Rporated several new safety features that include removing the Gag/Gag-Pol
Rporated 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 “supersplit” 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). Conclusion: 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.BackgroundThe genome of Human Immunodeficiency Virus Type 1 (HIV-1) is complex in that it employs overlapping reading frames to encode two essential polyproteins known asGag 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capsid (CAp17), nucleocapsid (NCp7), and p6 proteins while the Pol polyprotein precursor sup-Page 1 of(page number not for citation purposes)Retrovirology PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/27324125 2007, 4:http://www.retrovirology.com/content/4/1/plies the viral enzymes protease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 [3]. 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 BAY1217389 cost 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 noninfectious [7], and in the reverse case, when Gag-Pol is expressed without Gag, there is efficient PR processing but no production of virions [8]. 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 GagPol 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 PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/28212752 critical to ensure that not only the viral enzymes are.