Background Bortezomib is an FDA-approved proteasome inhibitor, and oncolytic HSV-1 (oHSV) is a promising therapeutic approach for cancer. We tested the impact of combining bortezomib with oHSV for anti-tumor efficacy. Methods The synergistic interaction between oHSV and bortezomib was calculated using Chou-Talalay analysis. Viral replication was evaluated using plaque assay and immune fluorescence. Western-blot assays were used to evaluate induction of ER stress and unfolded protein response (UPR). Inhibitors targeting Hsp90 were utilized to investigate the mechanism of cell killing. Anti-tumor efficacy in vivo was evaluated using subcutaneous and intracranial tumor xenografts of glioma and head and neck cancer. Survival was analyzed by Kaplan-Meier curves and two-sided log rank test. Results Combination treatment with bortezomib and oHSV, 34.5ENVE, displayed strong synergistic interaction in ovarian cancer, head & neck cancer, glioma, and malignant peripheral nerve sheath tumor (MPNST) cells. Bortezomib treatment induced ER stress, evident by strong induction of Grp78, CHOP, PERK and IRE1α (western blot analysis) and the UPR (induction of hsp40, 70 and 90). Bortezomib treatment of cells at both sublethal and lethal doses increased viral replication (p value <0.001), but inhibition of Hsp90 ablated this response, reducing viral replication and synergistic cell killing. The combination of bortezomib and 34.5ENVE significantly enhanced anti-tumor efficacy in multiple different tumor models in vivo. Conclusions The dramatic synergy of bortezomib and 34.5ENVE is mediated by bortezomib- induced UPR and warrants future clinical testing in patients.

Acyclovir (ACV) has been shown to inhibit the replication of herpes simplex virus (HSV) in vitro. We examined a wide variety of HSV clinical isolates for the presence of naturally occurring ACV-resistant (ACVD) (4,6,18). The specificity of action of ACV is based on the ability of the herpesvirus-specified thymidine kinase (TK) to phosphorylate the drug, which can then be converted to its active form, acycloguanosine triphosphate. Cellular TK, however, can phosphorylate ACV only inefficiently (6,12). The acycloguanosine triphosphate form inhibits viral DNA synthesis at the level of the viral DNA polymerase, which allows the drug to become incorporated into growing DNA chains and thereby effect chain termination (6, 11). Resistance to ACV can arise because of alterations in the herpes simplex virus (HSV) genes encoding either TK or DNA polymerase (1, 19). Viral TK gene mutations which result in resistance to ACV may be due to the loss of production of an active TK (1,8,19) or to the production of a TK with altered substrate specificity (7). Because the viral DNA polymerase gene is essential (14), alterations in its locus most likely lead to the production of a DNA polymerase with altered substrate or allosteric effector specificity. This is most evident by the fact that some ACV-resistant (ACVr) mutants which are TK+ are also resistant to phosphonoacetic acid (PAA), a competitive inhibitor of viral DNA polymerase (15), although the actual loci for ACV and PAA resistances may be separable (2, 10). In the laboratory, it is possible to derive ACV' mutants by growing HSV in the presence of ACV. In most instances, relatively low ACV concentrations (<10 puM) have been used to maintain selective pressure, resulting in the generation of mutants with alterations in the expression of the HSV TK phenotype (ACVr-TK mutants [1,19]

Pre-steady-state and steady-state kinetics of nucleotide incorporation and excision were used to assess potential mechanisms by which the fidelity of the herpes simplex virus type 1 DNA polymerase catalytic subunit (Pol) is enhanced by its processivity factor, UL42. UL42 had no effect on the pre-steady-state rate constant for correct nucleotide incorporation (150 s ؊1 ) nor on the primary rate-limiting conformational step. However, the equilibrium dissociation constant for the enzyme in a stable complex with primer-template was 44 nM for Pol and 7.0 nM for Pol/UL42. The catalytic subunit and holoenzyme both selected against incorrect nucleotide incorporation predominantly at the level of nucleotide affinity, although UL42 slowed by 4-fold the maximum rate of incorporation of incorrect, compared with correct, nucleotide. Pol, with or without UL42, cleaved matched termini at a slower rate than mismatched ones, but UL42 did not significantly alter the pre-steady-state rate constant for mismatch excision (ϳ16 s ؊1 ). The steady-state rate constant for nucleotide addition was 0.09 s ؊1 and 0.03 s ؊1 for Pol and Pol/UL42, respectively, and enzyme dissociation was the rate-limiting step. The longer half-life for DNA complexes with Pol/UL42 (23 s) compared with that with Pol (8 s) affords a greater probability for excision when a misincorporation event does occur, accounting predominantly for the failure of Pol/ UL42 to accumulate mismatched product at moderate nucleotide concentrations.The HSV-1 1 DNA polymerase holoenzyme is a stable heterodimer composed of a large, 134-kDa catalytic subunit (Pol) and a smaller, 51-kDa subunit, UL42 (1-4). Although Pol possesses inherent 5Ј to 3Ј polymerizing activity (5-7), its processivity is greatly enhanced by UL42 (3,8). Both subunits are absolutely essential for origin-dependent DNA synthesis and for productive viral replication (9 -13), indicating the importance of processive DNA synthesis to viral replication. However, the precise mechanism by which UL42 increases Pol processivity is not known and may differ from that utilized by ring-shaped processivity factors such as proliferating cell nuclear antigen and the ␤ subunit of Escherichia coli pol III (14).HSV-1 Pol, similar to pol ␦, possesses an inherent 3Ј to 5Ј exo activity that imparts proofreading ability to Pol (7, 15). Although the proofreading function of the HSV-1 DNA polymerase can reduce misincorporation frequency in vitro and in vivo (16 -18), the impact of UL42 on the exo activity of Pol and its effect on the fidelity of DNA synthesis have not been well studied. In fact, there has been much disagreement regarding the general role of processivity factors on the fidelity of their cognate polymerases. For example, proliferating cell nuclear antigen has been shown to increase base substitution errors and translesion synthesis by pol ␦ (19, 20). However, exodeficient T7 bacteriophage DNA polymerase has a decreased frequency of base substitutions but an increased frequency of frameshift mutations at reiterated sequences ...

Using a combination of conventional column chromatography and velocity sedimentation, we have purified the 65-kilodalton DNA-binding protein (65KDBp) encoded by herpes simplex virus (HSV) greater than 625-fold. The HSV type 1 (HSV-1)-encoded DNA polymerase (pol) cofractionated with 65KDBP through DEAE-Sephacel, Blue Sepharose, and Mono Q columns and was only separated from 65KDBP by sedimentation through a glycerol gradient. Immunoaffinity columns containing monoclonal antibody (MAb) 6898 immunoglobulin effectively bound most of the HSV-1 pol activity which coeluted with 65KDBP. The pattern of reactivities of HSV-1/HSV-2 recombinants with MAbs specific for HSV-1 65KDBP or the HSV-2-infected cell-specific protein ICSP34,35 strongly suggests that these two species are serotype equivalents of the same protein. Taken together, all these data indicate that 65KDBP is a pol-associated protein and the HSV-1 counterpart of HSV-2 ICSP34,35 previously reported to have similar properties (

No abstract