Individual herpes simplex virus 1 (HSV-1) particles exit by exocytosis and accumulate at preferential egress sites.

The human pathogen herpes simplex virus 1 (HSV-1) produces a lifelong infection in the majority of the world's population. While the generalities of alpha herpesvirus assembly and egress pathways are known, the precise molecular and spatiotemporal details remain unclear. In order to study this aspect of HSV-1 infection, we engineered a recombinant HSV-1 strain expressing a pH-sensitive reporter, gM-pHluorin. Using a variety of fluorescent microscopy modalities, we can detect individual virus particles undergoing intracellular transport and exocytosis at the plasma membrane. We show that particles exit from epithelial cells individually, not bulk release of many particles at once, as has been reported for other viruses. In multiple cell types, HSV-1 particles accumulate over time at the cell periphery and cell-cell contacts. We show that this accumulation effect is the result of individual particles undergoing exocytosis at preferential sites and that these egress sites can contribute to cell-cell spread. We also show that the viral membrane proteins gE, gI, and US9, which have important functions in intracellular transport in neurons, are not required for preferential egress and clustering in non-neuronal cells. Importantly, by comparing HSV-1 to a related alpha herpesvirus, pseudorabies virus, we show that this preferential exocytosis and clustering effect are cell type dependent, not virus dependent. This preferential egress and clustering appear to be the result of the arrangement of the microtubule cytoskeleton, as virus particles co-accumulate at the same cell protrusions as an exogenous plus end-directed kinesin motor.IMPORTANCEAlpha herpesviruses produce lifelong infections in their human and animal hosts. The majority of people in the world are infected with herpes simplex virus 1 (HSV-1), which typically causes recurrent oral or genital lesions. However, HSV-1 can also spread to the central nervous system, causing severe encephalitis, and might also contribute to the development of neurodegenerative diseases. Many of the steps of how these viruses infect and replicate inside host cells are known in depth, but the final step, exiting from the infected cell, is not fully understood. In this study, we engineered a novel variant of HSV-1 that allows us to visualize how individual virus particles exit from infected cells. With this imaging assay, we investigated preferential egress site formation in certain cell types and their contribution to the cell-cell spread of HSV-1.

Tumor Tropism of DNA Viruses for Oncolytic Virotherapy.

Oncolytic viruses (OVs) have emerged as one of the most promising cancer immunotherapy agents that selectively target and kill cancer cells while sparing normal cells. OVs are from diverse families of viruses and can possess either a DNA or an RNA genome. These viruses also have either a natural or engineered tropism for cancer cells. Oncolytic DNA viruses have the additional advantage of a stable genome and multiple-transgene insertion capability without compromising infection or replication. Herpes simplex virus 1 (HSV-1), a member of the oncolytic DNA viruses, has been approved for the treatment of cancers. This success with HSV-1 was achievable by introducing multiple genetic modifications within the virus to enhance cancer selectivity and reduce the toxicity to healthy cells. Here, we review the natural characteristics of and genetically engineered changes in selected DNA viruses that enhance the tumor tropism of these oncolytic viruses.

Nuclear Export Inhibitor Selinexor Enhances Oncolytic Myxoma Virus Therapy against Cancer.

Oncolytic viruses exploited for cancer therapy have been developed to selectively infect, replicate, and kill cancer cells to inhibit tumor growth. However, in some cancer cells, oncolytic viruses are often limited in completing their full replication cycle, forming progeny virions, and/or spreading in the tumor bed because of the heterogeneous cell types within the tumor bed. Here, we report that the nuclear export pathway regulates oncolytic myxoma virus (MYXV) infection and cytoplasmic viral replication in a subclass of human cancer cell types where viral replication is restricted. Inhibition of the XPO-1 (exportin 1) nuclear export pathway with nuclear export inhibitors can overcome this restriction by trapping restriction factors in the nucleus and allow significantly enhanced viral replication and killing of cancer cells. Furthermore, knockdown of XPO-1 significantly enhanced MYXV replication in restrictive human cancer cells and reduced the formation of antiviral granules associated with RNA helicase DHX9. Both in vitro and in vivo, we demonstrated that the approved XPO1 inhibitor drug selinexor enhances the replication of MYXV and kills diverse human cancer cells. In a xenograft tumor model in NSG mice, combination therapy with selinexor plus MYXV significantly reduced the tumor burden and enhanced the survival of animals. In addition, we performed global-scale proteomic analysis of nuclear and cytosolic proteins in human cancer cells to identify the host and viral proteins that were upregulated or downregulated by different treatments. These results indicate, for the first time, that selinexor in combination with oncolytic MYXV can be used as a potential new therapy. Significance: We demonstrated that a combination of nuclear export inhibitor selinexor and oncolytic MYXV significantly enhanced viral replication, reduced cancer cell proliferation, reduced tumor burden, and enhanced the overall survival of animals. Thus, selinexor and oncolytic MYXV can be used as potential new anticancer therapy.

