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The use of the human immunodeficiency virus type 1 (HIV-1)-based packaging system for gene transfer to a variety of cell types, such as neuronal, muscle, liver, and retinal cells as well as hematopoietic stem cells, is growing in popularity (13, 23, 24, 26, 37, 38, 40). This is due to the ability of HIV-1 to efficiently enter nondividing or quiescent cells (16, 17). Attempts are being made in many laboratories to optimize the packaging and vector constructs to ensure safety without compromising vector titer, so that HIV-1 vectors can eventually be used in a clinical setting. To create a packaging system with HIV-1, one needs a helper construct(s) that produces all necessary HIV-1 proteins, as well as a gene transfer vector. Both helper and gene transfer constructs will require sequences that ensure nucleocytoplasmic transport of their RNAs (34). In the natural context, HIV-1 structural-protein expression is regulated by the Rev protein and its cognate RNA recognition sequence, the Rev-responsive element (RRE), which forms part of the structural-protein gene message. Rev and RRE ensure nucleocytoplasmic transport and expression of the HIV-1 partially spliced and unspliced mRNAs (9, 20). Without a transport mechanism, viral protein expression from partially spliced and unspliced messages is severely reduced. This requirement by HIV-1 can be overcome by using the RNA transport elements from other viruses. For example, the constitutive transport element (CTE) from Mason-Pfizer monkey virus (MPMV) or simian retrovirus type 1 can substitute for this function of Rev and RRE in proviral HIV-1 clones as well as in subgenomic constructs (3, 39). We felt that it would be advantageous to use alternative or mutually exclusive transport systems to express the helper and gene transfer vectors. We rationalized that this would reduce the chance of recombination between helper and gene transfer vectors. Toward that end, we created helper plasmids that contain the CTE and gene transfer vectors that contain the RRE. We compared this combination packaging system with one that uses only CTE or Rev and RRE for expression of both packaging plasmid and gene transfer vector RNAs. The results with amphotropic-envelope-pseudotyped HIV-1 vectors indicated that the combination system was comparable to the CTE- or the Rev-RRE-based systems for production of retroviral vector stocks. There was no evidence for formation of replication-competent retrovirus (RCR) with any of the packaging systems, and prolonged transgene expression was evident even after extended in vitro culture of transduced cells.

We have previously described packaging cell lines which were used to define the requirement of HIV-1 Tat, Rev, and Nef proteins for gene transfer by HIV-1 vectors (34). In this study, using the combination packaging system approach, we evaluated novel HIV-1 packaging constructs that express one or more accessory proteins (Vif, Vpr, and/or Vpu) in addition to the Gag and Pol proteins for particle formation and for production of virus stocks for gene transfer studies. The results of our experiments indicated that packaging constructs that express all three accessory proteins (Vif, Vpr, and Vpu) produced larger numbers of particles and provided higher transduction levels than the one with just the gag-pol sequence. Amphotropic-envelope-pseudotyped virus stocks produced with all packaging constructs were able to transduce growth-arrested cells, suggesting that Vif, Vpr, and Vpu are dispensable for transduction of some cell types. The studies also revealed that the truncated Tat protein expressed from the first coding exon in some of the packaging constructs was as efficient as full-length (86-amino-acid) Tat in transactivation and transduction assays.

HIV-1-packaging plasmids. To introduce HIV-1 sequences into an expression vector, we created a plasmid vector, pCMC, containing the simian cytomegalovirus (sCMV) immediate-early promoter as well as the CTE and poly(A) signal of MPMV, with a multiple cloning site between the two. This plasmid expression vector was derived from another plasmid, pCMV-VSV-G.CTE (kindly provided by David Rekosh and Marie-Louise Hammarskjöld, University of Virginia, Charlottesville). The sCMV promoter-enhancer corresponds to bp 681 to 1349 of the IE94 gene (GenBank accession no. M16019), and the CTE-poly(A) sequence corresponds to bp 8007 to 8557 of MPMV (GenBank accession no. M12349).

