Human cytomegalovirus (HCMV) strain AD169 mutants carrying transposon insertions or large deletions in UL37 exon 1 (UL37x1) were recovered from modified bacterial artificial chromosomes by reconstitution in human fibroblasts expressing the adenovirus anti-apoptotic protein E1B19K. UL37x1 mutant growth was severely compromised in normal fibroblasts, with minimal release of infectious progeny. Growth in E1B19K-expressing cells was restored, but did not reach wild-type levels. Normal fibroblasts infected by UL37x1 mutants underwent apoptosis spontaneously between 48 and 96 h after infection. Apoptosis was inhibited by treatment of cells with the broad-spectrum caspase inhibitor z-Val-Ala-Asp(OMe)-fluoromethylketone, resulting in substantially increased release of virus. Inhibition of viral DNA replication by phosphonoformate or ganciclovir also inhibited apoptosis, implying that death was triggered by late viral functions or by replication and packaging of the viral genome. Immunofluorescent staining showed that although viral proteins accumulated normally during delayed-early phase and viral DNA replication compartments formed, viral late proteins were detected only rarely, suggesting that spontaneous apoptosis occurs early in late phase. These results demonstrate that anti-apoptotic proteins encoded by HCMV UL37x1 [pUL37x1 (vMIA), gpUL37 and gpUL37 M ] prevent apoptosis that would otherwise be initiated by the replication programme of the virus and are required for efficient and sustainable virus replication.

Host responses are increasingly considered important for the efficacious response to experimental cancer therapies that employ viral vectors, but little is known about the specific nature of host responses required. In this study, we investigated the role of host type I interferons (IFN-I) in the efficacy of virally delivered therapeutic genes. Specifically, we used a Semliki Forest virus encoding IL12 (SFV-IL12) based on its promise as an RNA viral vector for cancer treatment. Intratumoral injection of SFV-IL12 induced production of IFN-I as detected in serum. IFN-I production was abolished in mice deficient for the IFNβ transcriptional regulator IPS-1 and partially attenuated in mice deficient for the IFNβ signaling protein TRIF. Use of bone marrow chimeric hosts established that both hematopoietic and stromal cells were involved in IFN-I production. Macrophages, plasmacytoid, and conventional dendritic cells were each implicated based on cell depletion experiments. Further, mice deficient in the IFN-I receptor (IFNAR) abolished the therapeutic activity of SFV-IL12, as did a specific antibody-mediated blockade of IFNAR signaling. Reduced efficacy was not caused by an impairment in IL12 expression, because IFNAR-deficient mice expressed the viral IL12 transgene even more strongly than wild-type (WT) hosts. Chimeric host analysis for the IFNAR involvement established a strict requirement in hematopoietic cells. Notably, although tumor-specific CD8 T lymphocytes expanded robustly after intratumoral injection of WT mice with SFV-IL12, this did not occur in mice where IFNAR was inactivated genetically or pharmacologically. Overall, our results argued that the antitumor efficacy of a virally based transgene therapeutic relied strongly on a vector-induced IFN-I response, revealing an unexpected mechanism of action that is relevant to a broad array of current translational products in cancer research. Cancer Res; 75(3); 497–507. ©2014 AACR.

Drug-inducible systems allow modulation of the duration and intensity of cytokine expression in liver immuno-based gene therapy protocols. However, the biological activity of the transgene may influence their function. We have analyzed the kinetics of interleukin-12 (IL-12) expression controlled by the doxycycline (Dox)-and the mifepristone (Mif)-dependent systems using two long-term expressing vectors directed to liver: a plasmid administered by hydrodynamic injection and a high-capacity adenoviral vector. Daily administration of Dox or Mif was associated with a progressive loss of inducibility and a decrease of murine IL-12 production. This inhibition occurred at the transcriptional level and was probably caused by an interferon (IFN)-g-mediated downmodulation of liverspecific promoters that control the expression of transactivators in these systems. Genome-wide expression microarrays studies revealed a parallel downregulation of liverspecific genes in mice overexpressing murine IL-12. However, a promoter naturally induced by IL-12 was also inhibited by this cytokine when placed in a plasmid vector. Interestingly, treatment with sodium butyrate, a class I/II histone deacetylase inhibitor, was able to rescue liverspecific promoter activity solely in the vector. We conclude that biologically active IL-12 can transiently inhibit the function of drug-inducible systems in non-integrative DNA vectors by reducing promoter activity, probably through IFN-g and protein deacetylation-dependent mechanisms.