In Vitro-Synthesized Hepatitis Delta Virus RNA Initiates ... - Europe PMC

Vol. 64, No. 6

JOURNAL OF VIROLOGY, June 1990, p. 3104-3107

0022-538X/90/063104-04$02.00/0 Copyright © 1990, American Society for Microbiology

In Vitro-Synthesized Hepatitis Delta Virus RNA Initiates Genome Replication in Cultured Cells JEFFREY S. GLENN,' JOHN M. TAYLOR,2 AND JUDITH M.

WHITE'3*

Department of Biochemistry and Biophysics' and Department of Pharmacology,3 University of California, San Francisco, California 94143-0450, and Fox Chase Cancer Center, Philadelphia, Pennsylvania 191112 Received 11 January 1990/Accepted 23 February 1990

transcribed in vitro and then delivered was detected, but only in fibroblasts that stably expressed the delta antigen. Sequence analysis of the replicated products identified them as faithful copies of the hepatitis delta virus genome found in virions. Monomers of the genomic strand of hepatitis delta virus RNA

were

to NIH 3T3 fibroblasts by using a lipsome fusion technique. After 7 days, genome replication

fusion of the bound RNA-containing liposomes with the fibroblasts and thus leads to synchronous delivery of RNA into the cytoplasm. On the basis of protein delivery experiments, we estimate that -90% of the HA-expressing cells receive liposomal contents (6). The two cell lines used (GP4F and GAG) express HA from the Japan strain of influenza virus as a result of stable transfection of the HA structural gene. GP4F cells are the parental HA-expressing NIH 3T3 fibroblasts (6). GAG cells were made for this study by cotransfection of GP4F cells with a plasmid encoding hygromycin resistance (pTKHmr) and pSVL(Ag). The latter plasmid contains the 1.1-kilobase XbaI(781)-BglII(224) fragment of the HDV genome inserted under the control of the simian virus 40 late promoter so as to produce deltaantigen-encoding mRNA (9). Following selection with 300 ,ug of hygromycin per ml in Dulbecco modified Eagle medium containing 10% fetal calf serum, one of the resistant colonies, GAG, was chosen for further characterization. Figure 2 presents the results of Northern (RNA blot), Western (immunoblot), and immunofluorescence analyses. Confluent monolayers of GP4F and GAG cells were lysed in guanidinium, and total RNA was purified over a CsCl cushion essentially as described elsewhere (8). The RNA was glyoxalated, fractionated on a gel of 1.5% agarose, transferred to a nylon membrane, and probed for total antigenomic HDV RNA species as described previously (4), with minor modifications. GAG cells (Fig. 2A, lane 1) were shown to possess an RNA of the size and polarity expected from transcription of pSVL(Ag). Related RNA species were not observed in the parental GP4F cell line (Fig. 2A, lane 2). RNA from the liver of an HDV-infected chimpanzee (1 ,ug) served as a positive control (Fig. 2A, lane 3). Figure 2B shows a Western blot in which extracts of GAG and GP4F cells were subjected to sodium dodecyl sulfatepolyacrylamide gel electrophoresis, transferred to nitrocellulose, and probed with a rabbit polyclonal antiserum raised against a delta antigen-3-galactosidase fusion protein (10). The GAG cells (Fig. 2B, lane 1) were found to express a 24-kilodalton protein that reacted with anti-delta-antigen antibodies and comigrated with the lower of the two deltaantigen species seen in infected human liver (lane 3). The GP4F cell extract (Fig. 2B, lane 2) did not display any delta-antigen-related proteins. GAG cells were next analyzed by indirect immunofluorescence (Fig. 2C) by using the anti-delta-antigen antiserum described above. Prominent nuclear staining was observed, consistent with the previous localization of delta antigen to

