Oncolytic virotherapy is a promising avenue of cancer gene therapy. Current vectors include human viruses that have been engineered to replicate in tumor cells or nonhuman viruses that are naturally oncotropic and preferentially replicate in tumor cells harboring defects in innate immune pathways such as the type 1 interferon (IFN) pathway. Bovine herpesvirus type 1 (BHV-1) is a species-specific herpesvirus closely related to the human herpes simplex virus type 1 (HSV-1). Although BHV-1 does not efficiently replicate in and affect cellular viability of normal human cells, it is capable of infecting and killing various immortalized and transformed human cell types. Surprisingly, BHV-1 infection of human cells fails to elicit IFN production at the mRNA or protein level and the ability of BHV-1 to kill immortalized and transformed human cells does not correlate with defects in IFN pathways. Furthermore, although some cross-reactivity between BHV-1 and HSV-1 exists, the majority of human antibody or serum samples tested failed to neutralize BHV-1 despite possessing HSV-1 neutralizing capacity. Thus, BHV-1 is a novel candidate oncolytic virus with a distinct mechanism of tumor targeting.

Oncolytic viruses (OVs) are attractive avenues of cancer therapy due to the absence of toxic side effects often seen with current treatment modalities. Bovine herpesvirus 1 (BHV-1) is a species-specific virus that does not induce cytotoxicity in normal primary human cells but can infect and kill various human immortalized and transformed cell lines. To gain a better understanding of the oncolytic breadth of BHV-1, the NCI panel of established human tumor cell lines was screened for sensitivity to the virus. Overall, 72% of the panel is permissive to BHV-1 infection, with corresponding decreases in cellular viability. This sensitivity is in comparison to a sensitivity of only 32% for a herpes simplex virus 1 (HSV-1)-based oncolytic vector. Strikingly, while 35% of the panel supports minimal or no BHV-1 replication, significant decreases in cellular viability still occur. These data suggest that BHV-1 is an OV with tropism for multiple tumor types and is able to induce cytotoxicity independent of significant virus replication. In contrast to other species-specific OVs, cellular sensitivity to BHV-1 does not correlate with type I interferon (IFN) signaling; however, mutations in KRAS were found to correlate with high levels of virus replication. The knockdown or overexpression of KRAS in human tumor cell lines yields modest changes in viral titers; however, overexpression of KRAS in normal primary cells elicits permissivity to BHV-1 infection. Together, these data suggest that BHV-1 is a broad-spectrum OV with a distinct mechanism of tumor targeting. IMPORTANCECancer remains a significant health issue, and novel treatments are required, particularly for tumors that are refractory to conventional therapies. Oncolytic viruses are a novel platform given their ability to specifically target tumor cells while leaving healthy cells intact. For this strategy to be successful, a fundamental understanding of virus-host interactions is required. We previously identified bovine herpesvirus 1 as a novel oncolytic virus with many unique and clinically relevant features. Here, we show that BHV-1 can target a wide range of human cancer types, most potently lung cancer. In addition, we show that enhanced KRAS activity, a hallmark of many cancers, is one of the factors that increases BHV-1 oncolytic capacity. These findings hold potential for future treatments, particularly in the context of lung cancer, where KRAS mutations are a negative predictor of treatment efficacy.

Oncolytic viruses are attractive cancer therapeutics because of their unique mechanisms of tumor cell targeting and the absence of toxic side effects associated with current treatments. Bovine herpesvirus type 1 (BHV-1) is a species-specific herpesvirus that fails to induce cytopathic effects in normal human cells, but is capable of infecting and killing a variety of immortalized and transformed human cell types, including human breast tumor cell lines from luminal, basal A and basal B subtypes, representing a variety of receptor expression profiles. BHV-1 is capable of initiating replication in and killing both bulk and side population cells, the latter of which have enhanced tumor-initiating capacity. Despite the lack of a productive infection or secretion of cytotoxic factors, BHV-1 infection decreases cellular viability in long-term culture following low multiplicity of infection. Moreover, BHV-1-infected MCF7 cells are significantly diminished in their capacity to form tumors in vivo. Overall, these studies suggest that oncolytic BHV-1 targets bulk breast cancer cells and cancer-initiating cells from luminal and basal subtypes by a novel mechanism that is not contingent upon cellular receptor expression status.

Oncolytic human herpes simplex virus type 1 (HSV-1) shows promising treatment efficacy in late-stage clinical trials. The anticancer activity of oncolytic viruses relies on deregulated pathways in cancer cells, which make them permissive to oncolysis. To identify pathways that restrict HSV-1 KM100-mediated oncolysis, this study used a pooled genome-wide short hairpin RNA library and found that depletion of the splicing factor arginine-rich splicing factor 2 (SRSF2) leads to enhanced cytotoxicity of breast cancer cells by KM100. Serine/arginine-rich (SR) proteins are a family of RNA-binding phosphoproteins that control both constitutive and alternative pre-mRNA splicing. Further characterization showed that KM100 infection of HS578T cells under conditions of low SRSF2 leads to pronounced apoptosis without a corresponding increase in virus replication. As DNA topoisomerase I inhibitors can limit the phosphorylation of SRSF2, we combined a topoisomerase I inhibitor chemotherapeutic with KM100 and observed synergistic anticancer effect in vitro and prolonged survival of tumor-bearing mice in vivo.

Viruses interact intimately with the host cell at nearly every stage of replication, and the cell model that is chosen to study virus infection is critically important. Although primary cells reflect the phenotype of healthy cells in vivo better than cell lines, their limited lifespan makes experimental manipulation challenging. However, many tumor-derived and artificially immortalized cell lines have defects in induction of interferon-stimulated genes and other antiviral defenses. These defects can affect virus replication, especially when cells are infected at lower, more physiologically relevant, multiplicities of infection. Understanding the selective pressures and mechanisms underlying the loss of innate signaling pathways is helpful to choose immortalized cell lines without impaired antiviral defense. We describe the trials and tribulations we encountered while searching for an immortalized cell line with intact innate signaling, and how directed immortalization of primary cells avoids many of the pitfalls of spontaneous immortalization.