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. 1999 Aug;73(8):6937-45.
doi: 10.1128/JVI.73.8.6937-6945.1999.

Replication-competent rhabdoviruses with human immunodeficiency virus type 1 coats and green fluorescent protein: entry by a pH-independent pathway

E Boritz  1 , J Gerlach, J E Johnson, J K Rose
Affiliations

Replication-competent rhabdoviruses with human immunodeficiency virus type 1 coats and green fluorescent protein: entry by a pH-independent pathway

E Boritz et al. J Virol. 1999 Aug.

Abstract

We describe a replication-competent, recombinant vesicular stomatitis virus (VSV) in which the gene encoding the single transmembrane glycoprotein (G) was deleted and replaced by an env-G hybrid gene encoding the extracellular and transmembrane domains of a human immunodeficiency virus type 1 (HIV-1) envelope protein fused to the cytoplasmic domain of VSV G. An additional gene encoding a green fluorescent protein was added to permit rapid detection of infection. This novel surrogate virus infected and propagated on cells expressing the HIV receptor CD4 and coreceptor CXCR4. Infection was blocked by SDF-1, the ligand for CXCR4, by antibody to CD4 and by HIV-neutralizing antibody. This virus, unlike VSV, entered cells by a pH-independent pathway and thus supports a pH-independent pathway of HIV entry. Additional recombinants carrying hybrid env-G genes derived from R5 or X4R5 HIV strains also showed the coreceptor specificities of the HIV strains from which they were derived. These surrogate viruses provide a simple and rapid assay for HIV-neutralizing antibodies as well as a rapid screen for molecules that would interfere with any stage of HIV binding or entry. The viruses might also be useful as HIV vaccines. Our results suggest wide applications of other surrogate viruses based on VSV.

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Figures

FIG. 1
FIG. 1
Diagrams of recombinant VSV genomes. To generate VSV-GFP, the GFP gene was inserted into the VSV genome sequence preceded by the appropriate VSV transcription start and stop sequences (SS). The ΔG-gp160G-GFP clone was constructed by inserting the gp160G gene, again under the control of VSV transcriptional start and stop sequences, upstream of the GFP gene in the VSV-GFP clone. Diagrams represent the negative-sense RNA viral genome, extending 3′ to 5′ from left to right. XhoI, NheI, and MluI cleavage sites are indicated.
FIG. 2
FIG. 2
Protein expression by recombinant VSVs. BHK cells were infected with VSV, VSV-GFP, or VSVΔG-gp160G-GFP at an MOI of 5. Infected cells were incubated for 6 h at 37°C and labeled with [35S]methionine for 1 h. Cell lysates were then analyzed directly by SDS-PAGE. The positions of wild-type VSV proteins, L, G, N, P, and M, are indicated along with the GFP and gp160G proteins encoded by the recombinants.
FIG. 3
FIG. 3
GFP expression in infected cells. HeLa-CD4 cells were infected for 10 h with VSV-GFP or VSVΔG-gp160G-GFP at an MOI of approximately 0.75. All infections with VSVΔG-gp160G-GFP were performed in the presence of excess I1. The antibody eliminates a low level of infection resulting from carryover of traces of VSV G protein. A and B show the same field of cells infected with VSV-GFP. (A) Rhodamine immunofluorescence detecting VSV N. (B) GFP fluorescence. C and D show the same field of cells infected with VSVΔG-gp160G-GFP. (C) Rhodamine immunofluorescence detecting VSV N protein. (D) GFP fluorescence.
FIG. 4
FIG. 4
CD4-dependent infectivity of VSVΔG-gp160G-GFP. HeLa or HeLa-CD4 cells were infected with VSV-GFP or VSVΔG-gp160G-GFP at an MOI of 0.75 for 10 h. Infection was measured by detection of VSV N protein by using indirect immunofluorescence. (A) HeLa cells infected with VSV-GFP. (B) HeLa-CD4 cells infected with VSV-GFP. (C) HeLa cells infected with VSVΔG-gp160G-GFP. (D) HeLa-CD4 cells infected with ΔG-gp160G-GFP.
FIG. 5
FIG. 5
Inhibition of VSVΔG-gp160G-GFP infection through a block of receptor or coreceptor. (A) HeLa-CD4 cells were infected with approximately 100 infectious units of VSVΔG-gp160G-GFP in the presence of indicated dilutions of polyclonal sheep antiserum to human CD4, a monoclonal antibody to human CXCR4, or a monoclonal antibody to human CCR5. (B) HeLa-CD4 cells were infected with approximately 100 infectious units of ΔG-gp160G-GFP in the presence of the indicated concentrations of purified human chemokines SDF-1 or RANTES. After 12 h, GFP-positive cells were counted. Viral infectivity at each concentration of inhibitor is expressed as (number of infected cells per well with inhibitor/number of infected cells per well without inhibitor) × 100. Each dilution of antibody was tested in duplicate; error bars represent the range of results between duplicates.
FIG. 6
FIG. 6
Effect of chloroquine and ammonium chloride on the infectivity of VSV-GFP and ΔG-gp160G-GFP. HeLa-CD4 cells on 96-well plates were pretreated with either drug for 1 h then infected with either virus for 90 min in the presence of the drug. Cells were then incubated for an additional 5 h with chloroquine (A) or for an additional 2 h with ammonium chloride (B). At 10 h postinfection, GFP-positive cells were counted. Each drug concentration was tested in triplicate; error bars represent one standard deviation.
FIG. 7
FIG. 7
Neutralization of ΔG-gp160G-GFP by HIVIg. Approximately 100 infectious units of ΔG-gp160G-GFP were incubated with HIVIg or normal human serum (NHS) at the indicated dilutions for 15 min at 37°C. Virus was then applied to HeLa-CD4 cells in 96-well plates. After 10 h, GFP-positive cells were counted. Viral infectivity at each antibody dilution is expressed as (number of infected cells per well with antibody/number of infected cells per well without antibody) × 100. Each dilution of antibody was tested in duplicate; error bars represent the range of results between duplicates.
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