Design of a trans protease lentiviral packaging system that produces high titer virus
© Westerman et al; licensee BioMed Central Ltd. 2007
Received: 20 August 2007
Accepted: 28 December 2007
Published: 28 December 2007
The structural and enzymatic proteins of the human immunodeficiency virus (HIV) are initially generated as two long polyproteins encoded from overlapping reading frames, one producing the structural proteins (Gag) and the second producing both structural and enzymatic proteins (Gag-Pol). The Gag to Gag-Pol ratio is critical for the proper assembly and maturation of viral particles. To minimize the risk of producing a replication competent lentivirus (RCL), we developed a "super-split" lentiviral packaging system in which Gag was separated from Pol with minimal loss of transducibility by supplying protease (PR) in trans independently of both Gag and Pol.
In developing this "super-split" packaging system, we incorporated several new safety features that include removing the Gag/Gag-Pol frameshift, splitting the Gag, PR, and reverse transcriptase/integrase (RT/IN) functions onto separate plasmids, and greatly reducing the nucleotide sequence overlap between vector and Gag and between Gag and Pol. As part of the construction of this novel system, we used a truncated form of the accessory protein Vpr, which binds the P6 region of Gag, as a vehicle to deliver both PR and RT/IN as fusion proteins to the site of viral assembly and budding. We also replaced wt PR with a slightly less active T26S PR mutant in an effort to prevent premature processing and cytoxicity associated with wt PR. This novel "super-split" packaging system yielded lentiviral titers comparable to those generated by conventional lentiviral packaging where Gag-Pol is supplied intact (1.0 × 106 TU/ml, unconcentrated).
Here, we were able to create a true "split-function" lentiviral packaging system that has the potential to be used for gene therapy applications. This novel system incorporates many new safety features while maintaining high titers. In addition, because PR is supplied in trans, this unique system may also provide opportunities to examine viral protein processing and maturation.
The genome of Human Immunodeficiency Virus Type 1 (HIV-1) is complex in that it employs overlapping reading frames to encode two essential polyproteins known as Gag and Gag-Pol. The Gag polyprotein precursor supplies the structural components of the virus that include the matrix (MAp17), capsid (CAp17), nucleocapsid (NCp7), and p6 proteins while the Pol polyprotein precursor supplies the viral enzymes protease (PR, p11), reverse transcriptase/Rnase H (RT, p66/p51), and integrase (IN, p32) (for review see [1, 2]). The concentrations of Gag to Gag-Pol polyproteins are maintained at a ratio of 20:1 through a frameshift mechanism in which the ribosome slips by -1 on a heptanucleotide AU rich sequence located at the end of the NCp7 protein . The ensuing frameshift results in the ribosome reading through P6 to produce the full length Gag-Pol polyprotein. This 20:1 ratio of the Gag to Gag-Pol has been shown by many researchers to be critical for the production of "infectious" viral particles. Attempts to vary the 20:1 polyprotein ratio, has resulted in decreases in virus infectivity and stability [4–6]. In addition, the expression of Gag without Gag-Pol has been shown to result in the assembly of particles that are non-infectious , and in the reverse case, when Gag-Pol is expressed without Gag, there is efficient PR processing but no production of virions .
PR is essential for the processing of the viral polyprotein precursors and thus plays an important role in the maturation of viral particles and in the production of infectious particles [9–12]. During the assembly of the Gag and Gag-Pol polyproteins, PR is initially inactive. As the concentration of polyproteins increases and the virion components are confined in the budding particle, PR then dimerizes and becomes active [13–16]. Once PR is active, it then sequentially cleaves the assembled precursor polyproteins resulting in the transformation of the immature viral particle into a mature infectious virion [10, 12]. Hence, the correct balance of Gag to Gag-Pol is critical to ensure that not only the viral enzymes are incorporated into the viral particles but also that PR becomes activated at the appropriate time to prevent the production of defective particles with reduced infectivity due to premature processing of the Gag polyproteins [9, 14, 17].
Here we describe a novel lentiviral packaging system in which not only is Gag supplied separately from Pol, but PR is also supplied independently. One of the greatest concerns with the construction of retroviral and lentiviral packaging systems is the production of RCR (replication competent retrovirus) and RCL (replication competent lentivirus), respectively. As the production of RCR/RCL is believed to occur through homologous recombination between overlapping sequences, researchers have minimized this risk by dividing the functional components of the viral genomes onto separate expression plasmids. In the case of retroviruses, the vector, GagPol, and envelope have all been supplied separately in what was called a "split-function" packaging system . In the case of lentiviruses, which are more complex, it was found that not only can the Gag-Pol be separated from the vector and envelope, but that the accessory proteins (Vif, Vpr, Vpu, and Nef) and regulatory proteins (Rev and Tat) could also be either eliminated or supplied in trans [19–21]. The reasoning behind these split-function retroviral and lentiviral packaging systems is that it is much less likely that 2, 3, or even 4 recombinations would occur to generate a RCR/RCL, which in turn makes these split-function systems inherently safer. This is especially important for large-scale, clinical grade, vector production. In the case of lentiviral packaging systems, no RCL events have been detected to date, probably because the vesicular stomatitis virus glycoprotein G (VSV-G), which is widely used as pseudotyping envelope and is cytotoxic when constitutively expressed, makes it difficult to form a bona fide RCL that comprises and expresses the VSV-G gene. However RCRs have been detected in split-function retroviral packaging lines that make use of ecotropic or amphotropic retroviral envelopes [22, 23]. In view of the highly pathogenic nature of HIV-1, it is thus of the utmost importance to ensure that the safest possible lentiviral packaging systems are used for gene therapy applications to prevent the slightest possibility of RCL or even pre-RCL formation. Here we have devised a "super-split" 7-plasmid lentiviral packaging system with minimal loss of transducibility with which more than 4 recombination events would be required to produce a viable RCL.