View larger version (27K): [in this window] [in a new window]   FIG. 1.   Schematic representation of HIV-1-packaging constructs (A), the HIV-1 provirus (B), and the gene transfer vector used in this study (C). To create the packaging constructs pgp, pgp1v, pgp2v, and pgp3v, fragments from NL4-3 of different lengths (indicated by thick horizontal lines), extending from a restriction enzyme site upstream of gag to sites downstream of pol corresponding to nucleotides 5122 (NdeI), 5785 (SalI), 5999 (SacI) and 6399 (NdeI) of pNL4-3, were introduced into pCMC (described in the text) between the sCMV immediate-early promoter and the MPMV CTE and polyadenylation signal. All plasmids contain a deletion within the encapsidation sequence () between bp 751 and 779 of pNL4-3. In the "irin" plasmids, rev and nef cDNAs were positioned downstream of the IRESes of Ha-MSV and EMCV, respectively. The Ha-MSV IRES sequence corresponds to bp 205 to 794 of pHa-MSV (described by Makris et al. [19]) but was derived from pHaMDR1/A (30). The EMCV IRES sequence corresponds to bp 361 to 860 of EMCV (GenBank accession no. X74312). The Nef coding sequence corresponds to bp 8787 to 9407 of pNL4-3. The rev cDNA coding sequence corresponds to bp 981 to 1331 of pCV1 (GenBank accession no. M11840). The Ha-MSV IRES-Rev-EMCV IRES-Nef cassette was first assembled in an intermediate cloning vector and subsequently introduced downstream of pgp, pgp1v, pgp2v, and pgp3v to obtain the vectors pgpirin, pgp1virin, pgp2virin, and pgp3virin, respectively. The accessory and regulatory proteins encoded by the packaging constructs are summarized in the adjoining table. The HIV-1 gene transfer vector pN-FS-sCMVluc (C) was derived from pTR167 (31). The HIV-1 coding sequences between the proximal NsiI site in gag and the distal NsiI site in env (shown) have been deleted in this vector. It contains the firefly luciferase gene driven from an internal sCMV immediate-early promoter. The reporter cassette was positioned between the BamHI and XhoI sites, thereby interrupting the Nef coding sequence. A frameshift mutation (FS) was introduced 27 bp into the gag coding sequence by inserting an A residue between codons 9 and 10. pN-FS-sCMVluc-cte contains the MPMV CTE (GenBank accession no. M12349) (bp 8007 to 8240) downstream of the luciferase coding sequence but in other respects is identical to pN-FS-sCMVluc. The 5' splice donor site (5'ss) and 3' splice acceptor site (3'ss) are shown. Details of plasmid construction will be provided on request.

Coexpression of Vpr results in increased particle production. Previous studies had suggested that some of the accessory proteins might affect particle production by either enhancing transcription from the promoter (e.g., Vpr) (8) or increasing particle release (e.g., Vpu) (4, 15, 32). To test whether this occurred with our packaging constructs, pgp, pgp1v, pgp2v, and pgp3v were separately transfected into CMT3-COS cells or 293 cells (2). The supernatants and cell lysates were harvested 48 to 72 h posttransfection. The supernatants were cleared of cellular debris and assayed for HIV-1 p24 (CA) by using a commercial kit (Cellular Products, Buffalo, N.Y.). The cell lysates were assayed for p24 and for -galactosidase activity (Clontech, Palo Alto, Calif.). Particle production was determined by normalizing p24 levels to -galactosidase activity. The efficiency of particle release or export was determined by comparing the ratio of the particles in the supernatant to the particles associated with the cells.

View larger version (51K): [in this window] [in a new window]   FIG. 2.   (A and B) Particle production by (A) and efficiency of particle export into the medium of (B) 293 cells transfected with packaging constructs. A 3.75-µg quantity of each packaging plasmid was transfected into 293 cells together with 1 µg of pCMV-gal. (C and D) The effect of Vpr on particle production (C) and efficiency of particle export (D) by pgp in 293 cells. The indicated amounts of pVpr-wt or pVprAug() were cotransfected with a constant amount of pgp and pCMV-gal into 293 cells. The supernatants and cell lysates were harvested 72 h posttransfection and assayed for HIV-1 p24 antigen by using a commercial ELISA kit (Cellular Products, Buffalo, N.Y.) in accordance with the recommended protocol. The cell lysates were tested for -galactosidase (-gal) activity by using a luminescent -galactosidase detection kit (Clontech, Palo Alto, Calif.) according to the recommended procedure. p24 levels in the medium were normalized to -galactosidase activity in cell lysates and are shown in panels A and C. Efficiencies of particle export was estimated from the ratio of p24 in the medium to that seen in the cell lysates and are shown in panels B and D. The average amounts of p24 (in picograms per milliliter) found in the media of cells transfected with the different plasmids are shown above the respective bars. Error bars correspond to 1 standard deviation and were derived from duplicate experiments.

