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

Abstract

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.

Figure 1

Recovery, growth and analysis of rVSV-ΔG. (A) Diagram of the cDNA encoding the anti-genome of rVSV-ΔG-GFP. The conserved VSV transcriptional regulatory sequences (octagon = stop sequence; A_n= polyadenylation sequence; arrow = transcriptional promoter or start sequence) are shown above the diagram. The bacteriophage T7 RNA polymerase promoter (T7) and terminator sequences (Tϕ) and the hepatitis delta virus ribozyme (δ-RBZ) with cleavage site are indicated below the diagram. (B) A flow chart illustrating the key points involved in generating and characterizing recombinant rVSV-ΔG. (I) Initially cells are cotransfected with the plasmid pVSV-ΔG-GFP and the four support plasmids encoding the VSV N, P, G and L proteins. This is followed by virus propagation (II), with subsequent plaque-purification and growth of G-complemented rVSV-ΔG-GFP working stocks (III & IV). The G-complemented virus stocks produced in step IV are titrated, and then used to infect cells that express a heterologous glycoprotein for production of the ΔG-GFP pseudotypes. Figure courtesy of C.S. Robison (Robison, 2001).

Figure 2

Phase contrast micrograph of BHK-21 cells taken immediately before starting a recovery experiment for rVSV-ΔG viruses. The cells should be evenly distributed and should ~90–95% confluent. The image was collected using a digital camera connected to a phototube with a 2.5X lens on an Olympus CK2 microscope equipped with a 10X phase objective. The image brightness was adjusted in Canvas 11 using the “levels” function to optimize visualization of individual cells.

Figure 3

Phase contrast images of two fields of cells at 22 hours post-transfection with pCAGGS-G. Arrowheads indicate regions of small syncytia beginning to form. Images were captured and adjusted to optimize cell visualization as described for Fig. 2.

Figure 4

Immunofluorescence/phase contrast micrograph of ΔL-GFP transfected cells 24 hours post-transfection. Cells were imaged using low-level brightfield illumination and fluorescence was achieved by excitation using a FITC-filter set on a Zeiss Axiophot using Axiovision software. Quantification of GFP-positive cells revealed that 21.5% of the cells were replicating ΔL-GFP.

Figure 5

Phase contrast images of cells 24 or 48 hours post-transfer of two independent supernatants from a recovery experiment. The cells had been transfected with pCAGGS-G 24 hours prior to transfer of 0.2 µ filtered recovery supernatants. The top panels show cells after transfer of the supernatant from a culture that was infected with vvT7 and transfected with only the support plasmids. The middle and lower panels are from two independent recovery experiments. The middle panel has a small area of infected cells (arrow) near the middle of the image which are showing VSV-induced CPE. The lower panel also has a small, but much less obvious area of CPE (arrowhead). By 48 hours cells that were infected with the two recovery trial supernatants show extensive cell rounding, characteristic of VSV-induced CPE while the mock culture (top, right panel) does not.