The causative agent of delta hepatitis is composed of an envelope containing surface antigen from hepatitis B virus, a 1.7-kilobase, circular, single-stranded RNA genome, and the delta antigen, a protein encoded by the genome (1, 12). Current models for the replication of hepatitis delta virus (HDV) involve a rolling-circle mechanism such as that proposed for viroids (2). Considerable progress has been made in identifying and characterizing the cleavage and ligation activities required by the rolling-circle model (11, 14, 15, 19). Interestingly, in vitro, these activities have been found to be inherent properties of HDV RNA. However, the precise enzymatic machinery that transcribes HDV RNA as well as the cis-acting regulatory sequences on the RNA genome used for RNA-directed RNA synthesis have not yet been identified. Recently, HDV replication products were obtained by transfection of cells with a DNA plasmid containing three tandem head-to-tail inserts of the HDV genome under the control of a simian virus 40 promoter (9). In this study, we asked whether an in vitro-transcribed RNA monomer, of the polarity found in virions, could initiate genome replication when introduced into cultured fibroblasts. Production of monomeric HDV RNA. Monomeric RNA of genomic polarity was produced as outlined in Fig. 1. Plasmid pG4B(D3) contains three copies of the HDV genome in a tandem head-to-tail orientation (9). The monomer obtained by digestion of pG4B(D3) with NheI was cloned into the XbaI site of pSPT19, yielding pT7GM, in which the NheI insert is oriented so as to produce genomic RNA after cleavage with BamHI and transcription from the T7 promoter. Thus, the predicted RNA product would contain all 1,679 nucleotides of the HDV genome as well as 34 and 10 vector-encoded bases at the 5' and 3' ends, respectively. If the in vitro-generated RNA folds into the proposed collapsed, unbranched rod structure (18), the non-HDV sequences would lie in close proximity to each other, as diagrammed in Fig. 1, step F. Delivery of RNA. The basis for the RNA delivery protocol has been described previously (5, 6), and the details of the method will be presented elsewhere (J. S. Glenn, H. Ellens, and J. M. White, unpublished data). Briefly, RNA was encapsulated in glycophorin-containing liposomes and then bound to fibroblasts that express hemagglutinin (HA), the membrane fusion protein of influenza virus, at their surface. A transient lowering of the pH of the medium activates *

Corresponding author. 3104

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FIG. 1. Strategy for producing monomeric genomic RNA. A monomeric HDV genome was obtained by NheI (N) (position 430 on the HDV sequence [10]) digestion of a plasmid containing three HDV genomes inserted in tandem (step A) and cloned into pSPT19 (Pharmacia LKB Biotechnology, Inc., Piscataway, N.J.), a T7 expression vector (steps B and C). Following linearization with BamHI (B) (step D), and transcription with T7 RNA polymerase (step E), a complete monomeric HDV genomic RNA was obtained. The in vitro-transcribed RNA (step F) contains 34 and 10 bases of vector sequences at the 5' and 3' ends, respectively. Symbols: vector DNA; _, DNA encoding HDV genome; , HDV genomic RNA (transcribed vector sequences in bold; A\, T7 promoter. 9

the nuclei of infected cells (3, 9). Some subnuclear localization, as evidenced by the punctate staining, was also seen. No such staining was seen in the control GP4F cells. Taken together, the results in Fig. 2 show that GAG cells express delta antigen and that the constitutively expressed delta antigen is, as expected, localized in the nucleus.

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FIG. 3. Detection of antigenomic RNA in GAG cells following delivery of genomic RNA. Northern blot was probed for de novo antigenomic RNA and signal recognition particle receptor (SRPR) mRNA. The following samples were analyzed: total RNA from HDV-infected chimpanzee liver (lane 1); total GP4F cell RNA after delivery (day 0, lane 2; day 7, lane 3); total GAG cell RNA after delivery (day 0, lane 4; day 7, lane 5). The probe corresponded to positions 430 to 713 of the genomic strand.

Replication of delivered RNA. Monomeric genomic HDV RNA was transcribed in vitro, encapsulated in liposomes, and delivered to GP4F (Fig. 3, lanes 2 and 3) and GAG (lanes 4 and 5) cells as described above. Total cellular RNA was harvested at day 0 (Fig. 3, lanes 2 and 4) and at day 7 (lanes 3 and 5) and was subjected to Northern analysis with a probe designed to recognize only a sequence of antigenomic HDV RNA which is not already present in GAG cells; the probe would detect neither the input RNA nor that transcribed from pSVL(Ag). The probe therefore measures de novo antigenomic RNA production. A probe for the signal recog-

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FIG. 2. Characterization of GAG cells. (A) Northern blot probed for antigenomic RNA. Lane 1, Total GAG cell RNA; lane 2, total GP4F cell RNA; lane 3, total RNA from HDV-infected chimpanzee liver. Size markers at left are in kilobases. (B) Western blot probed with anti-delta-antigen antibody. Lane 1, GAG cell lysate; lane 2, GP4F cell lysate; lane 3, lysate of HDV-infected human liver. Size markers at left are in kilodaltons. (C) Immunofluorescence of GAG cells stained with anti-delta-antigen primary antibody and rhodamine-labeled goat anti-rabbit secondary antibody. Magnification, x 800.

