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. 2008 Oct;82(19):9369-80.
doi: 10.1128/JVI.01054-08. Epub 2008 Jul 30.

A hyperfusogenic F protein enhances the oncolytic potency of a paramyxovirus simian virus 5 P/V mutant without compromising sensitivity to type I interferon

Maria D Gainey  1 , Mary J Manuse, Griffith D Parks
Affiliations

A hyperfusogenic F protein enhances the oncolytic potency of a paramyxovirus simian virus 5 P/V mutant without compromising sensitivity to type I interferon

Maria D Gainey et al. J Virol. 2008 Oct.

Abstract

Viral fusogenic membrane proteins have been proposed as tools to increase the potency of oncolytic viruses, but there is a need for mechanisms to control the spread of fusogenic viruses in normal versus tumor cells. We have previously shown that a mutant of the paramyxovirus simian virus 5 (SV5) that harbors mutations in the P/V gene from the canine parainfluenza virus (P/V-CPI(-)) is a potent inducer of type I interferon (IFN) and apoptosis and is restricted for spread through normal but not tumor cells in vitro. Here, we have used the cytopathic P/V-CPI(-) as a backbone vector to test the hypothesis that a virus expressing a hyperfusogenic glycoprotein will be a more effective oncolytic vector but will retain sensitivity to IFN. A P/V mutant virus expressing an F protein with a glycine-to-alanine substitution in the fusion peptide (P/V-CPI(-)-G3A) was more fusogenic than the parental P/V-CPI(-) mutant. In two model prostate tumor cell lines which are defective in IFN production (LNCaP and DU145), the hyperfusogenic P/V-CPI(-)-G3A mutant had normal growth properties at low multiplicities of infection and was more effective than the parental P/V-CPI(-) mutant at cell killing in vitro. However, in PC3 cells which produce and respond to IFN, the hyperfusogenic P/V-CPI(-)-G3A mutant was attenuated for growth and spread. Killing of PC3 cells was equivalent between the parental P/V-CPI(-) mutant and the hyperfusogenic P/V-CPI(-)-G3A mutant. In a nude mouse model using LNCaP cells, the hyperfusogenic P/V-CPI(-)-G3A mutant was more effective than P/V-CPI(-) at reducing tumor burden. In the case of DU145 tumors, the two vectors based on P/V-CPI(-) were equally effective at limiting tumor growth. Together, our results provide proof of principle that a cytopathic SV5 P/V mutant can serve as an oncolytic virus and that the oncolytic effectiveness of P/V mutants can be enhanced by a fusogenic membrane protein without compromising sensitivity to IFN. The potential advantages of SV5-based oncolytic vectors are discussed.

