Institute of Medical Molecular Biology, Medical University of Lübeck, Ratzeburger Allee 160, D-23538 Lübeck, Germany1
Author for correspondence: Verena Gauss-Müller. Fax +49 451 500 3637. e-mail gaussmue{at}molbio.mu-luebeck.de
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Abstract |
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Introduction |
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Similar to other positive-strand RNA viruses, the picornaviral genome serves as template for both translation and replication. Initially, the viral RNA is translated into a polyprotein (P1P2P3), which is cleaved into the mature viral proteins by virus-encoded proteinases, making picornaviral gene expression mostly dependent on polyprotein processing. The viral structural proteins, VP1, VP2, VP3 and VP4, are released from their common precursor, P1. In a regulated fashion, the non-structural proteins, including the viral proteinase (3C) and polymerase (3D), are liberated from domains P2 and P3 of the polyprotein and are the major constituents of the virus replication complex (RC), which catalyses RNA genome replication. Once the RC is formed, viral RNA is used as the template for replication, which proceeds in the opposite direction to translation and most likely competes with it (Gamarnik & Andino, 1998 ). In addition to the viral proteins, cis-acting replication elements (CRE) on the viral RNA are essential for the formation of the RC and thus for viral genome replication. Involvement of individual viral and host components in this process has been investigated using either an in vitro translation/replication system (Molla et al., 1991
; Barton & Flanegan, 1993
) or viral replicons encoding a reporter gene in place of the viral capsid proteins. Autonomous replication of the replicon can be followed by measuring the increment of its reporter gene activity (Andino et al., 1993
).
Among the picornaviruses, hepatitis A virus (HAV) is unique in its protracted replication in cell culture. Weak translation initiation, protein processing, asynchronous uncoating and lack of host shutoff have all been suggested to contribute to this behaviour (Bishop & Anderson, 2000 ; Funkhouser et al., 1999; Jia et al., 1998; Michel et al., 2001
). Furthermore, nothing is known about the activity of either the HAV polymerase (3Dpol) or the molecular constituents of the virus RC. In order to assess HAV genome replication, we have constructed a replicon that lacks the genomic region of the structural proteins (P1) and encodes a reporter gene instead. As the replication capacity of the HAV replicon was found to be very poor in comparison with a replicon of poliovirus (PV), the prototype picornavirus (Andino et al., 1993
), its replication competence was demonstrated by the successful genetic recombination of the replicon with several non-viable HAV genomes, which encoded the capsid proteins as selection markers. Recombination resulted in infectious HAV particles. In contrast, a replication-deficient replicon was unable to produce infectious virus when co-expressed with P1-encoding non-viable HAV genomes. With the aid of the sensitive rescue system described here, we provide evidence that even very low levels of genome replication can be made detectable after genetic recombination.
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Methods |
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RNA transcription in vitro and transfection.
Plasmid DNA was linearized with appropriate restriction enzymes and in vitro-transcribed with the MEGAscript system (Ambion), basically as described previously (Kusov & Gauss-Müller, 1999 ). RNA (110 µg) was transfected into HuhT7 or Bsc-1 cells by liposome transfection or electroporation, respectively. For lipofection, the RNA was mixed with 3 µl of DMRIE-C (Life Technologies) before it was applied to a monolayer of 105 cells grown on 3·5 cm2. After a 3 h incubation, the transfection mixture was replaced by medium containing 10% foetal calf serum. Incubation was continued for the time periods indicated without passaging the cells. For determination of luciferase activity, transfected cells grown on 3·5 cm2 were lysed with 200 µl of cell culture lysis buffer (Promega) at room temperature. Luciferase activity was measured using a MicroLumat P96 and substrates (Berthold Detection Systems) and expressed in relative light units (RLU).
DNA transfection and recombination assay.
