Heterologous gene expression by infectious and replicon vectors derived from tick-borne encephalitis virus and direct comparison of this flavivirus system with an alphavirus replicon

Rainer Gehrke1, Franz X. Heinz1, Nancy L. Davis2 and Christian W. Mandl1

1 Institute of Virology, Medical University of Vienna, Kinderspitalgasse 15, A-1095 Vienna, Austria
2 Department of Microbiology and Immunology, School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA

Correspondence
Christian W. Mandl
christian.mandl{at}univie.ac.at


   ABSTRACT
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The flavivirus tick-borne encephaltis virus (TBEV) was established as a vector system for heterologous gene expression. The variable region of the genomic 3' non-coding region was replaced by an expression cassette consisting of the reporter gene enhanced green fluorescent protein (EGFP) under the translational control of an internal ribosomal entry site element, both in the context of an infectious virus genome and of a replicon lacking the genes of the surface proteins prM/M and E. The expression level and the stability of expression were measured by fluorescence-activated cell-sorting analysis and compared to an established alphavirus replicon vector derived from Venezuelan equine encephaltis virus (VEEV), expressing EGFP under the control of its natural subgenomic promoter. On the first day, the alphavirus replicon exhibited an approximately 180-fold higher expression level than the flavivirus replicon, but this difference decreased to about 20- and 10-fold on days 2 and 3, respectively. Four to six days post-transfection, foreign gene expression by the VEEV replicon vanished almost completely, due to extensive cell killing. In contrast, in the case of the TBEV replicon, the percentage of positive cells and the amount of EGFP expression exhibited only a moderate decline over a time period of almost 4 weeks. The infectious TBEV vector expressed less EGFP than the TBEV replicon at all times. Significant expression from the infectious vector was maintained for four cell-culture passages. The results indicate that the VEEV vector is superior with respect to achieving high expression levels, but the TBEV system may be advantageous for applications that require a moderate, but more enduring, gene expression.


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Positive-stranded RNA viruses belonging to various viral families have been used to develop vectors for heterologous gene expression. In principle, such viral vectors can be generated in two ways: either in the form of infectious mutants (Dufresne et al., 2002; Jia et al., 2002; Yun et al., 2003) or non-infectious replicons (Hewson, 2000; Khromykh, 2000; Lundstrom, 2003). In the first case, the foreign gene is inserted into the viral genome in addition to all of the viral genes that are necessary for a productive viral life cycle. In this approach, the vector spreads autonomously and thus can carry the gene of interest to a large number of potential host cells. Clear limitations of this approach derive from the potential pathogenicity of the vector. In contrast, replicons contain only the genomic information that is necessary to establish RNA replication and translation, but are incapable of forming infectious virus progeny. Replicons are commonly generated by deleting part or all of the genetic information that encodes viral structural proteins from the genome. By providing the missing structural proteins in trans, replicons can be packaged into virus-like particles (VLPs) that are capable of establishing only a single round of infection. Due to their safety and simplicity, RNA replicons have recently received much attention as molecular tools for a variety of in vitro and in vivo applications (Khromykh, 2000; Lundstrom, 2001; Schlesinger, 2001).

The most advanced replicon gene-expression systems are derived from members of the genus Alphavirus, family Togaviridae, i.e. Sindbis virus, Semliki Forest virus and Venezuelan equine encephalitis virus (VEEV) (Lundstrom, 2001; Perri et al., 2003; Schlesinger, 2001). Alphavirus expression systems take advantage of the efficient subgenomic promoter of these viruses, which normally drives the expression of the viral structural proteins. In alphavirus replicon vectors, the structural genes are replaced by the heterologous gene and this approach usually yields high expression levels of the foreign gene. Alphavirus vectors are used widely as vehicles for gene expression in cell culture and are being tested as experimental vaccines against various pathogens (Balasuriya et al., 2002; Davis et al., 2000; Gipson et al., 2003; Hanke et al., 2003; Hevey et al., 1998; Vajdy et al., 2001).

