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Recombinant Adenovirus (Adenovirus Vector/In Vivo Recombination/Tripartite Leader/Translational Regulation) ALAN R

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Recombinant Adenovirus (Adenovirus Vector/In Vivo Recombination/Tripartite Leader/Translational Regulation) ALAN R Proc. Natl. Acad. Sci. USA Vol. 82, pp. 7560-7564, November 1985 Biochemistry Expression of hepatitis B surface antigen with a recombinant adenovirus (adenovirus vector/in vivo recombination/tripartite leader/translational regulation) ALAN R. DAVIS, BEVERLEY KOSTEK, BRUCE B. MASON, C. L. HSIAO, JOHN MORIN, S. K. DHEER, AND PAUL P. HUNG Wyeth Laboratories, Inc., Microbiology Division, P.O. Box 8299, Philadelphia, PA 19101 Communicated by Irving M. Klotz, July 22, 1985 ABSTRACT Early region 1 of the adenovirus type . study we show that high-level production of HBsAg takes genome was replaced with a DNA sequence containing the gene place at late times in adenovirus-infected human cells when coding for the hepatitis B surface antigen (HBsAg) flanked by the HBsAg-specific mRNA is made from a DNA where the the major late promoter from adenovirus 2 and processing and adenovirus tripartite leader sequence precedes the HBsAg polyadenylylation signals from simian virus 40. In ono type of gene. hybrid virus only the adenovirus 2 major late pi amoter, including just 33 base pairs of the adenovirus type 2 ',-ipartite MATERIALS AND METHODS leader, preceded the coding region of the HBsAg ene. Iri another, this region was preceded by both the adenovir-.i Cells and Viruses. Cell line 293 (3) derived from human major late promoter and almost the entire tripartite leader. embryonic kidney was used for transfection. Wild-type The structure of the substituted sequence in each of the adenovirus type 5 (AdS) and the Ad5 mutant AEl (7, 8) were recombinant viral DNAs was identical to that in the plasmids grown and titrated on 293 cells. used to construct the viruses. Approximately equivalent DNA Transfections. The calcium phosphate method (9) was amounts of HBsAg-specific mRNA were produced late in used. With AEl or Ad5 viral DNA, transfection efficiencies infection with each recombinant virus. Although HBsAg pro- were approximately 1000 plaques per 1Lg of DNA. duction was detected late in infection of the hybrid virus not Radioimmunoassay (RIA) for HBsAg. Culture medium from containing the full tripartite leader sequence, its level V, as the adenovirus-infected cells was clarified by low-speed 1/70th of that obtained with the hybrid virus containing kimis centrifugation (3000 x g) before assay. Lysates were pre- sequence. One likely interpretation is that the presence of the pared from infected cells as follows: After washing with tripartite leader at the 5' end of this mRNA is critical for the Dulbecco's phosphate-buffered saline, cells were suspended synthesis of HBsAg polypeptide in the late stage of infection. in phosphate-buffered saline, frozen ans i thawed three times, HBsAg produced upon infection with the hybrid adenoviruses and then sonicated. The mixture was c -trifuged at 3000 x g was glycosylated and secreted into the culture medium as for 5 min and the supernatant was de ignated as lysate. An particles that were essentially indistinguishable from the 22-nm aliquot (100 A.l) from either the culturL medium or the lysate particles found in human serum. was assayed by using the HBsAg RIA kit from Nuclear Medical Laboratories (Irving, TX). Approximately 200,000 persons in the United States are infected each year with hepatitis B virus. Due to the lack of RESULTS an in vitro propagation system for hepatitis B virus, much of our knowledge of the viral genome comes from studies Construction of a "Cassette" for Expression of HBsAg. employing recombinant DNA methodology (1). The hepatitis First, a bacterial plasmid was constructed to contain a B surface antigen (HBsAg) is found in the sera of infected cassette, which is a sequence in this case composed of the individuals as a 22-nm particle (1, 2) and also as a 42-nm major late promoter from Ad2 and the HBsAg gene, followed particle (Dane particle) that is the infectious virion (2). by processing and polyadenylylation signals from simian HBsAg is the major antigen of the virus against which virus 40 (SV40). This plasmid, termed p6XH, contains the neutralizing antibody is directed. Using HBsAg, we describe HBsAg gene of the adw subtype in a DNA sequence from 26 here the further development of human adenovirus as a base pairs (bp) upstream of the HBsAg translation initiation system for the introduction of genes into mammalian cells. codon to 131 bp downstream of the translation termination The large size of the genome coupled with the existence of codon. It is flanked by upstream sequences from the