A key feature of this system is the use of the p6-binding domain of the accessory HIV protein Vpr to tether fusion proteins to the budding virions, an approach pioneered by Kappes' and Hahn's groups [24–26] and ourselves [27, 28]. In the past, we (unpublished data) as well as Wu, et al.  have designed split-function lentiviral packaging systems in which Gag-PR was supplied separately from RT-IN by means of Vpr-mediated tethering. However, these previous attempts either resulted in a substantial decrease in lentiviral titers or did not comprise a true split of the Gag-Pol gene. In the latter case, a stop codon was introduced at the start of RT and IN to prevent the expression of RT and IN, so that RT and IN sequences remained present in the Gag-PR expression plasmid . This configuration retains a residual risk of RCL formation by sequence read-through, reversion or recombination. Here, we have improved upon these systems by creating a true split-function lentiviral packaging system in which Gag, PR, and RT/IN are supplied by three independent plasmids. This "super-split" system affords an additional level of protection against RCL formation through a higher level of true plasmid separation while unexpectedly restoring useful lentiviral titers.
Delivering the Pol proteins in trans to the viral particles
Structure of the three lentiviral packaging systems
The data presented here compares 3 different lentiviral packaging systems. The first, referred to as the "5 plasmid system", is a conventional lentiviral packaging system where Gag-Pol is supplied from a single expression plasmid. In addition to the packaging plasmid, which contains both Gag-Pol and Vif (Vpr, Vpu, Tat, Rev, ENV, and Nef were all deleted), four other expression plasmids are used to generate virus: the first contains the lentiviral vector that encodes GFP, the second expresses Tat, the third Rev, and the fourth VSV-G. The second system, referred to as the "6 plasmid system", is a split-packaging system in which the Gag-Pol functions are expressed by two separate plasmids, one for Gag-PR and the other for RT-IN. The Gag-PR expression plasmid was derived from the aforementioned Gag-Pol plasmid in which all the RT, IN, and Vif sequences were deleted. The second packaging plasmid consists of Vpr fused to RT/IN-Vif, a splice donor site to allow for the proper splicing and expression of Vif, and the natural PR cleavage site for RT (33 bases before the start of RT) to allow for proper PR processing of the RT and IN proteins. The third system, referred to as the "7 plasmid system", is a "super-split" packaging system in which the functional components of the Gag-Pol are expressed from three separate plasmids. The first plasmid contains only the Gag gene from which the frameshift has been mutated and all the regions that encode the Pol proteins deleted. The second plasmid contains PR fused to Vpr along with the natural PR cleavage site (15 bases before the start of PR). The third plasmid is the same Vpr-RT/IN-Vif fusion plasmid used for the 6 plasmid system. Diagrams of the plasmids used for all three packaging systems are shown in (Figs. 1 and 2).
Titer analysis of the 5, 6, and 7 plasmid systems
Optimizing parameters, such as molar ratios of one plasmid to another, as well as comparing one system to another, were performed by means of a wt-LTR lentiviral vector that expresses GFP driven by an EF1α promoter. Since the 6 and 7 plasmid systems described here are not conventional, we suspected that p24 and RT assays may not accurately reflect viral titers. The p24 assay gives information about the amount of CAp24 present but does not discriminate infectious from non-infectious particles. In the same respect, the RT assay gives information on RT activity, but it may be difficult to interpret as the 6 and 7 plasmid systems supply RT in trans. We thus chose instead to measure functional infectious viral titers by scoring stable GFP expression in target cells upon chromosomal integration of the provirus. These titers were determined by transfecting 293T cells with 5, 6, or 7 plasmids, collecting the supernatants 48 h later, transducing NIH 3T3 and Jurkat cells with varying amounts of these viral supernatants, and then monitoring the transduced NIH 3T3 and Jurkat cells for the production of GFP by FACS.
Results from the split-packaging 6 plasmid system
Titer rescue of the 6 plasmid system by supplying PR in trans
To correct the processing problem detected with the 6 plasmid system, we decided to express PR separately from Gag, resulting in the development of a "super-split" 7 plasmid system. Before constructing this new system, there were three areas of concern that needed to be addressed: (i) What to do with the frameshift, (ii) How to deliver PR in trans without cytotoxicity or loss of infectivity, and (iii) How to minimize the sequence overlap between the packaging signal and Gag, and between Gag and Pol, see (Fig. 3B).
In confronting the first concern, we decided to remove the frameshift in order to completely separate Gag from Pol. This was performed using PCR to generate a fragment, between the Nsi I site (found in the CAp24) and the Bgl II site (just after NCp7), which encompasses the area of frameshift at the end of NCp7. This frameshift sequence was changed from AAT TTT TTA GGG to AAC TTC TTA GGG. A second PCR was performed from the Bgl II site (just after NCp7) to the stop codon of P6 in order to eliminate PR. The result was a Gag expression plasmid in which both the frameshift and PR had been eliminated.