In contrast to alphaviruses, flaviviruses, i.e. members of the genus Flavivirus, family Flaviviridae, do not make subgenomic RNAs, but express all structural (C, prM/M and E) and non-structural (NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5) proteins as a single polyprotein that is co- and post-translationally cleaved into the individual proteins by the action of viral and cellular proteases (Lindenbach & Rice, 2001). Heterologous gene expression in flavivirus vectors can be achieved either by inserting the foreign gene in-frame with the viral proteins, flanked by appropriate proteolytic cleavage sites, or by adding to the genome a second cistron that is translationally controlled by an internal ribosomal entry site (IRES). Replicons derived from Kunjin virus, a mosquito-transmitted Australian flavivirus that is related closely to West Nile virus, have been developed during the past few years into a well-established and practical expression system (Varnavski & Khromykh, 1999; Varnavski et al., 2000). The recent advance of an efficient packaging system has further contributed to the value of Kunjin virus vectors as vehicles for in vivo applications (Harvey et al., 2004). Heterologous genes have also been expressed from replicons of other flaviviruses, such as yellow fever virus (Molenkamp et al., 2003), West Nile virus (Lo et al., 2003a), dengue virus (Pang et al., 2001) and tick-borne encephalitis virus (Hayasaka et al., 2004). One study has highlighted the potential of such replicons not only for vaccination purposes, but also as screening systems for the development of antiviral substances (Lo et al., 2003b). The infectious-vector approach, on the other hand, has only in a single case been used to express foreign genes from a flavivirus, i.e. Japanese encephalitis virus (Yun et al., 2003).

In this study, we demonstrate expression of a reporter gene (enhanced green fluorescent protein, EGFP) by both infectious and replicon vectors derived from tick-borne encephalitis virus (TBEV), a tick-transmitted flavivirus that is endemic in many regions of Europe and Asia. Based on our studies with TBEV, we provide a direct comparison of the infectious-virus and replicon approaches with regard to the level and duration of heterologous gene expression. Moreover, we compare this flavivirus system with an established alphavirus replicon vector derived from VEEV (Balasuriya et al., 2000; Pushko et al., 1997). Quantitative evaluation reveals that heterologous gene expression from the TBEV replicon reaches its maximum level later and remains at least 10-fold lower than that from the VEEV vector. In contrast to the VEEV vector, however, which destroys its host cell within a few days, expression by the TBEV vector is maintained at a significant level over a long time period.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Plasmids.
All plasmids containing cDNA corresponding to the genome of TBEV were derived from Western subtype prototypic strain Neudoerfl (GenBank accession no. U27495). Plasmid pTNd/c contains a full-length copy of the genome under the control of a T7 promoter, from which infectious RNA can be transcribed in vitro (Mandl et al., 1997). Plasmid pTNd/3' contains cDNA corresponding to the 3'-terminal approximately two-thirds of the genome (Mandl et al., 1997). Plasmid pTNd/{Delta}ME is a derivative of pTNd/c lacking the genes encoding the two structural proteins prM/M and E and is described in detail elsewhere (Gehrke et al., 2003).

Plasmid pVR21-EGFP contains cDNA corresponding to a replicon derived from the alphavirus VEEV expressing EGFP under the control of the subgenomic promoter, as described originally by Pushko et al. (1997) with modifications described by Balasuriya et al. (2000). As is the case with the TBEV-specific plasmids, pVR21 contains bacterial plasmid sequences derived from pBR322 and a functional T7 promoter immediately upstream of the VEEV 5' non-coding region.

Plasmid pIRES2-EGFP was purchased from Clontech and contains the EGFP gene under the control of an encephalomyocarditis virus IRES sequence.

