Identification and characterization of a structural protein of hepatitis B virus: a polymerase and surface fusion protein encoded by a spliced RNA.
The hepatitis B virus (HBV) genome is known to contain four conserved and overlapped open reading frames (ORFs) encoding the viral core, polymerase (P), surface (S), and X proteins. Whether HBV encodes other proteins has long been a major interest in the field. Using (32)P-labeling of an introduced protein kinase A site attached to the N- or C-terminus of the HBV polymerase gene, a 43-kDa P-S fusion protein was detected in cell lysate, secreted virions, and 22-nm subviral particles. Immunobiochemical studies showed that the 43-kDa protein contains the epitopes of the N-terminus of polymerase and most parts of the surface proteins. This 43-kDa protein was shown to be a glycoprotein, similar to the surface protein. RT-PCR and sequence analyses identified a spliced mRNA which was derived from pregenomic RNA with a deletion of 454 nucleotides (nt) from nt 2447 to 2902. This splice event creates a P-S fusion ORF. This finding is consistent with the result obtained from an immunobiochemical study. Mutations at the splice donor or acceptor site on the HBV genome abrogated the production of the 43-kDa protein. These mutants had no effect on viral replication in transfected HuH-7 cells. However, this P-S fusion protein is able to substitute for the LS protein in virion maturation. On the basis of these results, we conclude that the 43-kDa protein is a polymerase-surface fusion protein encoded by a spliced RNA. Similar to the LS protein, the 43-kDa P-S fusion protein is a structural protein of HBV and might play a role in the HBV life cycle.
Huang
H L
HL
School of Life Science, Institute of Microbiology and Immunology, Shih-Pai, Taipei, 112, Taiwan, Republic of China.
Jeng
K S
KS
Hu
C P
CP
Tsai
C H
CH
Lo
S J
SJ
Chang
C
C
D016282:Aspartic Acid Endopeptidases / Q000235:genetics; D002460:Cell Line; D004591:Electrophoresis, Polyacrylamide Gel; D000939:Epitopes; D015684:Gene Products, pol / Q000235:genetics; D006023:Glycoproteins / Q000235:genetics / Q000378:metabolism; D006031:Glycosylation; D006514:Hepatitis B Surface Antigens / Q000235:genetics; D006515:Hepatitis B virus / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D008970:Molecular Weight; D016366:Open Reading Frames; D011233:Precipitin Tests; D012326:RNA Splicing; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014760:Viral Fusion Proteins / Q000235:genetics* / Q000378:metabolism; D015678:Viral Structural Proteins / Q000235:genetics* / Q000378:metabolism
D000939:Epitopes; D015684:Gene Products, pol; D006023:Glycoproteins; D006514:Hepatitis B Surface Antigens; C066027:P protein, Hepatitis B virus; D014760:Viral Fusion Proteins; D015678:Viral Structural Proteins; D016282:Aspartic Acid Endopeptidases; C022442:aspergillopepsin II