Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, UK1
Oxford BioMedica (UK) Ltd, The Medawar Centre, The Oxford Science Park, Oxford OX4 4GA, UK2
Author for correspondence: Alan Kingsman (at Oxford BioMedica). Fax +44 1865 783001. e-mail A.Kingsman{at}OxfordBiomedica.co.uk
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Abstract |
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Introduction |
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In retroviral vectors, the three components, genome, Gag/GagPol and envelope, are generally dissociated in order to minimize the possibility of production of replication-competent virus. This dissociation has traditionally been achieved by integrating the separate cassettes into the genome of a cell line to construct a producer cell. This strategy inevitably disrupts the regulatory systems that have evolved, presumably, to deliver optimum ratios of components that are present in a replication-competent virus. This may lead to suboptimal levels of vector produced from producer cells, a situation that is relatively complex to correct in an integrated expression system. In recent years, however, transient methods of retrovirus vector production have been developed. In these systems, viral components are segregated into three different plasmids that are used in short-term transfections (Soneoka et al., 1995 ). Typically, vector particles are harvested 2 days post-transfection. This technology now makes it possible to study the effect of the amount of each component on virus production simply by varying the amounts of the three plasmids used in the experiment.
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Methods |
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Cell culture.
293T and 293 cells were maintained in DMEM containing 10% foetal calf serum and 1% penicillin, streptomycin and L-glutamine. NIH3T3 cells were maintained in DMEM containing 10% newborn calf serum and 1% penicillin, streptomycin and L-glutamine. 293/12 cells were derived from 293 cells and expressed the murine ecotropic MLV receptor (Ragheb et al., 1995 ). They were a kind gift from Richard Tun and Paula Cannon (University of Southern California School of Medicine, Los Angeles, USA).
Virus production and titration.
Viral vectors were produced by a transient transfection system that has been described previously (Soneoka et al., 1995 ). The virus stocks were harvested 48 h post-transfection and titrated on NIH3T3 cells in 6-well plates. The transduced cells were stained for
-galactosidase 48 h post-transduction as described previously (Soneoka et al., 1995
).
Western blot analysis.
The anti-p15, anti-gp70 and anti--galactosidase antibodies were purchased from Serotec, Quality Biotech and Sigma, respectively. Five hundred µl virus supernatant was pelleted in a microfuge at 15000 r.p.m. for 30 min at 4 °C. The pellet was washed with PBS and centrifuged at 15000 r.p.m. for another 30 min. It was then boiled for 5 min in SDS loading buffer and loaded onto a 12·5% SDSPAGE gel. Cells were lysed in reporter lysis buffer from Promega. The lysate was clarified by centrifugation and 40 µg protein was loaded in each well. The separated protein bands were transferred onto an Immobilon-P membrane (Millipore) using the transfer apparatus from Bio-Rad. The membrane was blocked overnight in PBS Tween (PBS-T) containing 10% skimmed milk. It was then hybridized for 1 h at room temperature with the primary antibody, which had been diluted 1:1000 with PBS-T containing 5% skimmed milk. This was followed by four washes with PBS-T containing 0·5% skimmed milk before hybridization with the peroxidase-conjugated secondary antibody for 1 h at room temperature. Finally, the membrane was washed four times with PBS-T containing 0·5% skimmed milk and the bands were detected by using the chemiluminescence detection kit from Amersham.
Reverse transcriptase (RT) assay.
The assay for RT was performed according to Goff et al. (1981) . Briefly, 90 µl virus supernatant was added to 10 µl 10x RT buffer (500 mM TrisHCl, pH 8·3; 0·5% NP-40; 50 µg/ml oligodeoxythymidylic acid; 100 µg/ml polyadenylic acid; 200 mM DTT; 6 mM MnCl2; 600 mM NaCl) containing 10 µCi [
-32P]dTTP (Amersham) and incubated at 37 °C for 2 h. Five µl of each reaction was then spotted onto DEAE paper (DE-81, Whatman) and air-dried. The paper was washed twice in 0·6 M NaCl, 0·06 M sodium citrate for 15 min each and then in 95% ethanol for 15 min. It was then air-dried and exposed overnight to an X-ray film.
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Results |
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The negative effect of gag/gagpol on virus titre was not due to infection interference by defective particles
In order to test the hypothesis that the decrease in virus titre observed during increased Gag/GagPol expression was due to empty enveloped particles, which might have saturated the target-cell receptors, or to bald particles, which bound non-specifically to the target cells and obstructed binding of transducing particles, empty particles containing amphotropic and ecotropic envelopes were produced by transfecting 293T cells with 1 µg pHIT60 and 0·1 µg pHIT456 (amphotropic envelope) or pHIT123 (ecotropic envelope). Bald particles were also produced by transfecting 293T cells with 1 µg pHIT60. Five hundred µl of the defective virus stock was mixed with 500 µl of the virus stock produced by using 0·1 µg of all three plasmids (Table 2). The resulting virus mixture was titrated on NIH3T3 cells. The results showed that there was no decrease in titre when either bald, amphotropic or ecotropic empty particles were present in the virus stocks. This suggested that the decrease in titre was not due to obstruction of receptors by defective particles. Given this observation, it seemed likely that the reduced titre was due to events that occurred during virus production in the transfected cells and which affected the quality of the virus particles.
