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.