ATK Asp ASh Gly Pro Asn Phe LHs ASh Gln Lys M ~ Glu Gly Leu Leu Ash THr Met
Thr Leu $~r ~le A~n Thr AI~ II~ Ile Mls LHs Tyr Cys Leu GI~ Ile Phe Le~ beu ~le Phe His beu 01~ Ile 6291 6310 TAT AAA CAT GGC AGA C ~ GGG ATT TAC TCA TAA TCA ACA ATG GAT AGG ACC A G A Met Ala GI~ Gly Gly Phe Th= Bi3 ASh Gln Gln Trp Xle Gly Pro Glu
6345 6372 GC^ aOC 7GA ^(;a ^T7 GT7 C~,A TT~ TGA TAT ^~C ~O~ ^C^ AAT G^A TGA AG^ AGG Glu AI~ GIu GIu l.eu L~U Asp Phe A=p Ile Ala Val GIn Her Ash Glu Glu Gly 6399 6~26 TCC A~T AAA C C C AC~ AGT AAA CCC ATT TAG C~T ACe A C ~ C A T TAC T&~C ACA AGA ~ro I ~ u A~n Pro Gly Va). Ash Pro Fhe ArT V~I Pro G l y Ile T b r Set Gin Glu 6453 6480 AAA GGA TGA TTA T';G TAA GAT CTT ACA AAC ~ ACT A C A AGA A T T A A A G A A TGA Lys Asp A=p Tyr Cy~ Lys Ile Leu Gln Thr Lys Leu Gln G l u Leu Lys Ash Glu
fi534 6507 AGT CAA AGA GGT AAA AAT TGA AGA AGG AAA TGC AC~ T A A GTT TAG AAG GC-C AAG Val Lys GIu Val Lys Ile Glu Glu Gly Ash Ala G l y Lys Phe Arg A r g Ala Arg
7657 7884 b\A AGe TGT G ~ ATT OTA T~A TAT TGC TO0 A ~ TTG GTC ~rG CAC ATC GGA 7TT tys Ala Val Glu Leu Ty[ A~,I Ile A!a Gly A~n Trp Ser CyS Thr Ser Asp Leu 7911 7998 ACC KAA A & G ATG G G G ATA TAT G A A C T G T ~ TTG TAC AAA TGG GAC TGA TAA TAG P r o LyS G l y Trp G l y Tyr Her Ash Cys Ash Cy~ T h r Ash gly Thr A~p Ash Set 7965 7992 T G A A A C AAA AAT O G C ATG CCC TAK G A A C C A C ~ TAT TTT AAG A~, TTG GTA CAA GIU T h r Lys Met Ala Cys Pro Lya A = n Gln O l y Ile Leu ArT A~n T~p Tyr Asn 8019 8048 CCC AGT CC-C AGG GTT AAG ACA C ~ 8TT AAT A A A A T A T C A AGT A G T AAA ACA GCC Pro Val Ala Gly Leu Arg Gin Ala Leu Ile Lys Tyr Gln V~l Val Lys Gln Pro
6561 6589 ATA TTT AAG ATA TTC TGA TGA AAA TGT GCT ATC CAT A G T C T A TTT GCT AAT AC~ Tyr Leu Ar9 Tyr 8e# A~p Glu ASh Val Leu 5er ~le Val T y r Leu Leu Ile Gly
8073 8100 RGA G T A TTT GAT AGT GOC ¢~3A A G A A G T TAT GCA G T A T A A ATT C A A ACA AAA GAG Glu Tyr L~u Ile Val Pro glu Glu Val Met Gln T y r Ly~ Phe LyS Gln Lys Arg
6615 6842 A T A TCT AAG ATA TT~ AAT AGA TCA TAG G A G TTT A C ~ A T C T T T G A G A C A T G A TAT Tyr Leu Arg Tyr ~eu ~le ASp ~ s Arg Set Leu G l y Set Le~ A r g His A3p Ile