The third goal in constructing the 7 plasmid system, as seen in (Fig. 3B), was to minimize the sequence overlap between packaging signal and Gag and between Gag and Pol. The first overlap consisted of 542 bases and was minimized (from 542 to 55 bases) by optimizing the codons at the start of Gag, that is, by using alternate nucleotides for the codons while maintaining the originally encoded Gag amino acid sequence. The second overlap, located at the junction of Gag and Pol, was minimized in the two previous steps by removing the frameshift and separating Gag from PR (208 bases reduced to 54 bases). To determine whether the use of this optimized Gag had an impact on titers generated by the 7 plasmid system, we compared functional titers obtained with the original versus the Gag-optimized expression plasmids. Titer results for the 5 plasmid system, the 6 plasmid system, the 7 plasmid system after optimizing Gag, and the 7 plasmid system where Gag is not optimized, are shown in (Fig. 3A). These titers were obtained after optimizing transfections for variations in total DNA concentration and for molar differences in plasmids used to generate virus for the 6 (Gag-PR and Vpr-RT/IN-Vif) and 7 (Gag, Vpr-T26S PR, and Vpr-RT/IN-Vif) plasmid systems. As can be seen in (Fig. 3A), the 7 plasmid system in which PR is supplied independently of Gag and RT/IN generated titers that were about 2–3 fold higher than those obtained with the 6 plasmid system. Titers achieved with the 6 plasmid system averaged 2.4 × 105 TU/ml and were 9 fold lower than titers obtained with the 5 plasmid system, whereas titers obtained with the 7 plasmid system averaged 4.4 × 105 TU/ml with the optimized Gag, and 7.4 × 105 TU/ml with the non-optimized Gag, that is only about 3 to 5 fold lower than with the 5 plasmid system.
In addition to looking at the functional titers, we analyzed the viral particles generated by the 7 plasmid system to determine whether protein processing had improved by supplying PR independently of Gag. The results shown in (Fig. 4) demonstrate that the Vpr fusions are effective in supplying the Pol components in trans for both mutant PR and RT/IN. The virions produced by the 7 plasmid system, in which PR is delivered independently, showed more processed proteins (CAp24, RT, and IN) with less accumulation of both Pr55Gag and Vpr-RT/IN. Quantitative analysis of the CAp24 and Pr55Gag bands revealed that the ratio of CAp24 (CA from processed Gag) to Pr55Gag (unprocessed Gag precursor) had improved compared to the 6 plasmid system and was now just 2 fold lower than with the 5 plasmid system (Cap24/Pr55Gag; 5 plasmid system 6.1 and 4.3, 6 plasmid system 1.6 and 1.8, 7 plasmid system 2.6 and 2.1, without and with Vif respectively). In addition, because we generally saw a slight increase in titers in the presence of Vif (data not shown) we also looked at the processing in relation to the presence of Vif for all three systems. We were unable to establish conclusively that a change had occurred in the processing of the Gag precursor in the presence of Vif, although we detected an improvement in the processing of Vpr-RT/IN with the 6 plasmid system, as can be seen in Figure 4 by the concurrent reduction in Vpr-RT/IN and increase in RT (lanes 3 and 4).
Self-inactivating (SIN) vector improves viral titers
To demonstrate that the 7 plasmid system is capable of efficiently transducing other cell types, such as human T cells, we also transduced Jurkat cells using the GFP SIN vector along with the 5, 6, and 7 plasmid systems. As shown in (Fig. 6B), titers obtained with the 6 plasmid system averaged 2.7 × 106 TU/ml, once again 9 fold lower than titers obtained with the 5 plasmid system, titers obtained for the 7 plasmid system averaged 5.5 × 106 TU/ml with the optimized Gag and 6.9 × 106 TU/ml with the non-optimized Gag, these titers were 2–3 times higher than those obtained with 6 plasmid system and just 4 times lower than those obtained using the 5 plasmid system.
Here we describe a novel "super-split" lentiviral packaging system in which the overlapping Gag and Pol polyprotein precursors are completely separated and supplied independently to produce high titer virus. This approach also brings further evidence that Vpr can be used as a vehicle to incorporate the Pol components, PR and RT/IN, effectively into viral particles, as we and others have successfully used Vpr fusions to supply proteins in trans to viral particles [24–28]. Vpr has also been used to supply RT/IN as part of a safer lentiviral packaging system in which Gag-PR and RT/IN functions were delivered by separate plasmids . In this safer system, Wu, et al. showed that the lentiviral packaging functions could be supplied from separate plasmids, although they did not truly physically split the Gag-Pol gene. The Gag-PR plasmid they used had a stop codon at the start of RT and IN to prevent the expression of RT and IN, but the RT and IN sequences remained as part of their Gag-PR expression plasmid. This configuration was exposing to residual risk of RCL formation by sequence read-through, reversion or recombination. In contrast, the split packaging systems presented here establish the functionality of creating a true physical split of the Gag-Pol gene, where neither Gag-PR nor Gag expression plasmids contains RT or IN sequences. Tat and Rev are also provided from completely separated expression plasmids.
In our first attempt at constructing this split-packaging system, the Gag-Pol polyproteins were expressed using two expression plasmids: one for Gag-PR and the second expressing RT/IN. As was shown in the Results, this first generation system (the 6 plasmid system) produces infectious viral particles at titers 9 fold lower than those generated by the conventional lentiviral packaging system in which Gag-Pol is supplied intact from a single expression plasmid. After examining the profile of viral proteins from virions produced by the 6 plasmid system, we determined that RT/IN was not efficiently processed although it was incorporated into the viral particles. The same phenomenon was observed for the Gag p55 precursor. Because PR is central to processing the precursor polyproteins, the reduced processing of Gag and RT/IN suggested that the low titers might be explained by a defect in the activation and release of PR. Another contributing factor that could explain the low titers is the accumulation of uncleaved Vpr-RT/IN fusion proteins. We have previously shown that incorporation of Vpr fused heterologous amino-acid sequence affected the infectivity of HIV-1 viral particles .