pgp2v and pgp3v express a single-exon Tat that is functional in transactivation assays. The HIV-1 Tat protein in pNL4-3 is 86 amino acids long. The coding region of tat is separated into two exons. The first exon codes for 72 amino acids of the Tat protein. It was shown previously that almost all of the transactivation function of Tat is encoded in the first coding exon (5, 29). pgp3v and pgp3virin contain the entire first coding exon of tat and have the potential to express a functional Tat protein. pgp2v and pgp2virin encode the first 57 amino acids of Tat, but their sequences diverge downstream of amino acid 57. Since the Tat sequences encoded by pgp2v and pgp2virin retain the arginine-rich nucleic acid binding domain, pgp2v and pgp2virin should also have the potential to express a functional Tat protein. To determine if this is indeed the case, the different packaging constructs were transfected into CMT3-COS cells together with the reporter plasmid pLTR-luc-BGHpA, which expresses firefly luciferase under the control of the HIV-1 long terminal repeat (LTR). pCMVtat (which expresses full-length HIV-1 Tat) was included in parallel transfections with the packaging plasmids. All cells undergoing transfections, except the mock-transfected cells, also received pCMV-gal. Cell lysates were harvested and assayed for luciferase and -galactosidase activities. The results of this experiment are shown in Fig. 3. Cells transfected with the luciferase plasmid alone gave a mean value ± standard deviation of the mean of 1,652 ± 865 relative light units (RLU), which corresponds to the basal promoter activity in CMT3-COS cells. On addition of a full-length-Tat-expressing plasmid (pCMVtat), the luciferase activity increased 44-fold to 73,159 ± 5,426 RLU. Transfection of cells with pgp, which does not express any accessory proteins, did not significantly augment basal levels of transcription from the HIV-1 LTR. Cotransfection of pgp and pLTR-luc-BGHpA with a Tat-expressing plasmid, pCMVtat, resulted in luciferase activity levels comparable to those for the control transfection with the reporter and pCMVtat alone. Cotransfection of pLTR-luc-BGHpA with pg1v (which contains an intact Vif coding sequence) gave about twofold-higher basal levels of expression from the HIV-1 LTR. Again, cotransfection of pCMVtat and pgp1v gave RLU values similar to those obtained with pLTR-luc-BGHpA and pCMVtat. With pgp2v and pgp3v, very high levels of transactivation of the HIV-1 LTR were noted even in the absence of cotransfected pCMVtat. Inclusion of pCMVtat with these constructs did not augment expression from the HIV-1 LTR, in contrast to what was noticed with the pgp and pgp1v constructs. These results demonstrate that pgp2v and pgp3v express a functional single-exon Tat protein and that saturating levels of Tat must be produced under the conditions used, since the transactivation of the LTR was not augmented by inclusion of a full-length Tat-expressing plasmid. Interestingly, Tat also increased transcription from the sCMV promoter, as determined by measuring the -galactosidase activity in the cell lysates (Fig. 3B). This effect was not as dramatic as that seen with the HIV-1 LTR and resulted in an increase in -galactosidase activity of about fivefold.