LIVE-CELL FLUORESCENCE MICROSCOPY OF HSV-1 CELLULAR EGRESS BY EXOCYTOSIS.

The human pathogen Herpes Simplex Virus 1 (HSV-1) produces a lifelong infection in the majority of the world's population. While the generalities of alpha herpesvirus assembly and egress pathways are known, the precise molecular and spatiotemporal details remain unclear. In order to study this aspect of HSV-1 infection, we engineered a recombinant HSV-1 strain expressing a pH-sensitive reporter, gM-pHluorin. Using a variety of fluorescent microscopy modalities, we can detect individual virus particles undergoing intracellular transport and exocytosis at the plasma membrane. We show that particles exit from epithelial cells individually, not bulk release of many particles at once, as has been reported for other viruses. In multiple cell types, HSV-1 particles accumulate over time at the cell periphery and cell-cell contacts. We show that this accumulation effect is the result of individual particles undergoing exocytosis at preferential sites and that these egress sites can contribute to cell-cell spread. We also show that the viral membrane proteins gE, gI, and US9, which have important functions in intracellular transport in neurons, are not required for preferential egress and clustering in non-neuronal cells. Importantly, by comparing HSV-1 to a related alpha herpesvirus, pseudorabies virus, we show that this preferential exocytosis and clustering effect is cell type-dependent, not virus dependent. This preferential egress and clustering appears to be the result of the arrangement of the microtubule cytoskeleton, as virus particles co-accumulate at the same cell protrusions as an exogenous plus end-directed kinesin motor.

Nuclear export inhibitor Selinexor targeting XPO1 enhances coronavirus replication.

Nucleocytoplasmic transport of proteins using XPO1 (exportin 1) plays a vital role in cell proliferation and survival. Many viruses also exploit this pathway to promote infection and replication. Thus, inhibiting XPO1-mediated nuclear export with selective inhibitors activates multiple antiviral and anti-inflammatory pathways. The XPO1 inhibitor, Selinexor, is an FDA-approved anticancer drug predicted to have antiviral function against many viruses, including SARS-CoV-2. Unexpectedly, we observed that pretreatment of cultured human cells with Selinexor actually enhanced protein expression and replication of coronaviruses, including SARS-CoV-2. Knockdown of cellular XPO1 protein expression significantly enhanced the replication of coronaviruses in human cells. We further demonstrate that Selinexor treatment reduced the formation of unique cytoplasmic antiviral granules that include RNA helicase DHX9 in the virus-infected cells. These results, for the first time, show that the anti-cancer drug Selinexor enhances the replication of coronaviruses in human cells in vitro and thus should be further explored in vivo for the potential impact on the dual use for anticancer and antiviral therapy.

Role of cytokines in poxvirus host tropism and adaptation.

Poxviruses are a diverse family of double-stranded DNA viruses that cause mild-to-severe disease in selective hosts, including humans. Although most poxviruses are restricted to their hosts, some members can leap host species and cause zoonotic diseases and, therefore, are genuine threats to human and animal health. The recent global spread of monkeypox in humans suggests that zoonotic poxviruses can adapt to a new host, spread rapidly in the new host, and evolve to better evade host innate barriers. Unlike many other viruses, poxviruses express an extensive repertoire of self-defense proteins that play a vital role in the evasion of host innate and adaptive immune responses in their newest host species. The function of these viral immune modulators and host-specific cytokine responses can result in different host tropism and poxvirus disease progression. Here, we review the role of different cytokines that control poxvirus host tropism and adaptation.

Induction of tumor cell autosis by myxoma virus-infected CAR-T and TCR-T cells to overcome primary and acquired resistance.