8127 8154 A G C A G C T A T TCA TAT TAT GTT A G C T C T TGC G A C AGT G T T ATC TAT C43C TGG A C ~ Ala Ala Tle His Ile Met ~eu Al~ Leu ala T h r v a l Leu Set Met Ala G l y Ala
6669 6696 A G A CAT AGA AAC ACC TCA AGA C~A ACA TTA CAA T A A T A G T G A A A A C~C~ TAC CAC Asp Ile Glu Thr Pro Gln Glu Glu His Tyr Ash ASh Set G l u Lys Gly Thr Thr
6181 8208 AGG A A C ~ TGC C A C TGC TAT TGG A K T G G T G A C T C A A T A TCA T C A AGT TTT GC-C G l y T h r G l y Ala Thr Ala Ile G l y Met Val Thr Gln T y r His G l n Val Leu Kla
6728 6750 ATT AAA TAT AAA ATA TGG GAG AAG ATG TTG TAT TAG C A C A T T TAT TAT GTA TTT Leu ASh Ile Lys Tyr Gly Arq Arg Cys Cys Ile Set Th~ Phe Ile Met Tyr Leu
8235 8262 TAC TCA TCA ACA AGC ATT G G A AAA AAT AAC T G A ~ A C T G A ~ AKT AAA TAK TTT Thr His Gln Gln Ala Leu Glu Lys Ile Thr G1u Ala Le~ Lys ~le A=n ASh Leu
6777 6604 AAT CCT TTT TGC AGG AGT AGG CAT CTG CCT CGG AC~C TAG GGC G C A AGT AGT GTG Ile Leu Phe Ala Gly Val Gly Ile Trp Leu Gly A]a A r T Ala Gin Val Val Trp
8289 8316 AAG GCT AAT TAC CTT A G A GCA CCA AGT ATT AGT GAT A G G ATT AAG AGT AGA GGC A r g Leu Ile Thr Leu Glu His Gin Val Leu Val Ile G l y Leu A r g Val G1u Ala
683~ 6958 G A G ACT TCC CCC TTT AGT AGT TCC AGT AGA TGA TAC A G A ~ T AAT ATT TTG GGA ArT ~ u Pro Pro Leu v~l Val Pro Val A~p A 3 p T h r ~ l u Ile Xle Pbe T r p A = p
8343 %370 TAT A G A A]~% ATT C T T ATA TAC AC~C TTT TGC TAT C~CA A G A ATT AC-G ATG C A A TC~ Ile Glu Ly~ Pbe Leu Tyr Thr Ala ~he Ala Met Gln Glu Leu G l y Cys A~n Gln
68~8 6912 TTG TYG CC~ ACC AGA GGA ACC G~C TTG T~A AGA TTT C T T G G G A A C AAT OAT ATA Cys Trp AI~ Pro GIU Glu Pro A]a Cys Gln Asp Phe ]e~ G l y Thr Met Iie Tyr
8397 8424 AAA T C A ATT CTT TTG TAA AAT TCC TCC CAG CCT ATG G A G TAT G T A TAA CAT G A C Ash Gln Phe Phe Cys Ly8 Ile Pro Pro Set Leu Trp Set Met Tyr ASh Met Thr
6989 6966 TCT AAA AGC AAA TGT CAA TAT AAG TAT ACA AGA AC~ ACC TAC ATT C ~ AAA TTG Leu Lys Ala Ash Val Asn Ile Set Ile Gln Glu Gly Fro Thr Leu Gly Ash Trp 6993 7020 GGC AAG AGA AAT TTG GTC CAC ATT ATT TAA AAA C~C TAC AAG GCA ATG TAG AAG Ala Arg G]~ Ile Trp Set Thr Leu ?he Lys Lys Ala Tbr Arg Gln Cy~ ArT Arg
