Controlled Cell Killing by a Recombinant Nonsegmented Negative-Strand RNA Virus

Abstract

In most tissue culture cell lines tested, infection with the paramyxovirus simian virus 5 (SV5) results in very little cell death. To determine if SV5 could be used as a vector for controlled killing of tumor cells, a recombinant SV5 (rSV5-TK) was constructed to encode the herpes simplex virus thymidine kinase (TK) gene. MDBK cells infected with rSV5-TK showed a time-dependent loss of viability when infected cells were cultured in the presence of the prodrug acyclovir (ACV) or ganciclovir (GCV) while no significant toxicity was observed in the absence of prodrug. Cells infected with a control rSV5 expressing GFP and cultured with prodrug showed only a slight reduction in growth rate and little cell death. Time-lapse video microscopy of rSV5-TK-infected MDBK cells that were cultured in the presence of ACV showed an accumulation of cells with morphological effects characteristic of apoptotic cell death. An MDBK cell line persistently infected with rSV5-TK retained long-term expression of TK and sensitivity to prodrug-mediated cell killing that were comparable to those found in an acute infection. Titration experiments showed that the rSV5-TK plus GCV combination resulted in cell death for all mouse and human cell lines tested, although the kinetics and efficiency of cell death varied between cell types. Our results demonstrating controlled cell killing by a recombinant paramyxovirus support the use of negative-strand RNA viruses as therapeutic vectors for targeted killing of cancer cells.

Naturally Occurring Substitutions in the P/V Gene Convert the Noncytopathic Paramyxovirus Simian Virus 5 into a Virus That Induces Alpha/Beta Interferon Synthesis and Cell Death

“…Infection of most cell types with SV5 results in little if any cell death (8,20,38). An unexpected result of our experiments was that infection with rSV5-P/V-CPIϪ resulted in extensive cell death in a number of cell types (Fig.…”

“…Cell viability assays were performed as described previously (38). Briefly, cells were mock infected or infected at an MOI of 50 with WT rSV5-GFP or rSV5-P/V-CPIϪ, washed with PBS, and replaced with DMEM containing 2% FBS.…”

“…At 2 days p.i., cells remaining on the dish were trypsizined and viable cells were determined by trypan blue exclusion. Data are expressed as fold increase in cell number relative to the starting number of cells prior to infection (38). Time-lapse video microscopy was carried out as described previously (38) using 25-cm 2 flasks of A549 or MDBK cells that were mock infected or infected with rSV5-GFP or rSV5-V-CPIϪ.…”

“…Pictures were recorded at intervals on standard VHS tape and were viewed to monitor cytopathic changes in cell morphology. The accumulation of rounded cells that did not undergo cell division was determined and is expressed as a percentage of the total number of cells in the field (38).…”

“…8A, where the number of viable cells remaining at 48 h p.i. is expressed as a fold change relative to the initial number of cells in the culture (38). Infection of MDBK cells with rSV5-GFP did not significantly affect the change in the number of viable cells relative to the mock-infected control (Fig.…”