Manipulations of DNA and RNA.
The construction of EGFP-expressing TBEV vectors was performed in three steps. First, the variable region of the 3' non-coding region in plasmid pTNd/3' was replaced by an artificial multiple-cloning site (MCS), yielding plasmid pTNd/3'-MCS. This was achieved by taking advantage of unique cleavage sites for the restriction enzymes BssHII (at genome position 9880 in the NS5 gene) and AgeI (located at position 10796, exactly at the boundary between the variable and core regions of the 3' non-coding region). The BssHII–AgeI fragment was replaced by a PCR-generated fragment extending from the BssHII site to the stop codon terminating the long open reading frame (ORF; position numbers 10375–10377), followed by artificial recognition sites for the enzymes SacII, AflII, NotI and AgeI. The primer sequences for this PCR were 5'-CCCTGGTGGTGCCGTGCCGA-3' (corresponding to TBEV genome positions 9836–9855) and 5'-TTACCGGTGCGGCCGCTTAAGCCGCGGTTAGATTATTGAGCTCTCCA-3' (nucleotides complementary to genome positions 10358–10377 are shown in italics; the antisense stop codon is shown in bold; recognition sites for the restriction enzymes AgeI, NotI, AflII and SacII are underlined). In a second cloning step, an IRES–EGFP cassette obtained by SacII and NotI digestion of plasmid pIRES2-EGFP (Clontech) was introduced into the MCS of plasmid pTNd/3'-MCS via the corresponding restriction sites, yielding plasmid pTNd/3'-EGFP. Finally, the BssHII (9880)–NheI (11141) fragment from pTNd/3'-EGFP was recovered and used to replace the corresponding fragments of plasmids pTNd/c or pTNd/{Delta}ME to yield the final plasmids pTNd/c-EGFP and pTNd/{Delta}ME-EGFP, respectively.

All plasmids were amplified in Escherichia coli HB101 and purified with commercially available systems (Qiagen). Sequence analysis of all PCR-derived fragments and surrounding recognition sites for restriction enzymes was performed in both orientations with an automated DNA sequencing system (Applied Biosystems) to confirm the presence of the desired sequences.

RNA was transcribed from 1 µg aliquots of plasmids pTNd/c-EGFP, pTNd/{Delta}ME-EGFP and pVR-21-EGFP by T7 polymerase transcription, using commercially available reagents (Ambion) and conditions described in detail elsewhere (Mandl et al., 1997). RNA was introduced into BHK-21 cells by electroporation with a Bio-Rad Gene Pulser, as described previously (Gehrke et al., 2003; Mandl et al., 1997).

Cell culture.
BHK-21 cells were grown in minimal essential medium supplemented with 5 % fetal calf serum (FCS). After RNA transfection, cells were seeded into six-well cluster plates and analysed for the expression of EGFP either by fluorescence microscopy or fluorescence-activated cell-sorting (FACS) analysis. To monitor the stability of EGFP expression from replicons, cells were grown to confluence and split at a ratio of 1 : 5 every 4 days. To analyse EGFP-expressing virus, supernatants were cleared from insoluble material by low-speed centrifugation and 1 ml aliquots were transferred to fresh cells every 4 days.

FACS analysis.
EGFP expression was measured with a FACScalibur flow cytometer (Becton Dickinson; 15 mW argon laser, 488 nm) with a 530/30 bandpass filter (FL-1) analysing 10 000 events per sample. For FACS analysis, BHK-21 cells were detached with trypsin from six-well cluster plates after rinsing them once with PBS and resuspended in 1 ml PBS supplemented with 5 % FCS. Measurement of forward scatter versus side scatter was performed routinely to exclude alterations of cell sizes and to confirm viability of the cells. Commercially available BD FACS EGFP calibration beads (BD Biosciences, Clontech) yielding standardized fluorescence intensities were used as internal controls along with each dataset. FL-1 geometric mean values were calculated by using CellQuestPro Software (Becton Dickinson). For quantitative comparison of expression levels, mean values were calculated for the 5 % of the cell population that exhibited the brightest EGFP fluorescence. Calculations including larger percentages of cells yielded lower absolute numbers, but did not change the proportions between the individual samples. For comparison of the VEEV replicon with the TBEV vectors, the instrument settings were adjusted to keep the bright fluorescence obtained from the VEEV vector within the measurable range of the flow cytometer. In all other experiments, untransfected cells were used as a reference for instrument settings. To determine the percentage of EGFP-expressing cells in a cell population, the cut-off value was set to exclude at least 99 % of the untransfected control cells.


