Characterization of hepatitis delta antigen: specific binding to hepatitis delta virus RNA.
It has previously been shown that human hepatitis virus delta antigen has an RNA-binding activity (Chang et al., J. Virol. 62:2403-2410, 1988). In the present study, the specificity of such an RNA-protein interaction was demonstrated by expressing various domains of the delta antigen in Escherichia coli as TrpE fusion proteins and testing their RNA-binding activities in a Northwestern protein-RNA immunoblot assay and RNA gel mobility shift assay. Hepatitis delta virus (HDV) RNA bound specifically to the delta antigen in the presence of an excess amount of unrelated RNAs and a relatively high salt concentration. Both genome- and antigenome-sense HDV RNAs and at least two different regions of HDV genomic RNA bound to the delta antigen. Surprisingly, these two different regions of HDV genomic RNA could compete with each other for delta antigen binding, although they do not have common nucleotide sequences. In contrast, this binding could not be competed with by other viral or cellular RNA. Since both the genomic and antigenomic HDV RNAs had strong intramolecular complementary sequences, these results suggest that the binding of delta antigen is probably specific for a secondary structure unique to the HDV RNA. By expressing different subdomains of the delta antigen, we found that the middle one-third of delta antigen was responsible for binding HDV RNA. Neither the N-terminal nor the C-terminal domain bound HDV RNA. Binding between the delta antigen and HDV RNA was also demonstrated within the HDV particles isolated from the plasma of a human delta hepatitis patient. This in vivo binding resisted treatment with 0.1% sodium dodecyl sulfate and 0.5% Nonidet P-40. In addition, we showed that the antiserum from a human patient with delta hepatitis reacted with all three subdomains of the delta antigen, indicating that all of the domains are immunogenic in vivo. These studies demonstrated the specific interaction between delta antigen and HDV RNA.
Lin
J H
JH
Howard Hughes Medical Institute, University of Southern California, School of Medicine, Los Angeles 90033-1054.
Chang
M F
MF
Baker
S C
SC
Govindarajan
S
S
Lai
M M
MM
D000595:Amino Acid Sequence; D000956:Antigens, Viral / Q000235:genetics* / Q000302:isolation & purification; D001483:Base Sequence; D004926:Escherichia coli / Q000235:genetics; D003698:Hepatitis Delta Virus / Q000235:genetics* / Q000276:immunology; D035923:Hepatitis delta Antigens; D006801:Humans; D015151:Immunoblotting; D008969:Molecular Sequence Data; D010957:Plasmids; D011485:Protein Binding; D012367:RNA, Viral / Q000302:isolation & purification / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification; D015183:Restriction Mapping
D000956:Antigens, Viral; D035923:Hepatitis delta Antigens; D012367:RNA, Viral; D011993:Recombinant Fusion Proteins; C442244:hepatitis delta virus large antigen