To improve upon this first generation split-packaging system, we then developed a new "super-split" system (the 7 plasmid system) in which Gag is not only separated from Pol, but PR is separated from Gag and supplied independently in trans. It was our hope that, by supplying PR in trans, we could increase the amount of active PR and improve processing of the precursor proteins. This approach raised two theoretical concerns: the potentially enhanced cytotoxic effect of PR [9, 14, 17] and the possible premature processing of the precursor polyproteins [5, 14, 17]. To address the issue of cytotoxicity, we used a mutant PR with slightly reduced protease activity and none of the cytotoxic effects seen with the wt PR . We found that supplying PR in trans as part of the 7 plasmid system resulted in titer improvement comparatively to those obtained with the 6 plasmid system. Furthermore, the mutant PR supplied in trans yielded viral titers higher than those obtained with the use of wt PR. A concurrent improvement upon processing of both the Pr55Gag and RT/IN polyproteins was also observed. When the 7 plasmid system was compared to the conventional lentiviral packaging system, the viral titers were only 3 fold lower with a mean titer of 1.0 × 106 TU/ml for unconcentrated virus. In addition to the data presented here and to demonstrate that the viral particles generated by the 7 plasmid system can be concentrated and used to transduce dividing and non-dividing cells, the efficacy of this "super-split" packaging system with the PR supplied in trans was demonstrated by us in a published study where the 7 plasmid system was used to transduce human cord blood hematopoietic stem cells with a complex beta-globin expressing lentiviral vector assessed in NOD-SCID mouse transplant studies .
In order to generate the safest possible lentiviral packaging system, we incorporated several safety features into the 7-plasmid system. These features include: (i) splitting the Gag, PR, and RT/IN functions into separate plasmids, (ii) eliminating the frameshift, so that even in the event of a recombination event, Pol could not be produced, and (iii) minimizing the overlapping sequences that existed between the lentiviral vector (packaging signal) and the Gag expression plasmid (from 542 to 55 bases), and between the Gag and Pol (from 208 to 54 bases). In addition to the packaging systems, we also compared viral titers produced by the wt-LTR and SIN lentiviral vectors and found that the SIN vector produced equivalent or higher viral titers for all three packaging systems, possibly due to the benefit conferred by the "ideal" poly(A) sequence that we substituted for U5 within the 5' LTR. Iwakuma, et al., also reported an increase in viral titers when they replaced the U5 region of their SIN vector with a bGH poly(A) sequence . Most importantly, this increase resulted in viral titers for the 7 plasmid system in conjunction with the SIN vector that were only 2 fold lower than those obtained by the conventional system using a wt-LTR vector, 1.0 × 106 TU/ml and 2.2 × 106 TU/ml, respectively.
Here we presented a novel "super-split" lentiviral packaging system with the potential to be used for gene therapy applications. Since this system incorporates many new safety features while using a less cytotoxic mutant PR, it also presents new opportunities to develop better high titer HIV packaging cell lines.
Plasmid construction of transfer vectors
All the lentiviral components used in plasmid construction with the exception of Vpr  and RRE , were derived from PLNENV-1, accession # M19921 . Oligonucleotides used were purchased from Life Technologies and all PCR products were verified by sequencing. The basic vector design of the wt-LTR and SIN lentiviral vectors has been described previously . Briefly, both of the wt-LTR and SIN vectors contain a packaging signal, central polypurine tract, RRE (Rev response element), and an Ef1α promoter  driving the expression of eGFP (Clontech), which replaces the β-Globin cassette located between the BamHI and Kpn I restriction sites. The SIN vector contains a 400 bp deletion between EcoRV and Pvu II sites in the U3 region of the 5' LTR as well as a complete deletion of the U5, which was replaced by an "ideal" termination/polyadenylation sequence (ATG TGT GTG TTG GTT TTT TGT GT). In addition the SIN vector also contains two stops in the packaging signal placed at the 1st and 35th amino acid of Gag to prevent translation of Gag.
Plasmid construction of Gag expression plasmids
The backbone used for all three Gag expression plasmids, Gag-Pol-Vif, Gag-PR, and Gag alone, was "pCI Vector" (Promega) in which Nhe I site was replaced by a BssHII linker, the CMV promoter from the Bgl II to the Sac I sites was replaced by a Nhe 1 to Sac I fragment from cRev plasmid , this inserted the SV40 origin next to CMV promoter, and finally a RRE obtained from the Bgl II to Hind III sites of pgTatCMV  was inserted by blunt ligation into the Xba I to Sal I sites of the pCI Vector backbone. The Gag-Pol-Vif plasmid was made from digesting the backbone with BssHII and EcoRI and then inserting a 5032 bp BssHII to EcoRI Gag-Pol-Vif fragment from PNLENV-1. The Gag-PR plasmid was made by digesting the Gag-Pol-Vif plasmid with Bgl II and EcoRI to remove Pol-Vif, and then PCR was used to create a 463 bp fragment containing PR with a stop and EcoRI site. The primers used for PCR are 5' GGG AAG ATC TGG CCT TCC CAC 3' and 5' CGG AAT TCG GAT CCT TAA AAA TTT AAA GTG CAG CCA ATC TGA CT 3'. The Gag plasmid was made by first replacing the fragment from Nsi I to Bgl II with a PCR version (845 bp), in which the frameshift had been altered. The primers used are 5' TAA ATG CAT GGG TAA AAG TAG TA 3' and 5' CCA GAT CTT CCC TAA GAA GTT AGC CTG TCT CTC AGT ACA ATC 3'. Next the Bgl II to EcoRI fragment was replaced by a 208 bp PCR product, which contained P6 with an EcoRI site placed after the stop (all Pol components were removed). The primers used for PCR are 5' GGG AAG ATC TGG CCT TCC CAC 3' and 5' CGG AAT TCG CTA GCT ATC TTT ATT GTG ACG AGG GGT C 3'. The optimized version of the Gag plasmid was created by altering the coding sequence for the CAp24 and the start of MAp17 (502 bp), from BssHII to Nsi I sites, using PCR to first anneal 26 overlapping 40 mer oligonucleotides (sequence available upon request) and then a second PCR to create the 502 bp BssHII to Nsi I fragment from the annealed oligonucleotides. This protocol was described previously in [46, 47].
