Viral Purification

The goal of this protocol is to specifically tag and selectively isolate viral DNA from infected cells for the characterization of viral genome associated proteins.

Source: Fricano-Kugler, C. J., et al. Designing, Packaging, and Delivery of High Titer CRISPR Retro and Lentiviruses via Stereotaxic Injection. J. Vis. Exp. (2016).This video demonstrates the step-by-step process for concentrating and purifying lentiviral particles from a mixed suspension containing host cell debris. The procedure utilizes centrifugation, filtration, and PEG-salt precipitation to isolate viral particles while removing soluble impurities. The final product is resuspended in a...

Source: Gao, Y., et al. Generation and Assembly of Virus-Specific Nucleocapsids of the Respiratory Syncytial Virus. J. Vis. Exp. (2021)The video demonstrates the in vitro assembly of virus-specific nucleocapsids from a purified RNA-free nucleoprotein–phosphoprotein complex, their purification by size exclusion chromatography, and validation of full-length viral RNA within the assembled particles.

The genome of the influenza A virus consists of eight separate complexes of RNA and proteins, termed viral ribonucleoprotein complexes (vRNPs). This paper describes the glycerol gradient purification and transmission electron microscopy visualization of influenza A vRNPs.

Source: Lopez Jr., M. A., et al. Detection and Removal of Nuclease Contamination During Purification of Recombinant Prototype Foamy Virus Integrase. J. Vis. Exp. (2017)This video demonstrates the purification of polyhistidine-tagged viral integrase using nickel affinity chromatography. The protocol uses imidazole gradient elution to selectively isolate the integrase based on its affinity for nickel-charged resin.