View larger version (33K): [in this window] [in a new window]   FIG. 3.   (A and B) Transactivation of the HIV-1 LTR (A) or the sCMV promoter (B) by the different packaging plasmids. Five micrograms of each of the indicated packaging plasmids was transfected separately into CMT3-COS cells together with 5 µg of pLTR-luc-BGHpA and 2 µg of pCMV-gal. pLTR-luc-BGHpA contains the firefly luciferase gene under the control of the HIV-1 LTR as well as the bovine growth hormone poly(A) signal downstream of the luciferase coding region. Parallel transfections also received 2 µg of a CMV-tat plasmid (pCMVtat) (34). Transfected-cell lysates were prepared 72 h posttransfection and assayed for luciferase and -galactosidase activities. Cell lysates were prepared in 0.5 ml of a luciferase lysis buffer, and a 20-µl aliquot of a 1:100 dilution was used. Luciferase activity was assayed by using a kit and a luminometer (Analytical Luminescence Laboratory, Sparks, Md.) in accordance with the manufacturer's protocol. -Galactosidase activity was measured as described in the legend to Fig. 2. RLU are shown for transfections with (+) (cross-hatched boxes) and without () (boxes with wavy lines) cotransfected pCMVtat. The fold increase in the presence of Tat compared to the level in its absence is shown above the respective bar for each plasmid. Error bars correspond to 1 standard deviation and were derived from duplicate experiments.

Efficient transduction of target cells by virus stocks produced with single-exon-Tat-encoding packaging plasmids pgp2virin and pgp3virin. In a previous study (34), we found that Tat was essential for production of high-titer virus stocks. This was attributed to either transactivation of the HIV-1 LTR leading to the production of increased amounts of packagable RNA from the gene transfer vector or the effect of Tat on reverse transcription during infection (11). The above-described transactivation experiments with the packaging plasmids (Fig. 3) indicated that two of the constructs (pgp2v and pgp3v) and their "irin" counterparts expressed a functional Tat protein. It was therefore likely that the production of high-titer virus stocks from pgp2virin and pgp3virin constructs would not require cotransfection with a Tat-encoding vector. To test this premise directly, virus stocks were prepared separately for each packaging plasmid by transfection of CMT3-COS cells alone or with pCMVtat. All cells undergoing transfection also received a gene transfer vector (pN-FS-sCMVluc) (Fig. 1) and an amphotropic murine leukemia virus envelope-expressing plasmid (pSV-A-MLV-Env). The virus stocks obtained from the transfected-cell supernatants were then tested on HeLa cells. The lysates of transduced cells were assayed for luciferase activity. The results are shown in Table 1. Virus stocks produced with pgpirin and pgp1virin (which do not encode a functional Tat protein) exhibited low transduction levels in the absence of a Tat-expressing plasmid. The titers increased by 88- and 11-fold, respectively, when pCMVtat was included during the production of virus stocks. In contrast, for virus stocks prepared with pgp2virin and pgp3virin, transduction efficiencies were similar with and without cotransfected pCMVtat, as expected from the transactivation experiments (Fig. 3). The highest levels of transduction were noted with virus stocks made by using the pgp3virin plasmid; they were about twofold higher than those obtained with the pgp2virin plasmid. The increased level of transduction evident with pgpirin and pgp1virin in the presence of Tat was most likely due to increased production of vector RNA for packaging and is in part also explained by the increase in p24 levels. The increase in particle production was most likely due to transactivation of the sCMV promoter, which drives gag-pol expression, by Tat (Fig. 3B). These experiments indicated that the 72-amino-acid Tat was sufficient for efficient transduction of target cells and that Tat was being produced by pgp2virin and pgp3virin in the transfected cells in adequate amounts for the production of virus stocks. Although there was roughly a 21-fold or greater increase in particle production by pgpirin in comparison to pgp2virin or pgp3virin, their gene transfer efficiencies differed by only 3- to 6-fold. From this it appears that the limiting factor may be the amount or accessibility of gene transfer vector RNA available for packaging by the viral Gag or Gag-Pol proteins. Similar observations have been made by other investigators (27).

