A Herpes Simplex Virus Type 1 γ34.5 Second-Site Suppressor Mutant That Exhibits Enhanced Growth in Cultured Glioblastoma Cells Is Severely Attenuated in Animals

Abstract

We describe here the neurovirulence properties of a herpes simplex virus type 1 ␥34.5 second-site suppressor mutant. ␥34.5 mutants are nonneurovirulent in animals and fail to grow in a variety of cultured cells due to a block at the level of protein synthesis. Extragenic suppressors with restored capacity to replicate in cells that normally do not support the growth of the parental ␥34.5 deletion mutant have been isolated. Although the suppressor virus reacquires the ability to grow in nonpermissive cultured cells, it remains severely attenuated in mice and is indistinguishable from the mutant ␥34.5 parent virus at the doses investigated. Repairing the ␥34.5 mutation in the suppressor mutant restores neurovirulence to wild-type levels. These studies illustrate that (i) the protein synthesis and neurovirulence defects observed in ␥34.5 mutant viruses can be genetically separated by an extragenic mutation at another site in the viral chromosome; (ii) the extragenic suppressor mutation does not affect neurovirulence; and (iii) the attenuated ␥34.5 mutant, which replicates poorly in many cell types, can be modified by genetic selection to generate a nonpathogenic variant that regains the ability to grow robustly in a nonpermissive glioblastoma cell line. As this ␥34.5 second-site suppressor variant is attenuated and replicates vigorously in neoplastic cells, it may have potential as a replication-competent, viral antitumor agent.

ICP34.5 deleted herpes simplex virus with enhanced oncolytic, immune stimulating, and anti-tumour properties

“…Expression of US11 blocks phosphorylation of PKR which results in enhanced replication of ICP34.5 deleted viruses in tumour cells without compromising safety. 27,33 When tested in mouse xenograft tumour models, this mutation considerably enhanced the anti-tumour effect, as expected from previous work. 25 Increased expression of US11 is accomplished by deletion of the coding sequence for ICP47.…”

“…mutation. 5,33 US11 blocks PKR phosphorylation and expression as an IE gene allows this to occur before PKR phosphorylation would otherwise have been induced by virus infection. 34,35 To further improve the tumour cell killing properties of JS1/ICP34.5À the IE gene ICP47 gene was deleted such that the US11 coding sequence was placed under the control of the promoter which usually regulates the expression of ICP47.…”

“…Increased expression of US11 has previously been shown to enhance replication of HSV ICP34.5 mutants in tumours. [25][26][27] Additionally, the gene for GM-CSF was inserted to maximize the immune response generated following the release of tumour antigens by virus replication. This immune response was further enhanced as the method of increasing US11 expression also deletes the ICP47 gene which usually blocks antigen presentation in HSV infected cells.…”

Treatment of Human Thyroid Carcinoma Cells with the G47delta Oncolytic Herpes Simplex Virus

“…G47Δ is a thirdgeneration oHSV, which was constructed from G207, and contains 3 deletions/mutations that result in selective cytotoxicity to tumor cells (Todo et al, 2001). In addition, both copies of the γ34.5 gene are deleted in this vector, which removes the major HSV neurovirulence gene and precludes the shut-off of protein synthesis in response to viral infection in host cells (Mohr et al, 2001); thus, this mutation attenuates neurovirulence, decreases the chance of reverting to wild-type virus, and causes the virus to preferentially replicate in tumor cells. The ICP6 gene encodes the large subunit of ribonucleotide reductase, which is the key enzyme involved in deoxyribonucleic acid synthesis and viral DNA synthesis in nondividing cells (but not in dividing cells) (Goldstein and Weller, 1988).…”

Replication-competent herpes simplex vectors: design and applications

“…This mutant was also found to give improved antitumor activity in vivo, without compromising safety. 52 These improved characteristics were found to derive from a second site suppressor mutation in the unique short region, which determines the expression of the US11 gene as an IE rather than an L gene, and the deletion of US12, encoding the ICP47 protein, contributing the improvement of antitumor immune response. 53 This viral isolate was shown to exhibit enhanced antitumor effect.…”