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nition particle receptor mRNA was included in the hybridization to allow comparison of gel sample loading. One week after delivery of monomeric genomic RNA, a prominent antigenomic RNA species of monomeric length was present in GAG cells (Fig. 3, lane 5). Although it produced a less intense signal, an antigenomic species of dimeric length could also be seen in these cells. Probing similar blots with probes for HDV RNA of genomic polarity revealed that amplification of genomic RNA had also occurred (data not shown). These hallmarks of HDV replication were observed only in the GAG cells, which constitutively express the delta antigen. No antigenomic RNA was produced in the parental GP4F cell line (Fig. 3, lane 3). Some cross hybridization with 28S RNA occurred in all cellularRNA samples, independent of RNA delivery. RNA from the liver of an HDV-infected chimpanzee was included as a positive control (Fig. 3, lane 1). The requirement for delta antigen in this system may indicate the role of the antigen in some step of RNAdependent RNA replication or transcription, either directly, as part of a polymerase complex, or indirectly, as part of a ribonucleoprotein template, as has been suggested for the N protein of vesicular stomatitis virus (7). In addition, in our system as in a natural infection, it may be necessary for the genomic, HDV RNA to migrate to the nucleus for replication (17). The delta antigen, by virtue of its inherent nuclear localizing ability (Fig. 2C), may facilitate this transport or protect incoming HDV RNA from cytoplasmic nucleases or both. Analysis of replication products. There are two differences between the in vitro-transcribed HDV RNA and that found in virions. Firstly, the in vitro product is not a circular molecule but rather a linear molecule. Secondly, there are a total of 44 bases of non-HDV sequences (Fig. 1, step F) on the 5' and 3' ends. And yet, as shown above (Fig. 3), neither of these differences prevented the RNA from initiating genome replication. To determine the fate of the additional sequences, we used the polymerase chain reaction followed by direct dideoxy sequencing (13) (Fig. 4). The foreign sequences were removed precisely and completely, leading to an exact restoration of the original NheI site into which the vector sequences were cloned (Fig. 1). Only a homogeneous population of amplified sequences, each containing the fully repaired NheI site, would be expected to yield this result. On long exposures, such as the autoradiogram presented in Fig. 4, a minor but perfect "shadowing" of the major sequence pattern, shifted by exactly 1 base, was observed. This might have been the result of heterogeneity in the 5' end of the oligonucleotide 3 primer or of insertion of a single nucleotide by a stuttering polymerase (16). Nevertheless, even in this minor shadow population, the NheI site was fully restored. A small fraction of molecules, below our detection limit, may have been repaired to a different final sequence. At present, we do not know how the foreign vector sequences were removed. Several steps could have been involved, for example, beginning with a ligation of the 5' and 3' ends of the input RNA followed by repair. Perhaps only one step was required, such as a direct RNA transcription across the break in the HDV RNA sequences. In either scenario, we expect that the extensive intramolecular basepairing of the unbranched rod structure predicted for the HDV genome may have facilitated the removal of non-HDV sequences. In conclusion, we have shown that an in vitro-transcribed monomeric genomic HDV RNA can initiate genome repli-

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FIG. 4. Sequence analysis of antigenomic RNA produced in GAG cells. RNA from GAG cells was extracted 7 days after delivery of genomic RNA. Antigenomic RNA was reverse transcribed with oligonucleotide 1 as a primer. The resulting cDNA was amplified by the polymerase chain reaction with oligonucleotides 1 and 2, and the resulting product (230 base pairs) was subjected to direct dideoxy sequencing with oligonucleotide 3 as a primer. Oligonucleotide 3 was used as the sequencing primer, since it was in this region that we expected to find a change with respect to the input RNA. Note that the NheI site (GCTAGC) was restored in the entire population of sequenced products. Shown in the lower portion of the figure are the positions of the three synthetic oligonucleotides with respect to the HDV genome in Fig. 1, step F. Oligonucleotide 1 corresponds to positions 315 to 337 on the genomic strand. Oligonucleotide 2 corresponds to positions 523 to 542 on the antigenomic strand. Oligonucleotide 3 corresponds to position 363 to 383 on the genomic strand. (*, 3' ends of oligonucleotides).

cation in cultured cells. This finding corroborates two previous conclusions based on studies using DNA plasmids encoding multimeric HDV genomes (9). Firstly, the delta antigen is required for HDV replication, and secondly, no hepatitis B virus products are required for HDV genomic replication. Our results extend the earlier studies by showing that an engineered monomeric HDV RNA genome containing foreign sequences can be fully repaired to a species found in natural infections. Finally, and most importantly, because of the apparent ease with which the input RNA can be rapidly modified (for example, by mutation of the cloned DNA monomeric template), we believe that this will be a valuable system for studying the details of HDV replication as well as other aspects of the viral life cycle. We acknowledge Peter Walter, Pablo Garcia, and Katerina Strub for T7 RNA polymerase and for the plasmid encoding the signal recognition particle receptor mRNA and Ann Tsukamoto for pTKHmr. We thank Carl Blobel, Sam Green, and Don Ganem for helpful discussions and for critical reading of the manuscript. The work was supported by Public Health Service grants A122470 (J.M.W.) and A126522 (J.M.T.) from the National Institutes of Health. J. Glenn is supported by the Medical Scientist Training Program.

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