Cytotoxicity of tumor-specific T cells requires tumor cell-to-T cell contact-dependent induction of classic tumor cell apoptosis and pyroptosis. However, this may not trigger sufficient primary responses of solid tumors to adoptive cell therapy or prevent tumor antigen escape-mediated acquired resistance. Here we test myxoma virus (MYXV)-infected tumor-specific T (TMYXV) cells expressing chimeric antigen receptor (CAR) or T cell receptor (TCR), which systemically deliver MYXV into solid tumors to overcome primary resistance. In addition to T cell-induced apoptosis and pyroptosis, tumor eradication by CAR/TCR-TMYXV cells is also attributed to tumor cell autosis induction, a special type of cell death. Mechanistically, T cell-derived interferon γ (IFNγ)-protein kinase B (AKT) signaling synergizes with MYXV-induced M-T5-SKP-1-VPS34 signaling to trigger robust tumor cell autosis. CAR/TCR-TMYXV-elicited autosis functions as a type of potent bystander killing to restrain antigen escape. We uncover an unexpected synergy between T cells and MYXV to bolster solid tumor cell autosis that reinforces tumor clearance.

Oncolytic Myxoma virus infects and damages the tegument of the human parasitic flatworm Schistosoma mansoni.

Schistosomiasis is a devastating disease caused by parasitic flatworms of the genus Schistosoma. Praziquantel (PZQ), the current treatment of choice, is ineffective against immature worms and cannot prevent reinfection. The continued reliance on a single drug for treatment increases the risk of the development of PZQ-resistant parasites. Reports of PZQ insusceptibility lends urgency to the need for new therapeutics. Here, we report that Myxoma virus (MYXV), an oncolytic pox virus which is non-pathogenic in all mammals except leporids, infects and replicates in S. mansoni schistosomula, juveniles, and adult male and female worms. MYXV infection results in the shredding of the tegument and reduced egg production in vitro, identifying MYXV as the first viral pathogen of schistosomes. MYXV is currently in preclinical studies to manage multiple human cancers, supporting its use in human therapeutics. Our findings raise the exciting possibility that MYXV virus represents a novel and safe class of potential anthelmintic therapeutics.

Combination of LIGHT (TNFSF14)-Armed Myxoma Virus Pre-Loaded into ADSCs and Gemcitabine in the Treatment of Experimental Orthotopic Murine Pancreatic Adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is a deadly neoplasm. Oncolytic viruses have tumorolytic and immune response-boosting effects and present great potential for PDAC management. We used LIGHT-armed myxoma virus (vMyx-LIGHT) loaded ex vivo into human adipose-derived mesenchymal stem cells (ADSCs) to evaluate murine PDAC treatment in conjunction with gemcitabine (GEM). The cytotoxicity of this treatment was confirmed in vitro using human and murine pancreatic cancer cell cultures, which were more sensitive to the combined approach and largely destroyed. Unlike cancer cells, ADSCs sustain significant viability after infection. The in vivo administration of vMyx-LIGHT-loaded ADSCs and gemcitabine was evaluated using immunocompetent mice with induced orthotopic PDAC lesions. The expression of virus-encoded LIGHT increased the influx of T cells to the tumor site. Shielded virus followed by gemcitabine improved tumor regression and survival. The addition of gemcitabine slightly compromised the adaptive immune response boost obtained with the shielded virus alone, conferring no survival benefit. ADSCs pre-loaded with vMyx-LIGHT allowed the effective transport of the oncolytic construct to PDAC lesions and yielded significant immune response; additional GEM administration failed to improve survival. In view of our results, the delivery of targeted/shielded virus in combination with TGF-ß ablation and/or checkpoint inhibitors is a promising option to improve the therapeutic effects of vMyx-LIGHT/ADSCs against PDAC in vivo.

Identification of a Novel Myxoma Virus C7-Like Host Range Factor That Enabled a Species Leap from Rabbits to Hares.