Efficiency of gene transfer into nondividing target cells by virus stocks produced with the different packaging constructs. To determine if coexpression of Vif, Vpr, and Vpu affected transduction of growth-arrested cells, virus stocks produced with each of the packaging plasmids were tested on HeLa cells that were either growing or growth arrested. HeLa cells were treated with the DNA polymerase alpha inhibitor aphidocolin (15 µg/ml) to bring about growth arrest in G₁/S phase (12). A preliminary study was done to ensure that the aphidocolin concentration used was adequate for blocking cell cycling. For metabolic labeling of S-phase active cells, the cells were incubated with the thymidine analog 5-bromo-2'-deoxyuridine (BrdU) and then stained with an anti-BrdU antibody and a fluorescein isothiocyanate-conjugated secondary antibody. Flow cytometry demonstrated that the aphidocolin concentration used was effective in blocking the cell cycle in G₁/S (data not shown). As an independent biological control, we used virus stocks prepared with Moloney murine leukemia virus (MoMLV). MoMLV has been previously shown to infect dividing cells but not growth-arrested cells (17). MoMLV virus stocks were prepared by transfection of CMT3-COS cells with pSV-A-MLV-gagpol (5 µg), pSV-A-MLV-env (5 µg), and pLgSVluc (10 µg). pLgSVluc is an MoMLV vector that expresses the luciferase gene under the control of an internal simian virus 40 early promoter. (Details of the construction of this vector will be provided on request.) The results of the gene transfer experiments are shown in Table 2 and indicate that virus stocks from all of the different HIV-1-packaging constructs were equally efficient in transducing aphidocolin-treated and untreated HeLa cells. In contrast, the MoMLV vector transduced only growing HeLa cells. Interestingly, in several independent experiments, aphidocolin-treated cells transduced with the HIV-1 vectors exhibited about two- to fivefold-higher luciferase values than untreated cells. We have no explanation for this observation.

Comparison of packaging systems using different combinations of RNA transport mechanisms for expression of packaging and gene transfer vector RNAs. To reduce the chance of RCR formation, it may be advantageous to use alternative RNA transport systems for expression of helper and gene transfer vector RNAs (Fig. 4). We therefore compared our combination system, in which the packaging plasmid uses the CTE while the gene transfer vector uses Rev and RRE, with a system that exclusively uses either CTE or Rev and RRE for expression of both helper and vector RNAs. To this end, we transfected 293 cells with the following combinations of packaging and gene transfer vector plasmids to produce virus stocks: (i) pgp3virin and pN-FS-sCMVluc (combination system); (ii) pgp3v, pN-FS-sCMVluc-cte, and pCMVnef (CTE system); and (iii) pCMVR9 and pN-FS-sCMVluc (RRE-Rev system). Since pgp3v does not express either Rev or Nef, pCMVnef was added during production of virus stock with this packaging plasmid, to allow comparison with pgp3virin. This ensured that the only difference between pgp3v and pgp3virin would be the absence of Rev during virus stock production with pgp3v. pCMVR9 expresses all HIV-1 proteins with the exception of Vpu and Env and is regulated by the Rev-RRE system (26). This plasmid was kindly provided by Didier Trono, University of Geneva Medical School, Geneva, Switzerland. All transfections included an amphotropic MoMLV envelope-expressing plasmid. Virus stocks obtained from these transfections were then tested on HeLa cells, and the transduction efficiency was determined by the luciferase assay. The luciferase activity was normalized to p24 levels for each virus stock. The results are shown in Table 3. Virus stocks produced with pgp3v gave 8 to 12 RLU per pg of p24. Virus stocks produced with pgp3virin, in contrast, gave 127 to 167 RLU per pg of p24, an increase of 14- to 16-fold. Virus stock production with both of these helper plasmids is based on the combination RNA transport system. The difference was the absence of Rev during virus production with pgp3v while Rev was expressed by pgp3virin during virus stock production with the latter helper plasmid. The gene transfer vector is a Rev-dependent vector since it has the RRE but no CTE. The results obtained with virus stocks produced with the two different helper plasmids are in accordance with our previous results showing that Rev was essential for obtaining high transduction levels for vectors that were based on RRE (34). To determine if we could restore the titer by rendering the gene transfer vector Rev independent, we used a gene transfer vector that contained the CTE, pN-FS-sCMVluc-cte (Fig. 1), instead of pN-FS-sCMVluc for deriving virus stocks with pgp3v. The normalized titer of virus stocks produced with the CTE-containing vector was 22- to 27-fold higher than that obtained with the Rev-dependent vector in the absence of Rev in the same system.

