Attenuation of herpes simplex virus neurovirulence with picornavirus cis-acting genetic elements

Herpes simplex virus type 1 (HSV) and poliovirus (PV) are neuropathogens that target cells in the central nervous system (CNS). Both use cellular receptors belonging to the nectin family: nectin-1 or the PV receptor-related molecule 1 for HSV (15) and CD155 or the PV receptor for PV (35). Both viruses have been genetically engineered to attenuate neuropathogenicity. Nonneurovirulent variants of both HSV and PV selectively propagate in malignant glioma cells and are being investigated as oncolytic agents against glioblastoma multiforme (GBM). The HSV double-stranded DNA (dsDNA) genome of ∼152,250 bp encodes at least 84 gene products. Attenuation was achieved by deletion of the γ134.5 gene, abolishing neurovirulence in mice (2, 5) and primates (21). Moreover, Δγ134.5 HSVs, either with or without a mutation in another nonessential gene, have been safely administered to GBM patients (32, 48). A key function of the γ134.5 gene product, infected-cell protein 34.5 (ICP34.5), is to oppose host antiviral responses that culminate in protein synthesis shutoff. HSV infection results in dsRNA-dependent protein kinase (PKR) activation, which unchecked, arrests translation via phosphorylation of the α subunit of eukaryotic initiation factor (eIF-2α) (4, 6). ICP34.5 redirects cellular protein phosphatase 1α to dephosphorylate eIF-2α, maintaining a pool of active eIF-2α (19). Δγ134.5 mutants are unable to counter PKR, and viral growth is therefore inhibited in the CNS (4, 6). However, these mutants propagate efficiently in tumor cells with defective or suppressed PKR responses (1), spurring clinical application as oncolytic agents (32, 48). Δγ134.5 growth is impeded in some cancer cell lines (39, 50), indicating that ICP34.5 is required for robust propagation in these cell types. HSVs that conditionally express ICP34.5 in GBM would resolve this impasse, selectively supplying the protein in tumor cells resistant to Δγ134.5 viruses. This strategy has been pursued by selection for second-site suppressor mutants capable of PKR inhibition yet attenuated for neurovirulence (52) or by placing γ134.5 under transcriptional control of glioma-specific promoters (24, 25, 43). We report here engineering of HSVs specifically targeting GBM at the level of translation control. This was achieved by replacing noncoding regions driving translation of the γ134.5 transcript with picornavirus 5′ and 3′ untranslated regions (UTRs) conferring active gene expression in GBM, while thwarting ICP34.5 synthesis in the CNS. Neurovirulence of PV, a plus-strand RNA virus of the Picornaviridae family associated with paralytic poliomyelitis, depends on its internal ribosomal entry site (IRES). The ∼7,400-nucleotide (nt) PV genome consists of a single open reading frame (ORF) (27), which is translated in a 5′ end-, cap-independent fashion by an IRES in the ∼750-nt 5′UTR (22, 45). The PV IRES codetermines pathogenicity, because substitution with the corresponding segment of human rhinovirus type 2 (HRV2) eliminates neurovirulence in human PV receptor transgenic mice (16) and in primates (17). The HRV2 IRES produces neuron-specific growth defects (3, 16) but does not affect propagation in established (18) and primary (36) GBM cell lines. PV/HRV2 chimeras efficiently reduce the tumor burden in GBM xenograft animal models (18, 44). Specificity for GBM is attributed to neuronal repressors of HRV2 IRES function, the dsRNA binding protein 76 (DRBP76) and nuclear factor 45 (NF45) (37, 38). DRBP76 forms a heterodimer with NF45 in neuronal cells (but not in GBM cells) (38), which binds to the HRV2 IRES and produces a nonperforming ribonucleoprotein (RNP) complex (38). The heterodimer associates with the translation apparatus exclusively in neuronal cells and inhibits translation initiation at the HRV2 IRES (38). Placing the HSV γ134.5 transcript under translation control of the HRV2 IRES leads to efficient ICP34.5 expression in HRV2 IRES-permissive cell lines. Insertion of picornavirus UTR sequences does not compromise the integrity of the γ134.5 transcript in HSV-infected cells. HSV recombinants expressing ICP34.5 under HRV2 IRES control display vastly improved growth in glioma cells resistant to Δγ134.5 virus. Neurovirulence assessment in mice indicates that ICP34.5 is not expressed or is expressed at very low levels in neuronal cells, as the recombinant virus was safely administered to mice over a range of doses that were lethal with wild-type (wt) HSV.