Plasmid construction of VPR fusion plasmids
The backbone used to construct the Vpr fusion plasmids was "pCI Vector" (Promega) in which the CMV promoter from Bgl II to Mlu I had been replaced with an Hpa I to Mlu I fragment containing the Ef1α promoter . The Vpr-RT/IN-Vif fusion plasmid was made by first inserting the 1–88 truncated Vpr (276 bp) into the Xba I site of the pCI Vector backbone. The Bgl II site at the end of Vpr was ligated to a Bgl II to EcoRV 468 bp PCR fragment consisting of the start of RT, keeping RT in frame with Vpr and the PR cleavage site intact. The primers used for PCR were 5' GGA AGA TCTCTG TTG ACT CAG ATT G 3' and 5' GTA CTG ATA TCT AAT CCC TGG 3'. The EcoRV site from the PCR fragment was ligated to an EcoRV to Not I 3372 bp fragment from the Gag-Pol-Vif plasmid containing the rest of RT, IN, Vif, and the RRE. Finally, in the Vpr-RT/IN-Vif plasmid a splice donor site was placed before Vpr (to ensure proper expression of Vif) using annealed oligonucleotides spanning the Mlu I to Xba I sites. The annealed oligonucleotides used were 5' CGC GTG CTA GCG GCG ACT GGT GAG TAC GCC AT 3' and 5' CTA GAT GGC GTA CTC ACCAGT CGC CGC TAG CA 3'. The Vpr-PR plasmid was made from the Vpr-RT/IN-Vif plasmid by digesting this backbone with Bgl II and EcoRI and then ligating the annealed oligonucleotides from Bgl II to BspEI and a 231 bp BspEI to EcoRI PCR fragment generated from the Gag-PR plasmid. The annealed oligonucleotides consisted of 5' GAT CTG TAT CCT TTA GCT TCC CTC AGA TCA CTC TTT GGC AGC GA 3', 5' CCC CTC GTC ACA ATA AAG ATA GGG GGG CAA TTA AAG GAA GCT CTA TTA GAT T 3', 5' CCG GAA TCT AAT AGA GCT TCC TTT AAT TGC CCC CCT ATC TTT ATT GTG ACG A 3', 5' GGG GTC GCT GCC AAA GAG TGA TCT GAG GGA AGC TAA AGG ATA CA 3' and were used to fuse Vpr in frame to PR at the Bgl II site while maintaining the PR cleavage site and inserting a T26S mutation (ACA changed to TCC) thereby creating a BspEI site. The primers used to PCR the 5' portion of PR and to create a stop at the end of PR are 5' GAA GAT CTA CGC GTT CCG GAG CAG ATG ATA CAG TAT TAG AAG 3' and 5' CGG AAT TCG GAT CCT TAA AAA TTT AAA GTG CAG CCA ATC TGA GT 3'.
Human embryonic kidney (HEK) 293T cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 8% bovine growth serum, 2% fetal calf serum and 100-units/ml penicillin-streptomycin. NIH 3T3 were maintained in DMEM media supplemented with 10% calf serum and 100-units/ml penicillin-streptomycin. Jurkat cells were maintained in RPMI media supplemented with 10% fetal calf serum and 100-units/ml penicillin-streptomycin.
Virus production and titers
Virus was produced by transient transfection of 293T cells (10 cm dish) using Fugene (Roche). Twenty-four hours prior to transfection 293T cells were split 1:4 and at two hours prior to transfection media was removed and fresh media was added. Transfections were done using 5, 6, or 7 plasmids depending on the packaging system used. Plasmids transfected for the 5 plasmid system consisted of 3.2 μg lentiviral vector (wt-LTR or SIN, expressing GFP), 4 μg Gag-Pol-Vif packaging plasmid, 0.4 μg of each Rev, Tat, and VSV-G expression plasmids, for the 6 plasmid system the Gag-Pol-Vif plasmid was replaced by two plasmids one expressing Gag-PR (5.5 μg) and the other Vpr-RT/IN-Vif (2.0 μg), and for the 7 plasmid system the Gag-Pol-Vif plasmid was replaced by three plasmids expressing Gag (4.0 μg), Vpr-PR (1.8 μg) and Vpr-RT/IN-Vif (1.8 μg). Supernatants were collected and filtered through a 0.45 μM filter 48 h after transfection. Titers were determined by infecting either NIH 3T3 cells (2 × 105 cells/6 well dish) or Jurkat cells (3 × 106 cells/6 well dish) with serial dilutions of viral supernatants in a total volume of 900 μl in the presence of polybrene (8 μg/ml) or protamine sulfate (6 μg/ml), respectively. Transduced NIH 3T3 and Jurkat cells were analyzed, three or more days after infection for the expression of GFP by FACS. Transducing units (TU) were determined by multiplying the number of total cells at the time of infection by the percentage GFP positive cells by the dilution factor.