   RESULTS
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Construction of EGFP-expressing TBEV vectors
The genome of TBEV consists of a single, long ORF flanked by non-coding regions (Fig. 1). The 3' non-coding region has two distinct domains, referred to as the ‘core element’ and the ‘variable region’ (Wallner et al., 1995). The core element is located at the genomic 3' terminus and shows high sequence conservation among strains of TBEV. It probably forms specific RNA structures and presumably mediates crucial functions of the viral life cycle (Proutski et al., 1997; Rauscher et al., 1997). In contrast, the variable region, which is located between the ORF and the core element, exhibits high sequence variability, including the presence of an internal poly(A) tract in some strains (Wallner et al., 1995), and its removal was previously shown to have no detectable impact on viral growth properties in cell culture or mice (Mandl et al., 1998). In this study, we replaced the variable region of TBEV strain Neudoerfl by an IRES–EGFP expression cassette, which is about 900 nt longer than the replaced genomic fragment. This modification was performed both within the context of the full-length infectious cDNA clone pTNd/c (Mandl et al., 1997) and the recently described replicon clone pTNd/{Delta}ME (Gehrke et al., 2003). In vitro transcription of pTNd/c generates RNA that yields infectious TBEV when introduced into host cells, whereas RNA generated from pTNd/{Delta}ME replicates, but, due to a deletion of the surface-protein genes prM/M and E, is incapable of forming infectious virions. The corresponding IRES–EGFP-containing plasmids were named pTNd/c-EGFP and pTNd/{Delta}ME-EGFP, respectively.



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Fig. 1. Expression vectors derived from TBEV. Top, schematic drawing of the TBEV genome, showing the long ORF encoding the structural proteins C, prM and E and the non-structural proteins NS1–NS5, flanked by short non-coding regions (NCRs). In the replicon vector, the prM and E genes are deleted (shaded). Bottom, expanded view of the 3'-terminal part of the genome, depicting the two domains of the 3' NCR. The positions of the stop codon and restriction sites that were used during constructions of the vectors are shown (nucleotide numbers refer to the wild-type genome of TBEV, GenBank accession no. U27495). The expression vectors were generated by replacing the variable region of the 3' NCR by an IRES–EGFP cassette, as indicated. The schematic is not drawn to scale.

 
To test the ability of these TBEV vectors to express the heterologous gene in cell culture, RNA was transcribed in vitro from plasmids pTNd/c-EGFP and pTNd/{Delta}ME-EGFP and introduced into BHK-21 cells by electroporation. For comparison, a VEEV alphavirus replicon encoding the same reporter gene under the control of its own viral subgenomic promoter was transcribed from plasmid pVR21-EGFP (Balasuriya et al., 2000) and transfected into BHK-21 cells. Fluorescence microscopy of the cell cultures indicated that all three constructs expressed the reporter gene, albeit at apparently different levels (Fig. 2). In contrast to the two TBEV constructs, the VEEV vector induced a pronounced cytopathic effect (CPE).



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Fig. 2. EGFP expression from TBEV and VEEV vectors. The same microscopic field is shown, as viewed by phase-contrast light microscopy (top) or fluorescence microscopy (bottom). BHK-21 cells 3 days post-transfection with RNA synthesized from plasmids pTNd/{Delta}ME-EGFP (a), pTNd/c-EGFP (b) or pVR21-EGFP (c), and 3 days after inoculation with supernatant from cells harbouring RNA derived from pTNd/c-EGFP (d). (e) Untransfected negative-control cells.

 
To determine whether RNA transcribed from the full-length construct pTNd/c-EGFP was packaged into infectious virus particles, aliquots of supernatants of the corresponding cells were transferred to fresh cells. Expression of EGFP in these cells (Fig. 2) demonstrated that cells transfected with this construct did indeed produce infectious virus expressing the additional heterologous gene. In contrast, supernatants from cells transfected with the replicon RNA derived from pTNd/{Delta}ME-IRES were not able to initiate EGFP expression when transferred to fresh cells (data not shown).

Quantitative comparison of EGFP expression
To compare the efficiency of reporter-gene expression between the infectious and replicon TBEV vectors and the VEEV replicon vector, BHK-21 cells transfected with the corresponding RNAs were subjected to a standardized FACS analysis based on the quantitative determination of EGFP fluorescence for a constant number of cells. Fig. 3 shows results obtained at days 1, 2 and 3 post-transfection. Consistent with the visual intensity shown in Fig. 2, the highest expression level was achieved with the VEEV replicon. In comparison, EGFP expression by the TBEV vectors was significantly lower and exhibited a delayed onset, with an approximately 10-fold increase of mean fluorescence values from days 1 to 2. On day 1, the difference between the VEEV replicon and the TBEV replicon was 175-fold, whereas on days 2 and 3, the differences in expression were not as large, amounting to only 24- and ninefold, respectively. Infectious TBEV produced between two- and eightfold less EGFP than the TBEV replicon at all times but, again, the biggest difference was observed on the first day. A comparison of the two TBEV vectors over an extended time period is described below. A quantitative evaluation of the VEEV expression level at later time points was not possible because of the rapid destruction of cells by CPE and the resulting disappearance of expressing cells from the total cell population, as analysed in more detail in the following section.