Ad2 adenovirus-transformed cell lines (3) allows for insertion of major late promoter (-400 to +33 bp; ref. 10) and by large segments ofDNA. Also, during later stages ofinfection, downstream sequences from SV40 DNA (nucleotides 2770 to levels oftranscription from the major late promoter are high, 2533; ref. 11). This construct therefore contains only the first while cellular protein synthesis is inhibited (4). However, the 33 bp of the 200-bp Ad2 tripartite leader. The entire cassette majority of mRNAs produced at late times after infection is flanked by Xba I restriction sites. with adenovirus come from a long primary transcript that is Second, two bacterial plasmids were constructed to con- processed to yield five families of 3'-coterminal mRNAs. tain cassettes composed of the Ad2 major late promoter and These mRNAs all have a common 5' nontranslated region, the leftmost 168 bp of the 200-bp Ad2 tripartite leader, 200 nucleotides in length. This region is called the tripartite followed by the HBsAg gene and the processing and leader because it is generated by the splicing of sequences polyadenylylation signals from SV40. These plasmids, from three distinct regions of the viral genome (5, 6). In this termed pHM1 and pHM2, were derived from precursor plasmids pMy3l-3 and pMPCV2 (12). In plasmid pMy3l-3 the The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" Abbreviations: HBsAg, hepatitis B surface antigen; Ad2 and Ad5, in accordance with 18 U.S.C. §1734 solely to indicate this fact. adenovirus type 2 and type 5; SV40, simian virus 40; bp, base pair(s). 7560 Downloaded by guest on September 28, 2021 Biochemistry: Davis et al. Proc. Natl. Acad. Sci. USA 82 (1985) 7561 0 late promoter, beginning at the Sac II site at Ad2 map unit Cla I 16.5, the intact first and second Ad2 late leaders, and Ad2 MLP approximately two-thirds (56 of 88 bp) of the third leader. These leader sequences contain no introns because they were derived from a cDNA clone (pJAW) of Ad2 fiber mRNA (6). The Ad2 major late promoter and these leader sequences Leaders 1, 2, were flanked on the left by the leftmost 353 bp ofthe Ad5 viral and 3 genome and on the right by sequences from map positions 8 Yof to 15.5 of Ad5; all of these sequences were inserted into Xho I/Stu I pBR322. Plasmid pHM1 was formed by excision of HBsAg and 19 bp of SV40 sequences from plasmid pMy31-3, using Stu I and BamHI and ligation to the large fragment ofplasmid pMPCV2 extending from Bgl II to Xho I (Fig. 1). Plasmid pHM1 contains 19 bp of SV40 sequence (SV40 nucleotides 5190-5171) preceding the HBsAg gene. Plasmid pHM2 (not shown) is identical to pHM1 except that it does not contain Bgl II/BamHI this 19 bp of SV40 sequence. Insertion of the Cassette into the El Region of Ad5. To FIG. 1. Recombinant plasmid pHM1 used in the preparation of facilitate insertion of the cassette cloned in p6XH into the El hybrid adenoviruses. Plasmid pHM1 contains adenovirus sequences region of adenovirus, it was first cloned at the unique Xba I (open bars) including the Ad2 major late promoter (MLP) and site of plasmid pAC (not shown; ref. 7), which contains an tripartite leader, followed by HBsAg sequences (hatched bar) and insert ofAd5 DNA extending from an EcoRI linker at the left SV40 sequences (stippled bar), all inserted in pBR322 (solid line; terminus ofthe adenovirus genome to the HindIII site at map AmpR, ampicillin resistance). coordinate 17.0. In addition, adenoviral DNA between 1.4 and 9.1 map units (El region) has been deleted from pAC and SV40 early genes, large and small tumor (T) antigen, were the unique Xba I site has been placed at this junction. The replaced by the HBsAg gene described above. The HBsAg new plasmids pACH-2 and pACH-9 (Fig. 2) contained the gene was positioned behind the SV40 early promoter at a cassette positioned at the Xba I site in either orientation. unique Sal I site that juxtaposes SV40 sequences at nucleo- Cassettes from pACH-2, pACH-5, pHM1, and pHM2 plas- tides 5171 and 2770. Plasmid pMy3l-3 DNA was shown to mids were positioned at the El region ofAd5 by the technique direct SV40 virion formation and the expression ofHBsAg in of homologous recombination (14). Plasmids pACH-2 and Cos 1 cells (13) when pBR322 DNA sequences were excised pACH-9 were linearized with HindIII (Fig. 2A) and pHM1 with BamHI and SV40 was reformed by ligation (data not and pHM2 were linearized with EcoRI (Fig. 2B). Each shown). The plasmid pMPCV2 (12) contains the Ad2 major linearized DNA was combined with the large Xba I fragment A HindM - ": -. ~ SV40 uinU L pBR322 0 1.4Ad2 HBsA9 9 1 7 pACH-9 7% 0 1.4 HBsAg Ad29.1 177 pACH-2 SV40 9.1 17 "\ II V large XbaI fragment ~1 of AEI AEIH viruses B EcoRI Sac - 0 1.0 Ad2MLP+Ieaders HBSA9 SV40 9.1 15.5 I npl2D?) Eco RI .
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