Forty-eight hours post-transfection, cells were washed with PBS and radiolabeled with 250 μCi of [35S] methionine per 10 cm dish (Trans35S-Label; ICN, Irvine, CA) for 12 h. Following labeling, cell culture supernatants were collected and centrifuged at 3000 rpm for 30 min to remove any remaining cells or cell debris. Labeled viral particles in supernatants were isolated by ultracentrifugation through a 20% sucrose cushion at 32,000 rpm for 2 h using a Beckman Ti 61 rotor. Pelleted viral particles were then lysed in RIPA buffer (10 mM Tris-HCl (pH 7.4), 1 mM EDTA, 100 mM NaCl, 1% Triton X-100, 0.1% SDS, 0.25% sodium deoxycholate and 0.2% phenyl-methylsulfonyl fluoride (PMSF) and immunoprecipitated with the anti-HIV-1 serum (162) as described previously (53). Immunocomplexes were separated by SDS-12.5% polyacrylamide gel electrophoresis and analyzed by autoradiography. Densitometric analysis of autoradiograms was performed with a Molecular Dynamics Personal densitometer using the ImageQuant™ software version 3.22.
List of abbreviations
Human Immunodeficiency Virus Type 1
Gag precursor polyprotein, MAp17, matrix protein
capsid protein, NCp7, nucleocapsid protein
reverse transcriptase, IN, integrase
replication competent retrovirus
replication competent lentivirus
green fluorescent protein
We thank Patricia Morales for excellent technical assistance, Paul Allen for critical reading of the manuscript and Maria Denaro for helpful discussions. This work was supported by a grant from the NIH (RO1 DK065939) and Genetix Pharmaceuticals.
- Frankel AD, Young JA: HIV-1: fifteen proteins and an RNA. Annu Rev Biochem. 1998, 67: 1-25. 10.1146/annurev.biochem.67.1.1.View ArticlePubMedGoogle Scholar
- Luciw PA: Human Immunodeficiency Viruses and Their Replication, In B.N. Fields, D.M. Knipe, P.M. Howely, R.M. Chanock, J.L. Melnick, T.P. Monath, B. Roizman, and S.E. Straus (ed), Field Virology . 1996, Philadelphia, Pa , Lippincott-Raven Publishers, 1881-1975. 3rdGoogle Scholar
- Jacks T, Power MD, Masiarz FR, Luciw PA, Barr PJ, Varmus HE: Characterization of ribosomal frameshifting in HIV-1 gag-pol expression. Nature. 1988, 331 (6153): 280-283. 10.1038/331280a0.View ArticlePubMedGoogle Scholar
- Dulude D, Berchiche YA, Gendron K, Brakier-Gingras L, Heveker N: Decreasing the frameshift efficiency translates into an equivalent reduction of the replication of the human immunodeficiency virus type 1. Virology. 2006, 345 (1): 127-136. 10.1016/j.virol.2005.08.048.View ArticlePubMedGoogle Scholar
- Luukkonen BG, Fenyo EM, Schwartz S: Overexpression of human immunodeficiency virus type 1 protease increases intracellular cleavage of Gag and reduces virus infectivity. Virology. 1995, 206 (2): 854-865. 10.1006/viro.1995.1008.View ArticlePubMedGoogle Scholar
- Shehu-Xhilaga M, Crowe SM, Mak J: Maintenance of the Gag/Gag-Pol ratio is important for human immunodeficiency virus type 1 RNA dimerization and viral infectivity. J Virol. 2001, 75 (4): 1834-1841. 10.1128/JVI.75.4.1834-1841.2001.PubMed CentralView ArticlePubMedGoogle Scholar
- Wills JW, Craven RC, Weldon RA, Nelle TD, Erdie CR: Suppression of retroviral MA deletions by the amino-terminal membrane-binding domain of p60src. J Virol. 1991, 65 (7): 3804-3812.PubMed CentralPubMedGoogle Scholar
- Park J, Morrow CD: Overexpression of the gag-pol precursor from human immunodeficiency virus type 1 proviral genomes results in efficient proteolytic processing in the absence of virion production. J Virol. 1991, 65 (9): 5111-5117.PubMed CentralPubMedGoogle Scholar
- Rose JR, Babe LM, Craik CS: Defining the level of human immunodeficiency virus type 1 (HIV-1) protease activity required for HIV-1 particle maturation and infectivity. J Virol. 1995, 69 (5): 2751-2758.PubMed CentralPubMedGoogle Scholar
- Shehu-Xhilaga M, Kraeusslich HG, Pettit S, Swanstrom R, Lee JY, Marshall JA, Crowe SM, Mak J: Proteolytic processing of the p2/nucleocapsid cleavage site is critical for human immunodeficiency virus type 1 RNA dimer maturation. J Virol. 2001, 75 (19): 9156-9164. 10.1128/JVI.75.19.9156-9164.2001.PubMed CentralView ArticlePubMedGoogle Scholar
- Vogt VM: Proteolytic processing and particle maturation. Curr Top Microbiol Immunol. 1996, 214: 95-131.PubMedGoogle Scholar
- Wiegers K, Rutter G, Kottler H, Tessmer U, Hohenberg H, Krausslich HG: Sequential steps in human immunodeficiency virus particle maturation revealed by alterations of individual Gag polyprotein cleavage sites. J Virol. 1998, 72 (4): 2846-2854.PubMed CentralPubMedGoogle Scholar