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The negative effect of the gag/gagpol component on virus titre was only manifested at low levels of genome and envelope
The data in Table 1 (expts 3, 4, 7 and 8) show that increasing the level of the gag/gagpol plasmid from 0·1 µg to 1·0 µg did not lead to a reduction in titre when either the amphotropic envelope or genome component was present at high concentrations. The titre of around 104 was significantly greater than that observed when both amphotropic envelope and genome were low, but not as high as when both genome and amphotropic envelope were high. This is completely compatible with the hypothesis described above. Increasing either amphotropic envelope or genome would increase the probability of both being present on a particle, but increasing both would yield the product of those increased probabilities, with associated increases in titre.
The negative effect of gag/gagpol on virus titre was dependent on the type of envelope used
The amphotropic envelope was found to be limiting during virus production. One explanation for this observation was that a significant proportion of envelope protein was taken out of the pool that was available for incorporation due to premature interaction with their receptors in the producer cells. This could be tested by using an envelope protein the receptor of which was not expressed in the producer cells. If the hypothesis were true, this envelope would not be limiting during virus production.
The ecotropic envelope only mediates productive infection of murine cells. Its receptor has been identified and has been shown to be absent in human cells (Albritton et al., 1989 ). The absence of receptor for the ecotropic envelope in 293T cells was confirmed by transducing the cells with either amphotropic or ecotropic particles containing the pHIT111 genome. While the amphotropic particles yielded titres of 4·3±0·4x106, the cells were not transduced by the ecotropic particles, indicating the absence of the ecotropic receptor.
The transduction experiments described above were repeated with pHIT123 (ecotropic envelope expression plasmid) in place of pHIT456. The results are presented in Table 1. As with the amphotropic envelope, increasing the amount of genome plasmid from 0·1 to 1·0 µg resulted in a 24-fold increase in titre, indicating that the genome component was limiting. In addition, increasing the amount of genome plasmid brought the titre close to that achieved with 1 µg of all three plasmids, suggesting that genome was the only limiting component. This was confirmed by the fact that increasing the amount of envelope plasmid from 0·1 to 1·0 µg did not bring about a significant increase in titre. Hence, unlike the amphotropic envelope, the ecotropic envelope was not limiting during virus production, supporting the notion that there was less amphotropic envelope available for incorporation. These data are compatible with the idea that the presence of cognate receptor limits envelope availability.
Based on the observations made in the previous section, these results also predict that an increase in the gag/gagpol component would not result in a decrease in titre. This was indeed the case, as increasing the amount of pHIT60 from 0·1 to 1·0 µg did not reduce the titre at all. It was very likely that the absence of a reduction in titre was due to the ecotropic envelope being saturating. Hence, the probability of finding sufficient envelope on the particles was still high, even when particle formation was increased with increased Gag/GagPol expression.
Changes in envelope incorporation upon increasing the amount of envelope component
In order to attribute the lack of ability to increase the titre to the physical saturation of envelope proteins on the particles, the amounts of virion-associated amphotropic and ecotropic envelopes were investigated. Virus stocks were produced by transfecting 293T cells with 0·1 or 1·0 µg pHIT123 or pHIT456. Aliquots of 0·1 µg pHIT60 and pHIT111, respectively, were used to supply the gag/gagpol and genome components for all the samples. From the results described in the previous section, the Gag/GagPol component was expressed independently from the other components and there would be similar numbers of particles in all the samples.
Western blot analyses were performed on the lysates and culture supernatants of the transfected cells. As expected, a 3-fold increase in envelope expression was detected in the cell lysate for both ecotropic and amphotropic envelopes when more of the corresponding plasmid was used for transfection (Fig. 4, lanes 2 and 4). There was also a 2-fold increase in envelope detected in the supernatant when pHIT456 was increased in the transfection (Fig. 4
, lane 7), indicating that the amphotropic envelope was limiting at 0·1 µg pHIT456 and that the envelope found on the particles increased when the amount of pHIT456 was increased to 1 µg. This could account for the increase in titre brought about by increasing the amount of pHIT456 during virus production. In contrast, there did not seem to be a significant increase in the amount of ecotropic envelope detected in the virus supernatant when the amount of pHIT123 was increased from 0·1 to 1·0 µg, indicating that there was no significant change in the amount of virion-associated envelope (Fig. 4
, lane 9). This suggested that, at 0·1 µg pHIT123, the envelope was saturating on the particles. No more envelope proteins could be accommodated on the particles, despite increasing expression in the cells with 1 µg pHIT123. This would explain why there was no increase in titre upon increasing the amount of pHIT123 from 0·1 to 1·0 µg.