DNA transfection mix containing 14 µg of cDNA and 9 µl LipofectAmin (Life Technologies) in 200 µl OptiMEM (Life Technologies) was preincubated for 30 min at room temperature, diluted with OptiMEM to 1 ml and transferred to HuhT7 cells (5x105/10 cm2; Schultz et al., 1996 ). After incubation for 3 h at 37 °C, the transfection mix was replaced by growth medium containing geneticin (G418 sulfate, 400 µg/ml; Life Technologies). For in vivo recombination, acceptor and donor cDNA were mixed and transfected into HuhT7 cells, as depicted in Fig. 1
(lower panel). Transfected cells were incubated for 20 days (passage 0) without passaging, before extracts were harvested and tested for viral antigen, RNA and infectivity. In detail, transfected cells of multiple wells (10 cm2) were each harvested in 250 µl PBS containing 0·05% Tween-20 (PBS-Tw). After three cycles of freeze-thawing, the clarified supernatant was used for the detection of viral antigen (HAAg) by ELISA with the monoclonal antibody 7E7, as previously described (Kusov & Gauss-Müller, 1999
; Probst et al., 1998
, 1999
). For characterization of recombinant proteins, plasmid DNA was transiently expressed with the aid of vaccinia virus vTF7-3 (Elroy-Stein & Moss, 1990
). Extracts of trans-infected cells were analysed by immunoblot, as previously described (Kusov & Gauss-Müller, 1999
; Probst et al., 1998
, 1999
). To determine the infectivity of the virus rescued by genetic recombination, the PBS-Tw extract was diluted 1:100 and used to inoculate HuhT7 cells grown in 25 cm2 flasks (passage 1). After 1525 days of subpassage, infected cells were harvested and analysed by ELISA and RTPCR.
HAV RNA analysis by RTPCR.
Total RNA from transfected cells, and of cells infected with HAV strain 18f or with the virus rescued by genetic recombination (passage 1), was extracted by Trizol (LifeTechnologies). The genomic regions between HAV nucleotides 5310 and 5830 (Fig. 3) and between nucleotides 5908 and 7393 (Fig. 6
) were amplified using the C.therm. polymerase One-Step RTPCR system (Roche, Applied Science) and appropriate HAV-specific primers. The latter amplification product of 1·5 kb was digested with either XhoI, ClaI or Bsp1407I and analysed on an agarose gel.
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Results |
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In an initial experiment to assess the overall amplification competence of the HAV replicon, the kinetics of reporter gene expression for the replication-competent and -deficient replicons of HAV and poliovirus (PV) were compared after transfection of HuhT7 cells with equal amounts of run-off transcripts. Similar to comparable PV replicons whose expression kinetics have been shown elsewhere (Andino et al., 1993 ), maximal luciferase activity of the HAV replicons was reached approximately 15 h post-transfection (p.t.) under the applied conditions. Similarly, as reported recently (Yi & Lemon, 2002
), luciferase activity of the replication-competent HAV replicon was detectable up to 72 h p.t. (not shown). For a quantitative assessment of their function in translation and replication, the luciferase activities expressed from the replication-deficient HAV (pT7-18f-LUCmut) and PV (pRLuc-181) replicons were compared with the respective replication-competent replicons (Fig. 2A
). Whereas the replication-competent PV replicon (pRLuc-31) reached more than 100-fold higher levels than the deficient PV replicon (pRLuc-181) within 15 h of expression, the luciferase activity of the replication-competent HAV replicon (pT7-18f-LUC) was only 20% higher than that of the replication-deficient replicon (pT7-18-LUCmut). The luciferase activities of both deficient replicons were of the same order of magnitude. At later time-points (4060 h p.t.), the luciferase activity of the replication-competent HAV replicon was up to twofold higher than that of the replication-deficient replicon (not shown). These results clearly indicate that only a small fraction of the luciferase activity is translated from replicated HAV RNA and that the majority of reporter activity originates from translation of input RNA. Although we cannot completely rule out the possibility that PV and HAV RNA differ in their half-lives, our data are in accordance with other reports that showed similar translation efficiencies of the replication-deficient PV and HAV replicons in cell culture (Bergamini et al., 2000
; Michel et al., 2001
).