- Katoh I, Ikawa Y, Yoshinaka Y: Retrovirus protease characterized as a dimeric aspartic proteinase. J Virol. 1989, 63 (5): 2226-2232.PubMed CentralPubMedGoogle Scholar
- Krausslich HG: Human immunodeficiency virus proteinase dimer as component of the viral polyprotein prevents particle assembly and viral infectivity. Proc Natl Acad Sci U S A. 1991, 88 (8): 3213-3217. 10.1073/pnas.88.8.3213.PubMed CentralView ArticlePubMedGoogle Scholar
- Pearl LH, Taylor WR: A structural model for the retroviral proteases. Nature. 1987, 329 (6137): 351-354. 10.1038/329351a0.View ArticlePubMedGoogle Scholar
- Pettit SC, Gulnik S, Everitt L, Kaplan AH: The dimer interfaces of protease and extra-protease domains influence the activation of protease and the specificity of GagPol cleavage. J Virol. 2003, 77 (1): 366-374. 10.1128/JVI.77.1.366-374.2003.PubMed CentralView ArticlePubMedGoogle Scholar
- Wu X, Liu H, Xiao H, Kappes JC: Proteolytic activity of human immunodeficiency virus Vpr- and Vpx-protease fusion proteins. Virology. 1996, 219 (1): 307-313. 10.1006/viro.1996.0253.View ArticlePubMedGoogle Scholar
- Markowitz D, Goff S, Bank A: Construction of a safe and efficient retrovirus packaging cell line. Adv Exp Med Biol. 1988, 241: 35-40.View ArticlePubMedGoogle Scholar
- Dull T, Zufferey R, Kelly M, Mandel RJ, Nguyen M, Trono D, Naldini L: A third-generation lentivirus vector with a conditional packaging system. J Virol. 1998, 72 (11): 8463-8471.PubMed CentralPubMedGoogle Scholar
- Kim VN, Mitrophanous K, Kingsman SM, Kingsman AJ: Minimal requirement for a lentivirus vector based on human immunodeficiency virus type 1. J Virol. 1998, 72 (1): 811-816.PubMed CentralPubMedGoogle Scholar
- Zufferey R, Nagy D, Mandel RJ, Naldini L, Trono D: Multiply attenuated lentiviral vector achieves efficient gene delivery in vivo. Nat Biotechnol. 1997, 15 (9): 871-875. 10.1038/nbt0997-871.View ArticlePubMedGoogle Scholar
- Chong H, Starkey W, Vile RG: A replication-competent retrovirus arising from a split-function packaging cell line was generated by recombination events between the vector, one of the packaging constructs, and endogenous retroviral sequences. J Virol. 1998, 72 (4): 2663-2670.PubMed CentralPubMedGoogle Scholar
- Garrett E, Miller AR, Goldman JM, Apperley JF, Melo JV: Characterization of recombination events leading to the production of an ecotropic replication-competent retrovirus in a GP+envAM12-derived producer cell line. Virology. 2000, 266 (1): 170-179. 10.1006/viro.1999.0052.View ArticlePubMedGoogle Scholar
- Fletcher TM, Soares MA, McPhearson S, Hui H, Wiskerchen M, Muesing MA, Shaw GM, Leavitt AD, Boeke JD, Hahn BH: Complementation of integrase function in HIV-1 virions. Embo J. 1997, 16 (16): 5123-5138. 10.1093/emboj/16.16.5123.PubMed CentralView ArticlePubMedGoogle Scholar
- Wu X, Liu H, Xiao H, Conway JA, Hunter E, Kappes JC: Functional RT and IN incorporated into HIV-1 particles independently of the Gag/Pol precursor protein. Embo J. 1997, 16 (16): 5113-5122. 10.1093/emboj/16.16.5113.PubMed CentralView ArticlePubMedGoogle Scholar
- Wu X, Liu H, Xiao H, Kim J, Seshaiah P, Natsoulis G, Boeke JD, Hahn BH, Kappes JC: Targeting foreign proteins to human immunodeficiency virus particles via fusion with Vpr and Vpx. J Virol. 1995, 69 (6): 3389-3398.PubMed CentralPubMedGoogle Scholar
- Kobinger GP, Borsetti A, Nie Z, Mercier J, Daniel N, Gottlinger HG, Cohen A: Virion-targeted viral inactivation of human immunodeficiency virus type 1 by using Vpr fusion proteins. J Virol. 1998, 72 (7): 5441-5448.PubMed CentralPubMedGoogle Scholar
- Yao XJ, Kobinger G, Dandache S, Rougeau N, Cohen E: HIV-1 Vpr-chloramphenicol acetyltransferase fusion proteins: sequence requirement for virion incorporation and analysis of antiviral effect. Gene Ther. 1999, 6 (9): 1590-1599. 10.1038/sj.gt.3300988.View ArticlePubMedGoogle Scholar
- Wu X, Wakefield JK, Liu H, Xiao H, Kralovics R, Prchal JT, Kappes JC: Development of a novel trans-lentiviral vector that affords predictable safety. Mol Ther. 2000, 2 (1): 47-55. 10.1006/mthe.2000.0095.View ArticlePubMedGoogle Scholar
- Bryant M, Ratner L: Myristoylation-dependent replication and assembly of human immunodeficiency virus 1. Proc Natl Acad Sci U S A. 1990, 87 (2): 523-527. 10.1073/pnas.87.2.523.PubMed CentralView ArticlePubMedGoogle Scholar