Myxoma virus (MYXV) is naturally found in rabbit Sylvilagus species and is known to cause lethal myxomatosis in European rabbits (Oryctolagus cuniculus). In 2019, an MYXV strain (MYXV strain Toledo [MYXV-Tol]) causing myxomatosis-like disease in Iberian hares (Lepus granatensis) was identified. MYXV-Tol acquired a recombinant region of ∼2.8 kb harboring several new genes, including a novel host range gene (M159) that we show to be an orthologous member of the vaccinia virus C7 host range family. Here, to test whether M159 alone has enabled MYXV to alter its host range to Iberian hares, several recombinant viruses were generated, including an MYXV-Tol ΔM159 (knockout) strain. While MYXV-Tol underwent fully productive infection in hare HN-R cells, neither the wild-type MYXV-Lau strain (lacking M159) nor vMyxTol-ΔM159 (deleted for M159) was able to infect and replicate, showing that the ability of MYXV-Tol to infect these cells and replicate depends on the presence of M159. Similar to other C7L family members, M159 was shown to be expressed as an early/late gene but was translocated into the nucleus at later time points, indicating that further studies are needed to elucidate its role in the nucleus. Finally, in rabbit cells, the M159 protein did not contribute to increased replication but was able to upregulate the replication levels of MYXV in nonpermissive and semipermissive human cancer cells, suggesting that the M159-targeted pathway is conserved across mammalian species. Altogether, these observations demonstrate that the M159 protein plays a critical role in determining the host specificity of MYXV-Tol in hare and human cells by imparting new host range functions. IMPORTANCE The coevolution of European rabbit populations and MYXV is a textbook example of an arms race between a pathogen and a host. Recently, a recombinant MYXV (MYXV-Tol) crossed the species barrier by jumping from leporid species to another species, causing lethal myxomatosis-like disease. Given the highly pathogenic nature of this new virus in hares and the incidences of other poxvirus cross-species spillovers into other animals, including humans, it is important to understand how and why MYXV-Tol was able to become virulent in a new host species. The results presented clearly demonstrate that M159 is the key factor allowing MYXV-Tol replication in hare cells by imparting new host range functions. These results have the potential to improve current knowledge about the virulence of poxviruses and provide a platform to better understand the new MYXV-Tol, rendering the virus capable of leaping into a new host species.

Transplantation of autologous bone marrow pre-loaded ex vivo with oncolytic myxoma virus is efficacious against drug-resistant Vk*MYC mouse myeloma.

Multiple myeloma (MM) is a hematological malignancy of plasma cells that remains incurable despite significant progress with myeloablative regimens and autologous stem cell transplantation for eligible patients and, more recently with T cell redirected immunotherapy. Recently, we reported that ex vivo virotherapy with oncolytic myxoma virus (MYXV) improved MM-free survival in an autologous-transplant Balb/c mouse model. Here, we tested the Vk*MYC transplantable C57BL/6 mouse MM model that more closely recapitulates human disease. In vitro, the murine bortezomib-resistant Vk12598 cell line is fully susceptible to MYXV infection. In vivo results demonstrate: (i) autologous bone marrow (BM) leukocytes armed ex vivo with MYXV exhibit moderate therapeutic effects against MM cells pre-seeded into recipient mice; (ii) Cyclophosphamide in combination with BM/MYXV delays the onset of myeloma in mice seeded with Vk12598 cells; (iii) BM/MYXV synergizes with the Smac-mimetics LCL161 and with immune checkpoint inhibitor α-PD-1 to control the progression of established MM in vivo, resulting in significant improvement of survival rates and decreased of tumor burden; (iv) Survivor mice from (ii) and (iii), when re-challenged with fresh Vk12598 cells, developed acquired anti-MM immunity. These results highlight the utility of autologous BM grafts armed ex vivo with oncolytic MYXV alone or in combination with chemotherapy/immunotherapy to treat drug-resistant MM in vivo.

Systemic Delivery of mLIGHT-Armed Myxoma Virus Is Therapeutic for Later-Stage Syngeneic Murine Lung Metastatic Osteosarcoma.