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Besides other limiting steps in the virus life-cycle, inefficient and slow HAV RNA replication might contribute to the overall protracted infectious cycle of HAV in cell culture. To assess directly the onset of viral RNA biosynthesis, synthetic transcripts derived from cDNA containing the complete viral genome (Fig. 1, upper panel, A) were transfected into cells and RNA and capsid synthesis were followed by RTPCR and ELISA, respectively. A replication-deficient genome transcribed from a mutated cDNA (pT7-18f-
2B) was used as control for the half-life of the input RNA. Whereas the transfected non-viable HAV RNA derived from pT7-18f-
2B was degraded and no longer detectable by RTPCR 3 days p.t. (Fig. 3B
), newly synthesized viral RNA produced from infectious RNA was detectable in increasing levels starting 4 days p.t. (Fig. 3A
). Viral RNA synthesis was paralleled by antigen production. Combining these data on the relatively short half-life of transfected RNA and the delayed onset of viral RNA synthesis, it can be concluded that the study of HAV RNA replication is severely compromised, if not impossible, when RNA replication is initiated by short-lived in vitro RNA transcripts.
Replication of the HAV replicon after DNA transfection
In order to compensate for the rapid loss of transfected RNA molecules and based on the fact that the biological half-life of transfected DNA is much longer than that of synthetic RNA (Graeber et al., 1998 ), we next assessed replicon amplification after transfecting cDNA into HuhT7 cells. These cells constitutively express T7 RNA polymerase (Schultz et al., 1996
) and therefore allow the sustained in situ production of HAV RNA with high structural fidelity. The cells are susceptible to HAV infection and have been used to rescue infectious HAV from cDNA and RNA with equal efficiency (Kusov & Gauss-Müller, 1999
, and unpublished observation). As shown in Fig. 4
, the reporter gene activity of both competent and incompetent HAV replicons increased in parallel for about 20 h p.t. Whereas the activity of the incompetent replicon slowly decreased over the next 100 h, the activity of the replication-competent HAV replicon reached a peak about 70 h p.t. The data clearly show that amplification of the HAV replicon can be demonstrated after DNA transfection in appropriate cells. Through in vivo production of replicon RNA, a prolonged supply of templates for both translation and replication is produced, which might be an essential prerequisite for weakly replicating RNA to be rescued by recombination (see below). In addition, the data imply that the HAV P1 region is not required for autonomous HAV genome amplification and apparently does not contain a cis-active replication RNA element.
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In a first assessment of the recombination crossover point, we tested various mutants for their ability to be rescued after co-expression with the replication-competent replicon. Based on our earlier work, we were interested in investigating non-viable mutants encoding polyproteins that are not processed at the 3C cleavage sites within the P3 domain (Kusov & Gauss-Müller, 1999 ). In addition, we used pT7-18f-
VP4, pT7-18f-
3D and pT7-18f-3C-D98N as recombination acceptors, in which either VP4 was deleted, 3D was truncated, or the putative 3C RNA binding site was mutated, respectively. In Fig. 1
(upper panel, A), the locations of the lethal mutations in the HAV genome are marked. Table 1
lists the mutants that were co-expressed with both the replication-competent and -deficient donor replicon. Recombination was determined by subpassaging extracts of the transfected cells, followed by RNA amplification and restriction analysis of the P3 region. In none of the co-expression experiments with the replication-deficient replicon pT7-18f-LUCmut was a viable virus detected, again providing clear evidence that recombination directly correlates with the replicon's replication competence. Recombination with the HAV replicon pT7-18f-LUC occurred with all tested genomes that were defective in their polyprotein cleavage. HAV genomes carrying mutations in the RNA binding site of protein 3C or truncated in 3D also underwent recombination with the replicon, whereas the genome with a deletion of VP4 could not be rescued. This latter result is in line with our earlier observation that an HAV genome lacking VP4 is defective in particle formation and thus non-viable (Probst et al., 1999
). In conclusion, complete HAV genomes with an intact P1 domain, but carrying mutations in domain P3 can be rescued by recombination with an actively replicating replicon. Further experiments are necessary to map the crossover site in more detail.