- Paillart JC, Gottlinger HG: Opposing effects of human immunodeficiency virus type 1 matrix mutations support a myristyl switch model of gag membrane targeting. J Virol. 1999, 73 (4): 2604-2612.PubMed CentralPubMedGoogle Scholar
- Spearman P, Horton R, Ratner L, Kuli-Zade I: Membrane binding of human immunodeficiency virus type 1 matrix protein in vivo supports a conformational myristyl switch mechanism. J Virol. 1997, 71 (9): 6582-6592.PubMed CentralPubMedGoogle Scholar
- Spearman P, Wang JJ, Vander Heyden N, Ratner L: Identification of human immunodeficiency virus type 1 Gag protein domains essential to membrane binding and particle assembly. J Virol. 1994, 68 (5): 3232-3242.PubMed CentralPubMedGoogle Scholar
- Bachand F, Yao XJ, Hrimech M, Rougeau N, Cohen EA: Incorporation of Vpr into human immunodeficiency virus type 1 requires a direct interaction with the p6 domain of the p55 gag precursor. J Biol Chem. 1999, 274 (13): 9083-9091. 10.1074/jbc.274.13.9083.View ArticlePubMedGoogle Scholar
- Kondo E, Gottlinger HG: A conserved LXXLF sequence is the major determinant in p6gag required for the incorporation of human immunodeficiency virus type 1 Vpr. J Virol. 1996, 70 (1): 159-164.PubMed CentralPubMedGoogle Scholar
- Lu YL, Bennett RP, Wills JW, Gorelick R, Ratner L: A leucine triplet repeat sequence (LXX)4 in p6gag is important for Vpr incorporation into human immunodeficiency virus type 1 particles. J Virol. 1995, 69 (11): 6873-6879.PubMed CentralPubMedGoogle Scholar
- Di Marzio P, Choe S, Ebright M, Knoblauch R, Landau NR: Mutational analysis of cell cycle arrest, nuclear localization and virion packaging of human immunodeficiency virus type 1 Vpr. J Virol. 1995, 69 (12): 7909-7916.PubMed CentralPubMedGoogle Scholar
- Konvalinka J, Litterst MA, Welker R, Kottler H, Rippmann F, Heuser AM, Krausslich HG: An active-site mutation in the human immunodeficiency virus type 1 proteinase (PR) causes reduced PR activity and loss of PR-mediated cytotoxicity without apparent effect on virus maturation and infectivity. J Virol. 1995, 69 (11): 7180-7186.PubMed CentralPubMedGoogle Scholar
- Strisovsky K, Tessmer U, Langner J, Konvalinka J, Krausslich HG: Systematic mutational analysis of the active-site threonine of HIV-1 proteinase: rethinking the "fireman's grip" hypothesis. Protein Sci. 2000, 9 (9): 1631-1641.PubMed CentralView ArticlePubMedGoogle Scholar
- Imren S, Fabry ME, Westerman KA, Pawliuk R, Tang P, Rosten PM, Nagel RL, Leboulch P, Eaves CJ, Humphries RK: High-level beta-globin expression and preferred intragenic integration after lentiviral transduction of human cord blood stem cells. J Clin Invest. 2004, 114 (7): 953-962. 10.1172/JCI200421838.PubMed CentralView ArticlePubMedGoogle Scholar
- Iwakuma T, Cui Y, Chang LJ: Self-inactivating lentiviral vectors with U3 and U5 modifications. Virology. 1999, 261 (1): 120-132. 10.1006/viro.1999.9850.View ArticlePubMedGoogle Scholar
- Malim MH, Hauber J, Fenrick R, Cullen BR: Immunodeficiency virus rev trans-activator modulates the expression of the viral regulatory genes. Nature. 1988, 335 (6186): 181-183. 10.1038/335181a0.View ArticlePubMedGoogle Scholar
- Maldarelli F, Martin MA, Strebel K: Identification of posttranscriptionally active inhibitory sequences in human immunodeficiency virus type 1 RNA: novel level of gene regulation. J Virol. 1991, 65 (11): 5732-5743.PubMed CentralPubMedGoogle Scholar
- Pawliuk R, Westerman KA, Fabry ME, Payen E, Tighe R, Bouhassira EE, Acharya SA, Ellis J, London IM, Eaves CJ, Humphries RK, Beuzard Y, Nagel RL, Leboulch P: Correction of sickle cell disease in transgenic mouse models by gene therapy. Science. 2001, 294 (5550): 2368-2371. 10.1126/science.1065806.View ArticlePubMedGoogle Scholar
- Mizushima S, Nagata S: pEF-BOS, a powerful mammalian expression vector. Nucleic Acids Res. 1990, 18 (17): 5322-10.1093/nar/18.17.5322.PubMed CentralView ArticlePubMedGoogle Scholar
- Fuller M, Anson DS: Helper plasmids for production of HIV-1-derived vectors. Hum Gene Ther. 2001, 12 (17): 2081-2093. 10.1089/10430340152677421.View ArticlePubMedGoogle Scholar
- Kotsopoulou E, Kim VN, Kingsman AJ, Kingsman SM, Mitrophanous KA: A Rev-independent human immunodeficiency virus type 1 (HIV-1)-based vector that exploits a codon-optimized HIV-1 gag-pol gene. J Virol. 2000, 74 (10): 4839-4852. 10.1128/JVI.74.10.4839-4852.2000.PubMed CentralView ArticlePubMedGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.