Cancers that metastasize to the lungs represent a major challenge in both basic and clinical cancer research. Oncolytic viruses are newly emerging options but successful delivery and choice of appropriate therapeutic armings are two critical issues. Using an immunocompetent murine K7M2-luc lung metastases model, the efficacy of MYXV armed with murine LIGHT (TNFSF14/CD258) expressed under virus-specific early/late promoter was tested in an advanced later-stage disease K7M2-luc model. Results in this model show that mLIGHT-armed MYXV, delivered systemically using ex vivo pre-loaded PBMCs as carrier cells, reduced tumor burden and increased median survival time. In vitro, when comparing direct infection of K7M2-luc cancer cells with free MYXV vs. PBMC-loaded virus, vMyx-mLIGHT/PBMCs also demonstrated greater cytotoxic capacity against the K7M2 cancer cell targets. In vivo, systemically delivered vMyx-mLIGHT/PBMCs increased viral reporter transgene expression levels both in the periphery and in lung tumors compared to unarmed MYXV, in a tumor- and transgene-dependent fashion. We conclude that vMyx-mLIGHT, especially when delivered using PBMC carrier cells, represents a new potential therapeutic strategy for solid cancers that metastasize to the lung.

Oncolytic Viruses: Newest Frontier for Cancer Immunotherapy.

Cancer remains a leading cause of death worldwide. Despite many signs of progress, currently available cancer treatments often do not provide desired outcomes for too many cancers. Therefore, newer and more effective therapeutic approaches are needed. Oncolytic viruses (OVs) have emerged as a novel cancer treatment modality, which selectively targets and kills cancer cells while sparing normal ones. In the past several decades, many different OV candidates have been developed and tested in both laboratory settings as well as in cancer patient clinical trials. Many approaches have been taken to overcome the limitations of OVs, including engineering OVs to selectively activate anti-tumor immune responses. However, newer approaches like the combination of OVs with current immunotherapies to convert "immune-cold" tumors to "immune-hot" will almost certainly improve the potency of OVs. Here, we discuss strategies that are explored to further improve oncolytic virotherapy.

Convergent Loss of the Necroptosis Pathway in Disparate Mammalian Lineages Shapes Viruses Countermeasures.

Programmed cell death is a vital process in the life cycle of organisms. Necroptosis, an evolutionary form of programmed necrosis, contributes to the innate immune response by killing pathogen-infected cells. This virus-host interaction pathway is organized around two components: the receptor-interacting protein kinase 3 (RIPK3), which recruits and phosphorylates the mixed lineage kinase-like protein (MLKL), inducing cellular plasma membrane rupture and cell death. Critically, the presence of necroptotic inhibitors in viral genomes validates necroptosis as an important host defense mechanism. Here, we show, counterintuitively, that in different mammalian lineages, central components of necroptosis, such as RIPK3 and MLKL, are deleted or display inactivating mutations. Frameshifts or premature stop codons are observed in all the studied species of cetaceans and leporids. In carnivores' genomes, the MLKL gene is deleted, while in a small number of species from afrotheria and rodentia premature stop codons are observed in RIPK3 and/or MLKL. Interestingly, we also found a strong correlation between the disruption of necroptosis in leporids and cetaceans and the absence of the N-terminal domain of E3-like homologs (responsible for necroptosis inhibition) in their naturally infecting poxviruses. Overall, our study provides the first comprehensive picture of the molecular evolution of necroptosis in mammals. The loss of necroptosis multiple times during mammalian evolution highlights the importance of gene/pathway loss for species adaptation and suggests that necroptosis is not required for normal mammalian development. Moreover, this study highlights a co-evolutionary relationship between poxviruses and their hosts, emphasizing the role of host adaptation in shaping virus evolution.

Systemic delivery of TNF-armed myxoma virus plus immune checkpoint inhibitor eliminates lung metastatic mouse osteosarcoma.

Solid cancers that metastasize to the lungs represent a major therapeutic challenge. Current treatment paradigms for lung metastases consist of radiation therapy, chemotherapies, and surgical resection, but there is no single treatment or combination that is effective for all tumor types. To address this, oncolytic myxoma virus (MYXV) engineered to express human tumor necrosis factor (vMyx-hTNF) was tested after systemic administration in an immunocompetent mouse K7M2-Luc lung metastatic osteosarcoma model. Virus therapy efficacy against pre-seeded lung metastases was assessed after systemic infusion of either naked virus or ex vivo-loaded autologous bone marrow leukocytes or peripheral blood mononuclear cells (PBMCs). Results of this study showed that the PBMC pre-loaded strategy was the most effective at reducing tumor burden and increasing median survival time, but sequential intravenous multi-dosing with naked virus was comparably effective to a single infusion of PBMC-loaded virus. PBMC-loaded vMyx-hTNF also potentially synergized very effectively with immune checkpoint inhibitors anti-PD-1, anti-PD-L1, and anti-cytotoxic T lymphocyte associated protein 4 (CTLA-4). Finally, in addition to the pro-immune stimulation caused by unarmed MYXV, the TNF transgene of vMyx-hTNF further induced the unique expression of numerous additional cytokines associated with the innate and adaptive immune responses in this model. We conclude that systemic ex vivo virotherapy with TNF-α-armed MYXV represents a new potential strategy against lung metastatic cancers like osteosarcoma and can potentially act synergistically with established checkpoint immunotherapies.