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Fig. 3. Comparison of heterologous gene-expression levels achieved with VEEV alphavirus replicon (filled bars), TBEV flavivirus replicon (open bars) and TBEV infectious flavivirus (hatched bars). Expression of the reporter gene EGFP was determined by FACS analysis on days 1, 2 and 3 after introduction of the RNA vectors into BHK-21 cells by electroporation. Mean values from two independent experiments are shown (error bars represent minimum–maximum values). Fluorescence intensities of the 5 % of the total population of cells that exhibited the brightest fluorescence were evaluated. Instrument settings were adjusted to maintain the fluorescence values obtained from all three vectors within the measurable range of the flow cytometer.

 
Persistence of cells harbouring EGFP-expressing replicons
As already observed by light microscopy and in accordance with published data (Frolov & Schlesinger, 1994; Khromykh, 2000), the alphavirus replicon clearly appeared to be more cytopathic than the flavivirus replicon. For a direct and quantitative comparison, each replicon was introduced into BHK-21 cells as before and the percentage of EGFP-containing cells was monitored by FACS analysis over a prolonged time period (Fig. 4). To normalize values obtained from independent experiments with different electroporation efficiencies (typically, between 60 and 90 % of the cells were transfected with RNA by electroporation), the maximum number of EGFP-expressing cells obtained in each experiment was set as 100 % and the decline of the proportion of positive cells was calculated in relation to this number. As shown in Fig. 4, within 7 days post-transfection, the percentage of positive cells in the VEEV samples decreased rapidly to almost zero, due to extensive cell death. In contrast, in the case of the TBEV-replicon samples, the percentage of positive cells exhibited only a slow but steady decline, which may indicate that cells harbouring the replicon were dividing at a somewhat slower rate than cells without the replicon or that the replicon was not always passed on to both daughter cells during cell division. However, there was no distinct CPE observed in the TBEV-replicon samples, suggesting that, in this case, cell killing had little, if any, impact on the decline rate of positive cells.



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Fig. 4. Endurance of EGFP expression from the VEEV replicon (closed circles) or the TBEV replicon (open circles). BHK-21 cells were transfected with the corresponding RNAs, harvested at various time points and aliquots containing identical numbers of cells were subjected to FACS analysis to distinguish negative cells from cells expressing EGFP. Mean values derived from two independent experiments (VEEV replicon) or four independent experiments (TBEV replicon) are plotted (error bars depict minimum–maximum values). In the course of this experiment, cell cultures were split 1 : 5 three times at the time points indicated by arrows.

 
Stability of EGFP expression from TBEV vectors
RNA replication is known to yield random mutations that may affect the expression of the heterologous gene product. As expression of EGFP does not provide any selective advantage, such mutations are expected to accumulate over time and ultimately lead to the cessation of foreign-gene expression. This process was monitored by standardized FACS analysis for both the TBEV replicon and the infectious TBEV vector to assess the genetic stability of these two systems. The results of this analysis are shown in Fig. 5. The time points at which either the cells harbouring the replicon were split or the infectious virus was transferred (at a high m.o.i.) to fresh cells are indicated. In agreement with the data discussed above, the expression levels of both vectors increased within the first 2 days and the infectious vector expressed less EGFP per cell than the replicon vector at all times. After a plateau phase of approximately 1 week, the fluorescence intensities declined with time. In the case of the replicon, the decline rate stayed about the same over the entire observation period, up to day 27. In contrast, the infectious virus vector started with a similar decline rate but, after the third viral passage at day 12, the fluorescence decreased more rapidly to background value around day 20. At this time, 100 % of cells were still carrying infectious TBEV, as revealed by the transfer of supernatants to fresh cells and immunofluorescence staining of cells with antibodies recognizing viral proteins (data not shown). This virus, however, no longer expressed functional EGFP. An independent repetition of this experiment yielded essentially the same result, confirming that, in the case of the infectious vector, EGFP-negative mutants arise and outgrow the original virus in the course of several cell-culture passages.



