PMC:7464876 / 9007-12329 JSONTXT

{"project":"2_test","denotations":[{"id":"32806600-2153228-20296188","span":{"begin":258,"end":260},"obj":"2153228"},{"id":"32806600-10792995-20296189","span":{"begin":417,"end":419},"obj":"10792995"},{"id":"32806600-10792995-20296190","span":{"begin":523,"end":525},"obj":"10792995"},{"id":"32806600-2153228-20296191","span":{"begin":864,"end":866},"obj":"2153228"},{"id":"32806600-21129143-20296192","span":{"begin":1021,"end":1023},"obj":"21129143"},{"id":"32806600-24899172-20296193","span":{"begin":1282,"end":1284},"obj":"24899172"},{"id":"32806600-8392600-20296194","span":{"begin":1560,"end":1562},"obj":"8392600"},{"id":"32806600-7835336-20296195","span":{"begin":1745,"end":1747},"obj":"7835336"},{"id":"32806600-24614091-20296196","span":{"begin":1951,"end":1953},"obj":"24614091"}],"text":"2.1. Expression Constructs\nThe plasmid pHBV carries a 1.1 mer of the HBV genome (nt 2922 to 84, derived from genotype D, GenBank™ accession number V01460) under the transcriptional control of the human metallothionein (hMT) IIA promoter in a pUC19 backbone [32]. To prevent expression of the L, M and S envelope proteins (HBV.Envminus), their translational start codons were inactivated by mutagenesis, as described [33]. In the HBV.Lminus construct, only the L-specific start codon was substituted by a threonine residue [33]. To ablate the core expression from the HBV replicon (HBV.Cminus), a stop codon at triplet 38 of the core gene was created [34]. The mutant HBV.Polminus replicon carries an aspartic acid-to-histidine substitution at amino acid (aa) position 540 of the HBV polymerase and has been shown to be defective in reverse transcriptase activity [32]. For trans-complementation studies of the HBV.Cminus replicon, the vector pNI2.C containing the HBV core gene preceded by the hMT IIa promoter was used [35]. The core mutant C.K96A, defective in capsid envelopment, has been described [34]. The plasmid pT7HB2.7, which directs the synthesis of all three HBV envelope proteins under their authentic promoters, was kindly provided by C. Sureau (INTS, Paris, France) [13] and used for trans-complementation of the HBV.Envminus replicon. To inactivate the N-glycan acceptor site within the S domain of the three envelope proteins, the asparagine residue at aa position 146 (N146) was replaced by a glutamic acid residue (Env.N146D), as described [36]. For the sole expression of the S protein under the control of the hMT IIa promoter, plasmid pNI2.S.HA carrying the S gene with a C-terminal-fused haemagglutinin (HA) tag was used [10]. Where indicated, HBV replication was studied in cells transfected with pCEP4∆CMV∆SV40/1.1 × HBV (termed pHBV* herein), which carries the 1.1 mer of the HBV genome in a modified pCEP4 plasmid backbone [22]. The plasmid pEYFP-ER encodes an enhanced yellow fluorescent protein (EYFP) with an N-terminal-fused ER-targeting sequence of calreticulin plus a C-terminal-fused ER retrieval sequence and was purchased from Clontech (Clontech, Mountain View, CA, USA). The vector pGRASP65-GFP (green fluorescent protein) was kindly provided by E. Hildt (PEI, Langen, Germany) and encodes a fusion protein of GRASP65, a peripheral membrane protein that is localized to the cis-Golgi, and of the green fluorescent protein (GFP). The plasmid pDsRed-Golgi carries the DsRed-coding sequence fused to the N-terminal 81 aa of human β 1,4-galactosyltransferase, which targets the fusion protein to the trans-medial region of the Golgi (Clontech, Mountain View, CA, USA). The expression vector encoding human Vps4A with a C-terminal EGFP tag was generously provided by W. Sundquist (University of Utah, Salt Lake Cit, UT, USA). The vector pcDNA3.1(+)-ERGIC-53 carrying the human ERGIC-53 gene (NM_005570.3) under the control of the CMV promoter was purchased from GenScript (GenScript Biotech, Leiden, Netherlands). For tagging with the Myc epitope, the aa sequence 28–37 of ERGIC-53 was mutated to EQKLISEEDL by Q5® Site-Directed Mutagenesis (New England Biolabs, Ipswich, MA, USA) with the oligonucleotides 5′- CCGAGGAGGACCTCCTGGTGCAGAGCGACGG-3′ and 5′- AGATTAATTTTTGCTCGAAACGGCGATGTGGC-3′."}