8451 8478 TTT GAA TCA AAC AAT CTG GAA TCA TGG AAA TAT CTC ATT GGG G A A TTG GTA TAA Leu ASh Gin Thr Ile Trp ASh His Gly Asn Ile Ser Leu Gly Ash Trp Tyr ASh 8505 8532 TCA AAC AAG AGA TTT GCA AAA TAA ATT TTA TGA GAT AAT AAT CGA TAT AGA ACA Gln Thr Arg A~p Leu Gln A~n Lys Phe Tyr Glu Ile Ile Met A~p Ile Glu Gln
7047 7074 CC~ ~w%G AAT CTG GAG GAG ATG GAA TGA GAC TAT AAC A C ~ A C C A T T A G G ATG TGC Oly Arg lle Trp Arg Arg Trp ASh OIU Tbr Ile Thr G l y P r o Leu Gly Cy# Ale
8559 8886 A ~ TAA TGT ACA AC-G GAA AAC TC~ AAT A C A A C A ATT ACA GAA A T G G G A AAA TTG a~n A~n Val Gln G l y Ly~ Thr Gly Ile Gln Gln Leu Gln Lys Trp Glu Ash Trp
7101 7128 AAA CAA CAC CTG TTA TAA TAT CTC C~T AGT GGT ACC T G A T T A TCA ATG TTA TGT Ash Ash Thr Cys Tyr ASh Ile Ser Val Va] V81 Pro ASp Tyr G l n Cys Tyr Val
8640 8619 C~T C ~ G ATG GAT AC~ C A A AAT CCC TCA ATA TTT AAA AGG ACT T C T TGG TAG TGT Val Gly Trp Ile G!y Lys Ile Pro Gln Tyr Leu Lys Gly Leu Le~ Gly 5er val
7155 7182 AGA CAG AGT AGA TAC ATG GTT GCA AGG GAA AGT TAA TAT TTC A C T ATG TTT GAC A~p Arq val A~p Thr Trp Leu Gln Gly Lys Val AS~ ~le Set Leu Cys Leu Thr
8694 8667 GTT C ~ AAT AGG TCT AGG AAT CTT ACT ACT ACT TAT ATG CTT GCC TAC AT~ AGT ieu Gly lle Gly Leu Gly Ile Leu Leu Leu Leu Ile Cys Leu Pro Thr Leu Val
7209 7236 A~ AC~ AI~A GAT OCT ATA TAA TAA AGA AAC AAG ACA ATT A A G T T A TTG TAC A G A Gly Gly Lys Her Lau Tyr ASh Lys Glu Thr Arg Oln L~U Set Tyr Cys Thr A~p
8P21 8748 A G A TTG TAT AAG AAA C T G TAC G A A T A A AAT ACT GGG ATA TAC AGT TAT TGC AAT Asp Cys lle Arg Asn,Cys Thr ASh LyS lle Leu Gly Tyr Thr Val lle Ala Met
7263 7290 TCC ACT ~CA AAT T~C~C ATT GAT TAA TTA TAC ATT T ~ G C C TA.A T C A ~ C TT~ TAT Pro I~u GID Ile PrQ Leu ~le A~n Tyr Thr Pbe G l y P r o A S h Gln Thr Cys Met 7317 7344 GTG GAA CAC ATC TTT GAT C2~A AGA TTC TGA GAT ACC AAA A T G TC~ A T G GTG G A A T~p A~n Thr Set Imu Ile Lys ASp Set Glu Ile PrO Lys Cys G l y Trp Trp Ash 7371 7398 CCA AGT AGC T~A TTA CSA TAC TTG TAA ATG GGA AGA A ~ TAA TGT GAC ATT TCA GIn Val Ala Tyr Tyr Asp Thr Cys Lys Trp GiU Glu Ala ASh Val Thr Phe Gin 7425 7452 GTG TCA CAG AAC ACA A,AG TCA ATC AC~ ATC A~"X~ GAT TAG AAC AAT CTC CTC ATG Cys His Arg Thr Gin Set G)n Set Gly Set T~p Ile Arg Thr Ile Ser Ser Trp