RNA Helicase A/DHX9 Forms Unique Cytoplasmic Antiviral Granules That Restrict Oncolytic Myxoma Virus Replication in Human Cancer Cells.

RNA helicase A/DHX9 is required for diverse RNA-related essential cellular functions and antiviral responses and is hijacked by RNA viruses to support their replication. Here, we show that during the late replication stage in human cancer cells of myxoma virus (MYXV), a member of the double-stranded DNA (dsDNA) poxvirus family that is being developed as an oncolytic virus, DHX9, forms unique granular cytoplasmic structures, which we named "DHX9 antiviral granules." These DHX9 antiviral granules are not formed if MYXV DNA replication and/or late protein synthesis is blocked. When formed, DHX9 antiviral granules significantly reduced nascent protein synthesis in the MYXV-infected cancer cells. MYXV late gene transcription and translation were also significantly compromised, particularly in nonpermissive or semipermissive human cancer cells where MYXV replication is partly or completely restricted. Directed knockdown of DHX9 significantly enhanced viral late protein synthesis and progeny virus formation in normally restrictive cancer cells. We further demonstrate that DHX9 is not a component of the canonical cellular stress granules. DHX9 antiviral granules are induced by MYXV, and other poxviruses, in human cells and are associated with other known cellular components of stress granules, dsRNA and virus encoded dsRNA-binding protein M029, a known interactor with DHX9. Thus, DHX9 antiviral granules function by hijacking poxviral elements needed for the cytoplasmic viral replication factories. These results demonstrate a novel antiviral function for DHX9 that is recruited from the nucleus into the cytoplasm, and this step can be exploited to enhance oncolytic virotherapy against the subset of human cancer cells that normally restrict MYXV. IMPORTANCE The cellular DHX9 has both proviral and antiviral roles against diverse RNA and DNA viruses. In this article, we demonstrate that DHX9 can form unique antiviral granules in the cytoplasm during myxoma virus (MYXV) replication in human cancer cells. These antiviral granules sequester viral proteins and reduce viral late protein synthesis and thus regulate MYXV, and other poxviruses, that replicate in the cytoplasm. In addition, we show that in the absence of DHX9, the formation of DHX9 antiviral granules can be inhibited, which significantly enhanced oncolytic MYXV replication in human cancer cell lines where the virus is normally restricted. Our results also show that DHX9 antiviral granules are formed after viral infection but not by common nonviral cellular stress inducers. Thus, our study suggests that DHX9 has antiviral activity in human cancer cells, and this pathway can be targeted for enhanced activity of oncolytic poxviruses against even restrictive cancer cells.

Myxoma Virus Expressing LIGHT (TNFSF14) Pre-Loaded into Adipose-Derived Mesenchymal Stem Cells Is Effective Treatment for Murine Pancreatic Adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is a weakly immunogenic fatal neoplasm. Oncolytic viruses with dual anti-cancer properties-oncolytic and immune response-boosting effects-have great potential for PDAC management. Adipose-derived stem cells (ADSCs) of mesenchymal origin were infected ex vivo with recombinant myxoma virus (MYXV), which encodes murine LIGHT, also called tumor necrosis factor ligand superfamily member 14 (TNFSF14). The viability and proliferation of ADSCs were not remarkably decreased (1-2 days) following MYXV infection, in sharp contrast to cells of pancreatic carcinoma lines studied, which were rapidly killed by the infection. Comparison of the intraperitoneal (IP) vs. the intravenous (IV) route of ADSC/MYXV administration revealed more pancreas-targeted distribution of the virus when ADSCs were delivered IP to mice bearing orthotopically injected PDAC. The biodistribution, tumor burden reduction and anti-tumor adaptive immune response were examined. Bioluminescence data, used to assess the presence of the luciferase-tagged virus after IP injection, indicated enhanced trafficking into the pancreata of mice bearing orthotopically-induced PDAC, as compared to tumor-free animals, resulting in extended survival of the treated PDAC-seeded animals and in the boosted expression of key adaptive immune response markers. We conclude that ADSCs pre-loaded with transgene-armed MYXV and administered IP allow for the effective ferrying of the oncolytic virus to sites of PDAC and mediate improved tumor regression.