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Figures

FIG. 1.
FIG. 1.
Incorporation of the G3A mutation into the F protein of P/V-CPI− results in a virus with a hyperfusogenic phenotype. (A) Schematic of viruses used in this study. The genome structure of SV5 is shown with addition of the GFP gene between HN and L as described previously (20). Arrows indicate WT and mutant P/V and F genes occurring in rSV5-GFP, P/V-CPI−, and P/V-CPI−-G3A recombinant viruses. (B) Location of G3A mutation. The SV5 F protein is shown schematically as F1 and F2 subunits with the amino acid sequence at the proteolytic cleavage site. The location of the G3A substitution in the fusion peptide (hatched box) is indicated (adapted from Lamb et al. [32]). (C) P/V-CPI−-G3A is hyperfusogenic. BSR-T7 cells were mock infected or infected at an MOI of 0.05 with rSV5-GFP, P/V-CPI−, or P/V-CPI−-G3A, and microscopy pictures were taken at 48 h p.i. (D) Quantitative fusion assay. BSR-T7 cells were mock infected or infected at an MOI of 5 with rSV5-GFP, P/V-CPI−, or P/V-CPI−-G3A. At 21 h p.i., infected cells were overlaid with Vero cells that had been previously transfected with the pEMCLucβbAn plasmid, which encodes luciferase under the control of a T7 promoter. At 8 and 16 h postoverlay (po) luciferase activity was measured as the relative increase over mock-infected cells. Results are the average of three transfections, with error bars representing standard deviations.
FIG. 2.
FIG. 2.
Spread at low MOIs and IFN sensitivity of the hyperfusogenic P/V-CPI−-G3A mutant. (A) Virus spread at low MOIs. Vero cells were mock infected or infected at an MOI of 0.05 with the indicated viruses, and microscopy pictures were taken at 24, 48 and 72 h p.i. (B) IFN sensitivity. Vero cells were mock treated or treated for 24 h with 100 or 1,000 units of IFN before infection with the indicated viruses at an MOI of 0.05. At 30 h p.i., cells were examined by microscopy for GFP expression.
FIG. 3.
FIG. 3.
Cell killing by the hyperfusogenic P/V-CPI−-G3A mutant. Vero cells were mock infected or infected with the indicated viruses at an MOI of 0.05, 1, or 10. For cells infected with rSV5-TK, medium was used with or without 10 μg/ml GCA as described previously (39). Cell viability was determined at 24, 48, and 72 h p.i. using an MTS viability assay as described in Materials and Methods. Data are from quadruplicate samples and are expressed as a percentage of the value obtained with mock-infected cells, with error bars representing the standard deviation.
FIG. 4.
FIG. 4.
IFN induction and growth at low MOIs of the P/V-CPI−-G3A mutant in representative prostate cancer cell lines. (A) IFN production in human prostate cancer cell lines. LNCaP, DU145, or PC3 cells were mock infected or infected at an MOI of 10 with the indicated viruses. At 24 h p.i., medium was collected, and the amount of IFN-β was determined by an ELISA. Data are the average of three measurements, with error bars representing standard deviations. (B to D) Growth at low MOIs. LNCaP, DU145, or PC3 cells were infected at an MOI of 0.05 with rSV5-GFP, P/V-CPI−, or P/V-CPI−-G3A. Medium was harvested at the indicated times p.i., and the amount of infectious virus was determined by plaque assay. LNCaP and DU145 time courses represent one experiment done in triplicate. The PC3 time course represents the average of two independent experiments.
FIG. 5.
FIG. 5.
Growth at low MOIs and spread of P/V-CPI− and P/V-CPI−-G3A are restricted by IFN in the PC3 prostate cancer cell line. (A) Effect of IFN on viral spread. PC3 cells were mock infected or infected at an MOI of 0.05 with P/V-CPI− or P/V-CPI−-G3A. Cells were incubated in medium with (+) or without (−) neutralizing IFN-α and IFN-β antibodies as described in Materials and Methods. At 24, 48, and 72 h p.i., microscopy pictures were taken. (B) Effect of IFN on viral growth. The medium from PC3 cells treated as described in panel A was harvested to assay infectious virus by plaque assay. Results are representative of two experiments. Ab, antibody.
FIG. 6.
FIG. 6.
P/V-CPI−-G3A is more cytopathic than P/V-CPI− in two out of three prostate cancer cell lines tested. Results of cell viability assays are shown. LNCaP, DU145, or PC3 cells were mock infected or infected at an MOI of 0.05, 1, or 10 with the indicated viruses, and cell viability was measured by MTS assay at 24-h increments. In panel C, DU145 cells were treated overnight with 1,000 units/ml of IFN, washed, and infected as described for the other panels. Data are expressed as a percentage of the value obtained with mock-infected cells and are representative of two or more independent experiments done in quadruplicate. An asterisk denotes values from the P/V-CPI−-G3A samples that are statistically significant (P < 0.05; Students t test) compared to the corresponding P/V-CPI− samples.
FIG. 7.
FIG. 7.
P/V-CPI−-G3A is more effective at reducing LNCaP tumor burden than P/V-CPI−. (A) LNCaP tumor pictures. Groups of 10 male nude mice were implanted with LNCaP tumor cells. Mice that formed palpable tumors were injected intratumorally with PBS or 108 PFU of purified P/V-CPI− or P/V-CPI−-G3A at day 0 and day 8 of the study. On day 18 after the initial injection, tumors were harvested and fixed, and representative photographs were taken. (B) LNCaP tumor weight. The tumors harvested as described in panel A were weighed. Note that some symbols overlap. The bars indicate average tumor weights. vs, versus. (C) Mouse weight loss. Mice bearing LNCaP tumors and treated as described in panel A were monitored every other day for 18 days. Error bars represent ±95% confidence intervals.
FIG. 8.
FIG. 8.
Both P/V-CPI− and P/V-CPI−-G3A delay DU145 tumor growth. Groups of 3 or 4 male nude mice were implanted with DU145 tumor cells. Mice with palpable tumors were injected intratumorally with PBS or 108 PFU of P/V-CPI− or P/V-CPI−-G3A on days 0 and 8 of the study. Tumor size was measured by calipers every other day for 20 days. Error bars represent standard deviations from the average.
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