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Fig. 5. Stability of heterologous gene expression from the TBEV replicon (open circles) and the infectious TBEV vector (closed triangles). Expression of the EGFP reporter gene was monitored by FACS analysis and the mean fluorescence intensities of the brightest 5 % of the cells are plotted. Cell cultures transfected with replicon were split 1 : 5 and aliquots of supernatants from cells harbouring the infectious vector were transferred to fresh cells at the time points indicated by arrows. The level of autofluorescence exhibited by negative-control cells is indicated by a dashed line. Note that instrument settings are different from those used in the experiments shown in Fig. 3.

 

   DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Expression systems derived from alphaviruses and flaviviruses share some advantageous properties that make them attractive molecular tools. Their unsegmented, positive-stranded RNA genomes can be manipulated easily by established reverse-genetics techniques. They are capable of replicating in a wide range of host cells and replication and gene expression take place exclusively in the cytoplasm, excluding the possibility of an inadvertent chromosomal integration of foreign genetic material. Potential applications include not only the development of vaccines against various infectious agents, but also anti-cancer therapies (Anraku et al., 2002; Daemen et al., 2003), in vitro expression studies (Molenkamp et al., 2003), screening for antiviral substances (Lo et al., 2003b) and RNA interference (Uhlirova et al., 2003). The three alphavirus systems derived from Sindbis virus (Bredenbeek et al., 1993), Semliki Forest virus (Liljeström & Garoff, 1991) and VEEV (Pushko et al., 1997) have been extensively characterized and improved over the past several years, including the development of non-cytopathic Sindbis virus vectors (Agapov et al., 1998) and particle chimeras (Perri et al., 2003). VEEV replicon vectors, such as the one used in this study, have been used as experimental vaccine vectors against a wide variety of pathogens (Balasuriya et al., 2002; Davis et al., 2000; Gipson et al., 2003; Hevey et al., 1998). Expression vectors derived from mosquito-borne flaviviruses have been developed more recently; in particular, the Kunjin virus system has been tested in a variety of experimental applications (Hall et al., 2003; Harvey et al., 2003; Westaway et al., 2003).

In this study, we constructed and evaluated infectious and non-infectious expression vectors from TBEV, a tick-borne flavivirus that differs from its mosquito-transmitted relatives in a number of molecular and biological properties (Lindenbach & Rice, 2001; Mandl et al., 1993). We previously described the generation of subgenomic replicons of TBEV and demonstrated that a TBEV replicon lacking the proteins prM/M and E could be packaged into VLPs by trans-complementation using a cell line (CHO-ME cells) that expressed these two surface proteins constitutively (Gehrke et al., 2003). By using a different strain of TBEV (belonging to the Far Eastern subtype of TBEV), Hayasaka et al. (2004) recently also produced replicons and demonstrated expression of heterologous genes under the control of an IRES element inserted into the 3' non-coding region. Four days post-transfection, EGFP expression could be demonstrated in this system in <5 % of cells. Another replicon expressing a selectable marker protein (neomycin phosphotransferase) could be maintained under selection pressure in transfected cells for at least 50 days. In good agreement with our observations, the replicons derived from the Far Eastern subtype TBEV strain induced no apparent CPE in BHK-21 cells. Our study extends the evaluation of TBEV as a potential new expression vector by providing a quantitative comparison of the level and endurance of heterologous gene (EGFP) expression in the absence of a selectable marker in individual cells, achieved from infectious or non-infectious TBEV vectors, with expression of the same gene from an established non-infectious alphavirus vector.

