Background: 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.
Results: 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 x 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.
Download full-text PDF |
Source |
|---|---|
| http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2259377 | PMC |
| http://dx.doi.org/10.1186/1742-4690-4-96 | DOI Listing |
Publication Analysis
Top Keywords
Similar Publications
Enabling icIEF Peak Identification of AAV Capsid Proteins by Fractionation on MauriceFlex and Subsequent Analysis by LC-MS.
Electrophoresis
January 2025
Pfizer, Analytical Research and Development, Chesterfield, Missouri, USA.
A significant limitation of imaged capillary electric focusing (icIEF) is the inability to identify and characterize specific species in the electropherogram. This has led to the development of complementary ion-exchange chromatography (IEX)-based methods that are amenable to either fraction collection and subsequent characterization or online IEX coupled to mass spectrometry. To overcome this limitation while maintaining the use of icIEF, novel approaches, including an icIEF separation and fractionation technology (MauriceFlex, ProteinSimple), have been developed.
View Article and Find Full Text PDFBananaImageBD: A comprehensive banana image dataset for classification of banana varieties and detection of ripeness stages in Bangladesh.
Data Brief
February 2025
Department of Computer Science and Engineering, East West University, Dhaka, Bangladesh.
Bananas are among the most widely consumed fruits globally due to their appealing flavor, high nutritional value, and ease of digestion. In Bangladesh, bananas hold significant agricultural importance, being one of the most extensively cultivated fruits in terms of land coverage and ranking third in production volume. The banana image dataset presented in this article includes clear and detailed images of four common banana varieties in Bangladesh: Sagor Kola (), Shabri Kola (), Bangla Kola ( sp.
View Article and Find Full Text PDFThe Cost of Primary Care: An Experience Analysis in an Urban Setting.
Acta Med Philipp
December 2024
Department of Medicine, College of Medicine and Philippine General Hospital, University of the Philippines Manila, Manila, Philippines.
Objectives: PhilHealth's present health benefit scheme is largely centered on in-patient services. This inadvertently incentivizes hospital admissions for increased access to benefit coverage. To address this problem, this study proposes a costing method to comprehensively finance outpatient care.
View Article and Find Full Text PDFThe molecular pathways leading to GABA and lactic acid accumulation in florets of organic broccoli rabe ( subsp. ) stored as fresh or as minimally processed product.
Hortic Res
January 2025
Institute for Biological Systems, National Research Council (CNR), Via Salaria Km 29,300, 00015 Monterotondo, Rome, Italy.
In the context of organic farming, the introduction of a local product to wider markets and an evaluation of storage effects, metabolic and transcriptomic variations in two broccoli rabe genotypes from production cycles of two different years were studied by comparing florets of stored fresh (SF) and packaged (P) for 4 days with those harvested fresh from the field (H). Twenty-five hydrosoluble compounds, including amino acids, carbohydrates, and organic acids, were quantified by untargeted nuclear magnetic resonance (NMR). Principal component analysis produced a neat separation among the three commodity statuses with P being the most divergent and SF closer to H.
View Article and Find Full Text PDFCurrently, the most common approach for manufacturing GMP-grade adeno-associated virus (AAV) vectors involves transiently transfecting mammalian cells with three plasmids that carry the essential components for production. The requirement for all three plasmids to be transfected into a single cell and the necessity for high quantities of input plasmid DNA, limits AAV production efficiency, introduces variability between production batches, and increases time and labor costs. Here, we developed an all-in-one, single-plasmid AAV production system, called AAVone.
View Article and Find Full Text PDFWant AI Summaries of new PubMed Abstracts delivered to your In-box?
Enter search terms and have AI summaries delivered each week - change queries or unsubscribe any time!