Construction and properties of a herpes simplex virus 1 designed to enter cells solely via the IL-13alpha2 receptor

Abstract

Current design of genetically engineered viruses for selective destruction of cancer cells is based on the observation that attenuated viruses replicate better in tumor cells than in normal cells. The ideal virus, however, is one that can infect only cancer cells by virtue of altered host range. Such a virus can be made more robust than the highly attenuated viruses used in clinical trials. Earlier, we reported the construction of a recombinant herpes simplex virus 1 (R5111) in which the capacity to bind heparan sulfate was disabled and which contained a chimeric IL-13-glycoprotein D that enabled the virus to infect cells expressing the IL-13alpha2 receptor (IL-13Ralpha2) commonly found on the surface of malignant glioblastomas or high-grade astrocytomas. In the earlier report, we showed that the recombinant R5111 was able to enter and infect cells via the interaction of the chimeric glycoprotein D with IL-13Ralpha2 but that the virus retained the capacity to bind and replicate in cells expressing the natural viral receptors HveA or nectin-1. Here, we report the construction of a recombinant virus (R5141) that can only enter and replicate in cells that express the IL-13Ralpha2. The recombinant R5141 does not depend on endocytosis to infect cells. It does not infect cells expressing HveA or nectin-1 receptors or cells expressing IL-13Ralpha2 that had been exposed to soluble IL-13 before infection. The studies described here show that the host range of herpes simplex viruses can be altered by genetic manipulation to specifically target cancer cells.

Fig. 1.

Schematic representation of the sequence arrangements of the wild-type parent virus HSV-1(F) and R5111 and the chimeric gD contained in recombinant viruses R5121, R5113, R5123, R5141, and R5144. In all recombinant viruses, residues 68–78 of gB were deleted and IL-13 was inserted in place of residues 1–140 of gC. In R5111, R5121, and R5113, the signal sequence and residues 1–24 of gD were retained. In R5123, the residues 1–33 were deleted and only the signal peptide sequence of gD was retained. In R5141 and R5144, the signal peptide sequence of gD was replaced by that of IL-13. In addition, these two chimeric gDs contain additional amino acid substitutions as shown. The IL-13 inserted in R5121 and R5113 recombinant viruses carried the E13Y mutation, in which the glutamic acid was substituted by tyrosine. E13Y substitution restricts IL-13 binding to the IL-13Rα2 (52). R5121, R5141, and R5144 carried a wild-type IL-13 copy in gD.

Fig. 2.

Replication of R5141, R5144 virus, and HSV-1(F) in J-nectin, J-HveA, and J-13R cells. Cells grown in 25-cm² flasks were exposed to 0.1 pfu of the recombinant virus or wild-type HSV per cell and harvested 24 h after infection. Progeny virus was titrated on Vero-13R cells.

Fig. 3.

Preexposure of J-13R or Vero-13R cells to IL-13 precludes the infection and replication of R5141 mutant virus in both cell lines. Replicate cultures of J-13R cells (A) or Vero-13R cells (B) were mock-treated or exposed for 1 h to IL-13, IL-2, or IL-4 (1 ng/μl or 10 ng/μl). Then the cells were infected with 0.1 pfu of HSV-1(F) or R5141 per cell and harvested 24 h after infection. Progeny virus was titrated on Vero-13R cells.

Fig. 4.

R5141 recombinant virus does not depend on endocytosis for entry and replication in J-13R or Vero-IL-13R cell lines. Vero, Vero-13R, and J-13R cells were exposed to a 30 μM concentration of PD98059 inhibitor for 1 h. The cells were infected with 0.1 pfu of HSV-1(F) or R5141 per cell and then harvested 24 h after infection. Progeny virus was titrated on Vero-13R cells.

Fig. 5.

Comparison of the accumulation of selected R5141 and wild-type parent proteins in Vero cells (Left) and Vero-13R cells (Right). Vero and Vero-13R cells grown in 25-cm² flasks were exposed to 1.0 pfu of HSV-1 or R5141 virus per cell. The cells were harvested 24 h after infection, solubilized, subjected to electrophoresis in 10% denaturing polyacrylamide gels, electrically transferred onto a nitrocellulose sheet, and reacted with antibody against ICP0, gC, gM, gD (H170 and ZC25), gG, U_L38, or U_S11. The trace amount of gD reacting with anti-gD(H170) represents spillover from an adjacent lane and is not reproducible.