The growing number of expression vectors calls for a direct comparison of their properties to enable a rational choice of the most appropriate vector for each particular application. Therefore, we performed a direct comparison between a well-established VEEV replicon and the newly developed TBEV replicon. From these results, it is clear that the alphavirus replicon used in this study is superior with respect to short-term expression, yielding a significantly higher expression level within the first 3 days (the difference decreased from 180-fold at day 1 to 10-fold at day 3). However, with respect to long-term expression, the TBEV-based system has the advantage that host cells carrying this replicon were able to survive and maintain expression for almost 4 weeks, and our data suggest that even longer expression times can be achieved even in the absence of a selection marker. Cells carrying the replicon underwent cell division at a rate similar to that of untransfected cells and distributed the replicon to their daughter cells. This indicates that the flavivirus causes little disturbance of fundamental host-cell functions. Expression levels were found to decline moderately at a steady rate, possibly reflecting inactivation of the IRES–EGFP cassette by randomly occurring mutations. The alphavirus replicon would thus be the vector of choice for achieving a transiently high level of expression, whereas the TBEV vector might be considered for applications where long-term expression and prolonged survival of the host cells are required. Sustained expression, however, has also been achieved with modified, non-cytopathic alphavirus replicons (Agapov et al., 1998; Frolov et al., 1999; Kong et al., 2002; Perri et al., 2000). Studies on flavivirus replicons derived from Kunjin virus have shown that insertion of an IRES–heterologous gene cassette into the 3' non-coding region reduces replication efficiency of the replicon RNA (Khromykh & Westaway, 1997). Moreover, expression of heterologous genes from the Kunjin viral promoter by replacing the genes encoding the structural proteins with the foreign gene yielded expression levels that were comparable to those obtained with two alphavirus systems, derived from Sindbis virus and Semliki Forest virus (Varnavski & Khromykh, 1999; Varnavski et al., 2000). Thus, it may also be possible to construct TBEV-derived vectors with higher expression efficiencies than those described in this study. Clearly, a larger number of comparative studies among the various vector systems will be necessary to provide a rational basis for choosing the optimal system for a particular application.

The infectious TBEV expressing EGFP was genetically stable for a few cell-culture passages. This finding demonstrated that substitution of the variable region of the 3' non-coding region by a foreign genetic element, which is more than three times larger and increases the total genome length by approximately 8 %, is still compatible with replication and packaging of the genome. Interestingly, the expression level achieved by infectious virus was somewhat lower than that observed with the replicon and increased more slowly to its maximum value. This suggests that the replicon RNA had possibly multiplied faster and to higher copy numbers within the cell than the full-length genome. In contrast to the replicon, expression by the infectious virus did not decline at a constant rate but, after a period of steady decline, expression vanished rapidly. We hypothesize that mutants carrying spontaneous deletions within the IRES–EGFP cassette may have emerged and, due to somewhat better replication kinetics and/or packaging efficiency, outgrew the original vector virus. In contrast, similar mutations may also have occurred in the replicon but, as these could not spread to other cells, this had little effect on the overall EGFP-expression level in the total cell population. This leads to the conclusion that the stability of expression with the infectious TBEV vector depends on both the mutation rate and the impact that the insert has on the evolutionary fitness of the vector. In contrast, with the replicon vector, the stability of genes that do not provide a selective advantage or disadvantage for the host cell is determined mainly by the RNA-mutation rate of the viral polymerase. Recent work with yellow fever chimeric virus suggests that the flavivirus RNA polymerase exhibits a relatively high fidelity, compared with those of other RNA viruses (Pugachev et al., 2004); this may turn out to be a relevant advantage of flavivirus RNA-expression systems.

The TBEV expression system described in this study provides a tool for introducing heterologous genes into a wide variety of cells that support replication of this virus. In particular, the tropism of TBEV for ticks and tick-derived cell lines (Lawrie et al., 2004) can make this expression system a valuable tool for studying these arthropod vectors that, in addition to TBEV, also transmit other relevant human diseases, such as Lyme disease. Due to its inherent pathogenicity, the use of infectious TBEV vectors will certainly be restricted in terms of clinical applications, but will nevertheless be useful for in vitro and in vivo studies on assembly, tropism and pathogenesis of this flavivirus, as well as for the development of new antiviral agents.


   ACKNOWLEDGEMENTS
 
We are grateful to Ursula Sinzinger for expert technical help with FACS analysis. This work was supported by the Austrian ‘Fonds zur Förderung der wissenschaftlichen Forschung’, FWF project no. P16376-B04.


   REFERENCES
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
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Received 7 October 2004; accepted 21 December 2004.