8775
8802
G C C T G A AAT AGA TC~% TGA A ~ A A G T A C A C C T A T C AGT G G A A T T GAG GAG AAA TC~ Pro GI~ Ile ASp A s p GIu GI~ V & I His Leu Set Val Glu Leu Arg ArT A~n G l y Met Ala 8829 8856 CAG C ~ A ATG TGG CAT ATC TGA A 2 ~ AGA G G A G G A ATG ATG AAG CAT CTC CGG ACT Arg Gin Cys Gly Ile Set GIu Lys Glu Glu Glu . G l y Ash Val Ala Tyr Leu Lys Dys Arg Arg Ash Asp GIU AIm Set Pro Asp Cys
8888
~&A
GTA AAA TAC AGG AGA GAT GCT GAG CTG ATT TCT TCC CTT TGA Lys Ile Gln Glu Arg Cy~ •
8937 7479 7506 OAA ACA AAG AAA TAG ATG GGA ATG GAG GCC AGA CTT T G A AAG T G A G A A AGT A A A Ly3 Gin Arg A5~ Arg Trp Glu Trp Arg ~ro Asp ?he Glu Set G!u Lys Val Lys 7833 7560 AAT ATC AT? ACA ATG TAA TAG TAC AAA AAA CTT AAC G T T TGC AAT G A G AAG TTC Ile Set I~u Gln Cys Ash Set Thr Lys Asn I~u Thr ?he Ala Met Arg Ser Set
G~GC 8964
R T A T~% ATC CAT TTC A A A T C A A A A TAA C A T TAA AAT CTG ~AT TGC AA A AAC Met Ash Pro Phe Gln Ile Ly3 lle Thr Leu Lys Ser Val Le~ Gin Gly Ly~ Arg 8981 ~018 GTA A G A GAC AAC G~A G A A G A A C ~ A A G A A G A A C ~ C C T TCA AAC ATA TGA TGG CAA Lys Arg Gin Arq Arg Arg A~g Lys Lys Ly8 Ala Phe Lys His He% Set AIa ASh
90q5 7557 7614 AAG TGA TTA T?A TGA TGT ACA AGG A&C ATG GAT AGA ATT TG~ ATG TCA TAG GAA Set ASp Tyr Tyr ASp Val Gin G]y A]8 Trp Ile Giu ~he Gly Cys His Arg Asn 764~ 7668 TAA ATC AAA AAG GCA CTC CGA GGC ~ G ATY TAG AAT A A G ATG TAA ATG GAA TGA Lys Set Lys Arg NiS Ser Glu Ala Arg Phe Arg ~le Arg Cya Lys Trp ASh Glu 7695 7722 AGG A~%A TAA TAT CTC ACT TAT TGA TAC GTG TC~G GAC A ~ TCC A A A TGT CAC ACX~ Gly A=n Ash I!e Se~ L~u lle Asp Thr Cy5 Gly Thr ASh ?to A=n Val Thr Gly 77~9 7776 ACC CAA CCC C~T AGA TTG TAC TAT GAA GCC AAA CAC TAT G T A C A A TTG CTC TTT Ala Asm ?to Val ASp CyS Thr MET Lys Ale A~n Th/ M E T Tyr Ash Cy8 Set L~u 7803 783O ACA AGA TAG TT~ TAC TAT GAA AAT AGA GGA TCT TAY TGT ACA TTT TAA TAT GAC Gln ASp 5er Phe Th~ Met Lys lie Glu As~ Leu ~le Val His Phe Ash Her Thr
8910 C~A TGT GTC Met Cys His
9072
ATC TAG AGA ACC GCT TTA AGA TGC TGT TTG GCA CAC CGT CAA CCA CAG ACG AAA Leu Glu ASh Arg Phe Lys Her Leu Phe Gly Thr Pro Set Thr Thr Asp 01u Thr 9099 9126 CA~ A~G AAA A~kA CAT CTG C ~ i ~ AAA AAA C ~ TG
