Translational modulation in hepatitis B virus preS-S open reading frame expression.
A series of hepatitis B virus open reading frame (ORF) preS-S variants, including mutants in which the relative order of the in-frame start codons (AUG1, AUG2 and AUG3) and nearby sequences had been altered, was expressed both in vivo (in HepG2 hepatoblastoma cells) and in vitro (by T7 promoter-driven transcription followed by translation in a rabbit reticulocyte lysate). The ratio of the synthesis of the large, middle (M) and major (S) proteins or their chimeric counterparts was analysed to study the translational regulation of ORF expression. As expected on the basis of the ribosome scanning model, the AUG sequence context was found to be a prominent factor in determining the different translational behaviour of the two preS-S-specific mRNAs of 2.4 kb (predominantly translated from AUG1) and 2.1 kb (which includes AUG2 and/or AUG3 and can be translated from either). Results from both experimental systems suggested that initiation at internal AUGs in the 2.4 kb RNA is possible. In experiments in vitro, preS-S mutants bearing lesions in a region 5' to AUG2, which has been implicated in AUG2/AUG3 cis repression, showed no increase in the utilization of internal AUGs. In addition, the chimeric envelope polypeptides produced in transfected HepG2 cells in this study were informative with respect to preS-mediated endoplasmic retention: replacement of the preS2 N terminus with that from preS1 generated a chimeric M protein that was glycosylated within the putative preS1 retention sequence ad was not secreted. Thus, the preS1 retention sequence most likely acts inside the lumen of endoplasmic reticulum and its function is insensitive to glycosylation. A similar element might be active at the N terminus of M protein.
Gallina
A
A
Istituto di Genetica Biochimica ed Evoluzionistica, Consiglio Nazionale delle Ricerche, Pavia, Italy.
De Koning
A
A
Rossi
F
F
Calogero
R
R
Manservigi
R
R
Milanesi
G
G
D001483:Base Sequence; D002460:Cell Line; D003062:Codon / Q000235:genetics; D015967:Gene Expression Regulation, Viral / Q000235:genetics*; D006514:Hepatitis B Surface Antigens / Q000235:genetics* / Q000378:metabolism; D006515:Hepatitis B virus / Q000235:genetics*; D008969:Molecular Sequence Data; D016366:Open Reading Frames / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D011233:Precipitin Tests; D014176:Protein Biosynthesis / Q000235:genetics*; D011498:Protein Precursors / Q000235:genetics* / Q000378:metabolism; D014162:Transfection / Q000235:genetics
D003062:Codon; D006514:Hepatitis B Surface Antigens; D011498:Protein Precursors; C050806:presurface protein 1, hepatitis B surface antigen; C050807:presurface protein 2, hepatitis B surface antigen