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Discussion |
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Various steps in the virus life-cycle, including uncoating, translation initiation and particle maturation, are thought to contribute to the restricted replication of HAV in cell culture, a striking feature that distinguishes the Hepatoviruses from other genera of the Picornaviridae. Here we provide experimental proof that HAV RNA replication is inefficient and delayed, as evidenced by the in vivo amplification of a replicon and the complete HAV RNA, and might therefore restrict the overall HAV life-cycle. By sensitive RTPCR, de novo-synthesized RNA could be clearly distinguished from input RNA (Fig. 3). Furthermore, low but extended luciferase activity was observed after transfection of either HAV replicon RNA or DNA (Fig. 4
). This is in accordance with a recent report (Yi & Lemon, 2002
) showing that the replication-dependent luciferase activity of an HAV replicon reaches maximal levels 70 h p.t. It can be hypothesized that maximal translation precedes RNA biosynthesis by several hours and possibly with little temporal overlap. In contrast to the HAV replicon, the expression kinetics reported for PV replicons clearly showed that the reporter activity translated from input RNA was surpassed by reporter activity derived from progeny RNA (Andino et al., 1993
) within a few hours p.t. The delayed onset and low efficiency of HAV RNA replication might be due to an inefficient template switch from translation to replication (Gamarnik & Andino, 1998
). Only a small portion of HAV RNA either transfected into cells or in vivo-transcribed from transfected DNA seems to be used as template for negative-strand synthesis with the RNA serving primarily and mostly as template for translation. We favour the notion that RNA synthesis is both poorly initiated and proceeds in a rather inefficient manner. For HAV, neither the putative steps regulating the template switch (e.g. specific binding of viral and host proteins at the 5' end of viral RNA, circularization of the RNA genome) nor the activity of the HAV RC and its components (e.g. 3Dpol) itself have been addressed at the molecular level.
The biological half-life of transfected in vitro RNA transcripts is limited due to unspecific intracellular RNA degradation, and thus their functioning as templates for translation and replication is impaired. As demonstrated in Fig. 3, the apparent half-life of transfected HAV RNA is shorter than the lag period after which significant RNA replication can be detected. In contrast, transfected DNA is highly stable and functional in transcription for several days p.t. (Graeber et al., 1998
). For viral and non-viral cDNAs, we confirmed the long-term expression of T7-promoted genes in cells either constitutively expressing T7 RNA polymerase or infected with a helper virus expressing T7 RNA polymerase (e.g. vaccinia virus MVA-T7; Kusov et al., 2002
). Besides its longer biological half-life, it can be assumed that the RNA in vivo-transcribed from transfected DNA has improved translation efficiency compared with transfected RNA that had been produced by in vitro transcription. In order to circumvent the conflict of low RNA stability with the delayed onset of RNA replication, DNA transfection was used for the studies on genetic recombination. This experimental approach is also supported by a report that emphasizes the need for sufficiently high concentrations of RNA for recombination (Kirkegaard & Baltimore, 1986
). As deduced from detailed studies described earlier (Evans et al., 1988
), plasmid DNA recombination does not occur due to the lack of extrachromosomal DNA polymerization. Our recombination experiments showed a direct correlation between the rescue of viable virus and the replication competence, clearly indicating that the genetic recombination is an event tightly associated with replication.
A model for the in vivo HAV recombination system presented here is shown in Fig. 7 with the rescued genetically marked genome in the middle. Assuming homologous recombination as the most common mechanism, and based on studies on poliovirus (Jarvis & Kirkegaard, 1992
), we propose that during negative-strand synthesis, the RC formed after translation of the donor RNA (replicon) switches templates and continues to copy the acceptor RNA encoding capsid proteins. In contrast to recombination systems described elsewhere (Duggal et al., 1997
; Tang et al., 1997
), we identified the recombination product by its ability to form viral particles, i.e. its competence to complete the full virus life-cycle and to spread from cell to cell. This property is a crucial prerequisite for the analysis of HAV replication described here, since the detection of newly formed viral genomes has been difficult under single-cycle growth conditions (Beard et al., 2001
).
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We thank B. Kölsche for excellent technical assistance and R. Andino for the poliovirus replicons. pT7-18f-2B was provided by M. Jecht. We are grateful to S. Lemon for HuhT7 cells and to T. Morris for critical reading of the manuscript. This work was supported by a grant (SFB 367, project B7) from the Deutsche Forschungsgemeinschaft (DFG).
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References |
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Received 28 February 2002;
accepted 8 May 2002.