1.Introduction :

      Hepatitis delts virus(HDV) is a satellite of the hepatitis B virus(HBV)

which provide the surface antigen for the viral coat.The genome of the 

hepatitis delta virus consists of a single-stranded,circular RNA of 1679

nucleotide which forms a rod structure due to extensive self homology.HDV

replicates through synthesis of an antigenomic RNA via a rolling circle

mechanism .This mechanism is governed by autocatalytic cleavage and ligation

reactions. HDV encodes two proteins,the small delta-antigen and the large

delta-antigen. The latter resembles the former except for the presence in

the latter of additional 19 amino acids at the C terminus. While the small

delta-antigen is required for HDV RNA replication,the large delta-antigen

inhibits replication. HDV delta-antigen contains mainly three

subdomains,covering residues 1 to 88,89 to 143,and 144 to 212. The middle

domain was shown to be responsible for RNA binding. Analysis of a variety of

large delta-antigen mutants revealed that the RNA-binding domain is

essential for viral RNA packing. By increasing the incorporation of small

delta-antigen,it is also found a three to four fold enhancement of HDV RNA

packing.

     By amino acid sequence homology search,Lee et al. identified two

stretches of arginine rich motif(ARM) wuthin the HDV delta-antien RNA

binding domain . THe first one is KERQDHRRRKA, and the second is

EDEKRERRIAG, and they are separated by 29 amino acids. Deletion of either

one of three ARM sequences resulted in the total loss of the in vitro

RNA-binding activity of HDV delta-antigen. It was shown by Poisson et

al.that peptides corresponding to residues 2~27 and 79~107 of HDV

delta-antigen can also bind specifically to HDV RNA. Recently,another RNA

binding region of HDV delta-antigen was mapped to be residues 1~88.

Later,truncated peptides covering residues 14~59 or residues 24~75 were also

shown to have RNA binding activity. Thus,HDV delta-antigen differs from

other RNA-binding proteins in that this antigen contains multiple regions

that mediate RNA-binding. Furthermore. from a close inspection of HDV

delta-antigen sequence and from structural prediction method(Fig.1),we found

that residues 24~60 of HDV delta-antigen may form an alpha-helical structure

with a possible coil-coil interaction. An alpha-helical structure may be

required for the RNA binding activity of HDV delta-antigen. To study the RNA

chaperone activity of HDV delta-antigen and to investigate the interaction

of delta-antigen with HDV RNA, we will determine the complete structure and

study the activity of the RNA-binding domain(residues 1~88) of HDV

delta-antigen using CD and NMR spectroscopic techniques. 

          .   10    .   20    .   30    .   40    .   50    .   60
      MSRSERRKDRGGREDILEQWVSGRKKLEELERDLRKLKKKIKKLEEDNPWLGNIKGIIGK
helix HHHHH           HHHHHH    HHHHHHHHHHHHHHHHHHH     H         
sheet               EE                                     EEEEEE 
turns      TTTTT                                   TT TT TT      T
coil            CCCC        CCCC                     C            

          .   70    .   80    .   90    .  100    .  110    .  120
      KDKDGEGAPPAKKLRMDQMEIDAGPRKRPLRGGFTRKERQDHRRRKALENKRKQLSSGGK
helix         HHHHHHHHHHHHHH               HHHHHHHHHHHHHHHH       
sheet                            EE                        EE     
turns  TTTTT                   T   TTTT   T                   TT  
coil  C     CC              CCC C      CCC                   C  CC

          .  130    .  140    .  150    .  160    .  170    .  180
      SLSREEEEELKRLTEEDEKRERRIAGPSVGGVNPLEGGSRGAPGGGPVPSMQGVPESPFA
helix    HHHHHHHHHHHHHHHHHHHH
sheet                                EEE              EEEE  
turns                        T           TTT T   T              T
coil  CCC                     CCCCCCC   C   C CCC CCCC    CCCCCC C

          .  190    .  200    .  210
      RTGEGLDIRGSQGFPWDILFPADPPFSPQSCRPQ
helix
sheet        E        EEEE 
turns    T  T TTT   TT    TT  TT TTTT TT
coil  CCC CC     CCC        CC  C    C

Residue totals: H: 81   E: 24   T: 49   C: 60
       percent: H: 40.9 E: 12.1 T: 24.7 C: 30.3
 

pir|S|JC1062 delta large antigen - hepatitis delta vir (214 aa)
initn: 1044  init1: 1044  opt: 1044 z-score: 1484.7 E():      0
Smith-Waterman score: 1044;    98.6% identity in 214 aa overlap

pir|S|A53175 delta large antigen - hepatitis delta vir (214 aa)
initn:  993  init1:  993  opt:  993 z-score: 1412.8 E():      0
Smith-Waterman score: 993;    87.9% identity in 214 aa overlap

pir|S|SAVLDV delta large antigen - hepatitis delta vir (214 aa)
initn:  974  init1:  974  opt:  974 z-score: 1386.1 E():      0
Smith-Waterman score: 974;    86.0% identity in 214 aa overlap

pir|S|SAVLDN delta large antigen - hepatitis delta vir (214 aa)
initn:  964  init1:  964  opt:  964 z-score: 1372.0 E():      0
Smith-Waterman score: 964;    84.1% identity in 214 aa overlap

pir|S|SAVLWC delta large antigen - hepatitis delta vir (205 aa)
initn:  929  init1:  929  opt:  929 z-score: 1323.0 E():      0
Smith-Waterman score: 929;    85.9% identity in 205 aa overlap