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Production of ecotropic particles in 293 cells expressing the ecotropic receptor
In order to investigate the effect of premature envelopereceptor interaction on virus production, a study on the production of ecotropic particles was performed in 293/12 cells. These cells expressed the ecotropic receptor and could be transfected with high efficiencies (Ragheb et al., 1995 ). As a control, the same set of experiments was also conducted in 293 cells, which did not express the ecotropic receptor. The results are presented in Table 3
. In general, the titres were lower compared with the virus stocks that were produced in 293T cells. This was expected, since there was a lack of plasmid amplification due to the absence of the large T antigen. The profile of the titres produced in the control 293 cells mirrored that of 293T cells: there was an increase in titre only when the genome component was increased, and no decrease in titre was observed upon increasing the amount of gag/gagpol component, indicating that only the genome, and not the envelope component, was limiting. In the 293/12 cells, however, the profile was more similar to the production of amphotropic particles in 293T cells: apart from increasing the amount of genome component, an increase in the amount of envelope component also brought about an increase in titre, indicating that the ecotropic envelope, in addition to the genome component, was limiting in the 293/12 cells. A reduction in titre was also observed when the amount of gag/gagpol component was increased, which could reflect the reduction in particles with sufficient envelope proteins. Furthermore, the titre produced in 293/12 cells by using equal amounts of all three plasmids was at least 5-fold lower compared with production in the absence of the receptor. An obvious conclusion from these observations was that the presence of the ecotropic receptor resulted in the envelope component becoming limiting during virus production. This was probably due to the sequestration of envelope proteins by the receptors, thereby reducing the pool of envelope available for incorporation. The reduction of available envelope resulted in a decrease in the number of virus particles that contained sufficient envelopes, leading to a reduction in the number of transducing particles, which was subsequently manifested as a decrease in virus titre.
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Discussion |
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The limitation by envelope was shown to be due to the presence of its receptor in the producer cell, presumably through premature envelopereceptor interaction. Premature interaction between virus envelope and the cognate receptor in the infected cell has been reported previously (Delwart & Panganiban, 1989 ; Matano et al., 1993
). Our results on the production of ecotropic particles in 293/12 cells were consistent with the notion that this interaction might limit the pool of envelope available for incorporation. In addition, the incorporation of heterologous proteins into virus particles was shown to be reduced when their cognate ligands were co-expressed in the producer cells (Henriksson & Bosch, 1998
; Henriksson et al., 1999
). It was shown subsequently that interaction between those proteins and their receptors precluded their incorporation into virions. In human immunodeficiency virus, the envelopereceptor interaction in infected cells was shown to be prevented by the action of the vpu gene product, which down-regulated the CD4 receptor (Willey et al., 1992
).
The strategies used by other retroviruses have not been reported (Swanstrom & Wills, 1997 ). In simple retroviruses such as MLV, which lack any accessory genes, it is conceivable that this could be achieved by expressing more envelope than is required for incorporation during virus production. The importance of production of sufficient envelope has been noted before, when it was observed that there was a threshold number of provirus copies required for efficient virus production (Odawara et al., 1998
). Interestingly, there seemed to be a correlation between the numbers of provirus copies required for virus production and to achieve interference. In the light of our results on the envelopereceptor interaction, this could reflect the need for production of sufficient envelope to down-regulate the receptors. Efficient virus production was achieved when there were enough provirus copies to down-regulate receptor expression. This also led to virus interference through the reduction of available receptors for the entry of viruses with similar receptor usage. The importance for all viral genes to be present in the right proportions was also noted by Odawara et al. (1998)
. This is the first report of a systematic study describing the effects of each component on virus production. Our results suggest that it is essential to ensure that there is never an excess of gag/gagpol when designing an optimal vector production system. Although the study was performed by using a transient transfection system, it would be reasonable to extrapolate the result to include vector production from stable packaging cell lines. In the latter system, the ratio of the number of copies of the gag/gagpol gene to the env gene has never been taken into account during the selection of clones when constructing packaging cell lines. The results of this study indicate that this might be important for obtaining clones that produce virus stocks with higher titres.
Finally, the results of this study have revealed that a large number of empty particles lacking genome were present when all three plasmids were used in equal amounts. These particles could be filled by increasing the amount of the genome component, resulting in a virus stock with a high infectivity to particle ratio. One possible explanation is that there is a lack of proximity between the newly synthesized Gag protein and the genome transcript that is to be packaged. This proximity could be achieved in the wild-type virus because the gag gene products are translated from the full-length transcript, which can also be packaged. Alternatively, the wild-type packaging signal could be suboptimal. A packaging signal with the maximum affinity for the Gag proteins might not have evolved, as the strength of interaction would exclude the binding of ribosomes and hence down-regulate Gag expression. A study of these possibilities would shed light on the relationship between translation and packaging and might provide useful information that could be used to produce better-quality virus stocks with higher infectivity to particle ratios.
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Acknowledgments |
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Footnotes |
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References |
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Received 31 January 2000;
accepted 3 May 2000.
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