View larger version (21K): [in this window] [in a new window]   FIG. 4.   Strategies for construction of HIV-1-based packaging systems. (A) In the RRE-Rev-based packaging system, expression of RNA from both helper plasmid and gene transfer vector is regulated by Rev and RRE. (B) In the CTE-based system, helper and vector RNA expression is regulated by the MPMV CTE. (C) In one combination packaging system (1), the helper plasmid is regulated by the CTE while the gene transfer vector is controlled by RRE and Rev. In an alternative scenario (2), the helper plasmid is regulated by RRE and Rev while the gene transfer vector is controlled by the CTE. Possible sites of recombination between helper and vector constructs are indicated with bidirectional arrowheads or arrows. For clarity, the transgene expression cassette in the gene transfer vector and accessory and regulatory proteins in the helper constructs are not depicted.

Assay for RCR and persistence of transgene expression. We checked the virus stock products of the packaging systems described in the previous section (Table 3) for RCR formation by using marker rescue assays and an enzyme-linked immunosorbent assay (ELISA) for the HIV-1 CA antigen. For these experiments, we used an extremely sensitive reporter gene (luciferase) in combination with 293 target cells, which allow high levels of expression from the internal sCMV immediate-early promoter in the HIV-1 vector. The transduction of 293 cells for marker rescue was done as follows. 293 cells (2.5 × 10⁶) in T25 flasks were transduced with 1-ml volumes of virus stocks produced by the three different types of packaging systems shown in Table 3. This corresponded to 10- and 3- to 6-fold-higher transducing units of virus stocks produced by the combination system than by the RRE-Rev-CTE-based systems, respectively. Following infection, for the marker rescue assay, the primary transductants were cultured in the presence of 4 µg of Polybrene/ml (to aid the spread of any replication-competent virus in the culture) for at least 2 days. The medium was then replaced with fresh medium lacking Polybrene, and culture supernatants were harvested the following day for use in infection of fresh 293 cells (secondary transductants). The secondary transductants were assayed for luciferase activity. The results of this experiment are shown in Table 4. While the primary transductants showed abundant levels of luciferase expression, no luciferase activity significantly above background was observed in the secondary transductants. This was true for virus stocks prepared by all three of the different packaging systems (the combination system, the CTE-based system, and the Rev- and RRE-based system). The primary transductants were maintained in culture by subculturing cells approximately twice a week at a density of 10⁶ per flask (which corresponded to a 1:5 to 1:10 split at each passage). No selection agent was used in the maintenance of these cells, since the vectors did not contain any antibiotic resistance genes. After 10 passages, the cells were harvested and assayed for luciferase activities, and the resultant values were compared to those of the cell lysate obtained from the first passage. The results are shown in Table 4. Luciferase activity could be readily detected in transduced cells at the 10th passage. To determine if the persistence of the transgene could be explained by the spread of the marker within the culture by a replication-competent virus, the supernatants from passages 1, 2, 5, and 8 were assayed for HIV-1 p24 by using a commercial ELISA kit. The results of the p24 assay indicated that the residual input p24 detected at the early passages from the initial inoculum quickly decreased to below detectable levels by passage 5 and remained so even at passage no. 8 (data not shown). This was true for virus stocks derived from the combination packaging system, which was tested at as large of an inoculum as possible (resulting in up to 10-fold-higher p24 levels than those of virus stocks produced with the other packaging systems). To further rule out the possibility that a virus not detectable by the p24 assay was spreading the transgene within the culture, the supernatant of the 10th passage was harvested and used for infection of naive 293 cells in the presence of 10 µg of DEAE-dextran/ml. Cell lysates of these secondary transductants were prepared 60 h postinfection and assayed for luciferase activity. As shown in Table 4, only background levels of luciferase activity could be detected in these secondary transductants. Taken together, these results suggest that no RCR was produced during culture of transduced 293 cells, that marker gene expression was maintained at high levels, and that this level of expression remained virtually unchanged in the transduced cells for over 10 passages. Finally, the results also suggested that there was no negative selection against the transduced cells in culture.