The Oncolytic Activity of Myxoma Virus against Soft Tissue Sarcoma Is Mediated by the Overexpression of Ribonucleotide Reductase.

BACKGROUND: Myxoma virus (MYXV) is an oncolytic poxvirus that lacks the gene for 1 of the subunits of ribonucleotide reductase (RR), a crucial DNA synthesis and repair enzyme. The overexpression of RR has been implicated in the invasiveness of several cancers, including soft tissue sarcomas (STS). The purpose of the study was to investigate the oncolytic efficacy of MYXV in STS with different levels of RR expression. METHODS: The oncolytic effect of recombinant MYXV was evaluated in 4 human STS cell lines, LS141 (a dedifferentiated liposarcoma), DDLS8817 (a dedifferentiated liposarcoma), RDD2213 (recurrent dedifferentiated liposarcoma), and HSSYII (a synovial sarcoma) using infectivity and cytotoxicity assays. Following the overexpression of RRM2 by cDNA transfection and silencing of RRM2 by siRRM2 in these STS cell lines, the RRM2 expression levels were analyzed by Western blot. RESULTS: We observed a direct correlation between viral oncolysis and RRM2 mRNA levels (R = 0.96) in STS. Higher RRM2 expression was associated with a more robust cell kill. Silencing the RRM2 gene led to significantly greater cell survival (80%) compared with the control group (P = .003), whereas overexpression of the RRM2 increased viral oncolysis by 33% (P < .001). CONCLUSIONS: Our results show that the oncolytic effects of MYXV correlate directly with RR expression levels and are enhanced in STS cell lines with naturally occurring or artificially induced high expression levels of RR. Myxoma virus holds promise in the treatment of advanced soft tissue cancer, especially in tumors overexpressing RR.

A class of viral inducer of degradation of the necroptosis adaptor RIPK3 regulates virus-induced inflammation.

The vaccine strain against smallpox, vaccinia virus (VACV), is highly immunogenic yet causes relatively benign disease. These attributes are believed to be caused by gene loss in VACV. Using a targeted small interfering RNA (siRNA) screen, we identified a viral inhibitor found in cowpox virus (CPXV) and other orthopoxviruses that bound to the host SKP1-Cullin1-F-box (SCF) machinery and the essential necroptosis kinase receptor interacting protein kinase 3 (RIPK3). This "viral inducer of RIPK3 degradation" (vIRD) triggered ubiquitination and proteasome-mediated degradation of RIPK3 and inhibited necroptosis. In contrast to orthopoxviruses, the distantly related leporipoxvirus myxoma virus (MYXV), which infects RIPK3-deficient hosts, lacks a functional vIRD. Introduction of vIRD into VACV, which encodes a truncated and defective vIRD, enhanced viral replication in mice. Deletion of vIRD reduced CPXV-induced inflammation, viral replication, and mortality, which were reversed in RIPK3- and MLKL-deficient mice. Hence, vIRD-RIPK3 drives pathogen-host evolution and regulates virus-induced inflammation and pathogenesis.

Methods for the Construction of Recombinant Oncolytic Myxoma Viruses.

Myxoma virus (MYXV) has proven to be an effective candidate for oncolytic virotherapy in many preclinical cancer models. As a nonhuman pathogen, MYXV does not need to overcome any preexisting antiviral immunity, and its DNA cannot integrate into the host genome, making it an extremely safe vector. Moreover, the large dsDNA genome of MYXV allows the insertion of multiple transgenes and the design of engineered recombinant oncolytic viruses (OVs) with enhanced immunostimulatory or other desired properties. In this chapter, we describe detailed protocols for the generation and characterization of transgene-armed recombinant MYXV vectors.