Generation of VSV pseudotypes using recombinant ΔG-VSV for studies on virus entry, identification of entry inhibitors, and immune responses to vaccines

• Published in: Journal of virological methods 2010-11, Vol.169 (2), p.365-374
• Main Author: Whitt, Michael A.
• Format: Article
• Language: English
• Subjects: Animals; Antiviral Agents - pharmacology; Biological and medical sciences; Cell Line; Envelope glycoprotein; Fundamental and applied biological sciences. Psychology; Gene Deletion; High-throughput screening; Humans; Membrane Glycoproteins - genetics; Microbiology; Pseudotypes; Receptor identification; Recombinant Proteins - genetics; Recombinant Proteins - immunology; Recombinant Proteins - metabolism; Techniques used in virology; Transfection; Vaccines; Vesiculovirus - genetics; Viral Envelope Proteins - genetics; Viral Envelope Proteins - physiology; Viral Load; Viral Vaccines - genetics; Viral Vaccines - immunology; Virology; Virus Cultivation; Virus entry; Virus Internalization - drug effects; VSV
• ISSN: 0166-0934; 1879-0984
• DOI: 10.1016/j.jviromet.2010.08.006

▶ Methods for recovery and amplification of ΔG-VSV pseudotypes. ▶ Recombinant vesicular stomatitis virus lacking the envelope glycoprotein (G) for cell entry studies. ▶ Vaccinia-T7 recovery of recombinant rhabdoviruses. Vesicular stomatitis virus (VSV) is a prototypic enveloped animal virus that has been used extensively to study virus entry, replication and assembly due to its broad host range and robust replication properties in a wide variety of mammalian and insect cells. Studies on VSV assembly led to the creation of a recombinant VSV in which the glycoprotein (G) gene was deleted. This recombinant (rVSV-ΔG) has been used to produce VSV pseudotypes containing the envelope glycoproteins of heterologous viruses, including viruses that require high-level biocontainment; however, because the infectivity of rVSV-ΔG pseudotypes is restricted to a single round of replication the analysis can be performed using biosafety level 2 (BSL-2) containment. As such, rVSV-ΔG pseudotypes have facilitated the analysis of virus entry for numerous viral pathogens without the need for specialized containment facilities. The pseudotypes also provide a robust platform to screen libraries for entry inhibitors and to evaluate the neutralizing antibody responses following vaccination. This manuscript describes methods to produce and titer rVSV-ΔG pseudotypes. Procedures to generate rVSV-ΔG stocks and to quantify virus infectivity are also described. These protocols should allow any laboratory knowledgeable in general virological and cell culture techniques to produce successfully replication-restricted rVSV-ΔG pseudotypes for subsequent analysis.