Translational modulation in hepatitis B virus preS-S open reading frame expression

Abstract

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 scanningmodel, 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, preSS 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 preS 1 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.

Intracellular retention of hepatitis B virus surface protein mutants devoid of amino-terminal pre-S1 sequences

“…Another work demonstrated that, in HepG2 cells, N-terminal myristylation can block secretion of HBsAg even in the absence of any preS sequence (Prange et aI., 1991). In a previous report, we described a chimeric L protein mutant lacking the preS1 N-terminal sequence and still being substantially retained (Gallina et al, 1992). Here, we re-examine this issue, analysing a wider series of mutations in the preS region for their effects on envelope protein secretion.…”

“…3, pRPAUG1.3ayw, pRPAUGI1.3adw2 and pRPpreS2A. Mutants AUG2.2.3, AUG1.3ayw and AUG1.3adw2 have been described previously (Gallina et al, 1992). Plasmids pRPpreS1A and pRPpreS2A express Y-terminally deleted versions of ORF preS-S. Mutant preS1A fuses a strong context AUG (Kozak, 1989) to preS1 codon 19 (ayw), thus eliminating codons 1 to 18 of the ORF.…”

“…1 b), placed under the transcriptional control of the Rous sarcoma virus (RSV) long terminal repeat (LTR), into the cloning site of vector pRPRSV (Manservigi et al, 1990;Gallina et al, 1992; Fig. 1 c).…”

“…2. 3, pRPAUG1.3ayw and pRPAUG1.3adw2 have been described (Gallina et al, 1992). The remaining plasmids were constructed with standard recombinant DNA techniques (Sambrook et al, 1989), as follows.…”

“…Two days posttransfection, cells were labelled with a 3 h [35S]methionine pulse (140 gCi; Amersham) in methionine-free DMEM, and dissolved in lysis buffer (50 mM-Tris-HC1 pH 8, 0.1% SDS, 1% Triton X-100, 1% sodium deoxycholate, 20 mM-disodium-EDTA, 1 mM-PMSF). The soluble extract was subjected to immunoprecipitation with sheep anti-HBsAg-Sepharose, followed by reducing SDS-PAGE and fluorography, as described (Gallina et al, 1992).…”