Myxoma and Vaccinia Viruses Bind Differentially to Human Leukocytes

Dunlap

Clinical Lymphoma Myeloma and Leukemia

2016

Introduction Multiple myeloma is a clonal malignancy of plasma B cells. While recent advances have improved overall prognosis, virtually all myeloma patients still succumb to relapsing disease. Therefore, novel therapies to treat this disease remain urgently needed. We have recently demonstrated that treatment of human multiple myeloma cells with an oncolytic virus known as myxoma results in rapid cell death even in the absence of viral replication; however, the specific mechanisms and pathways involved remain unknown. Materials and Methods To determine how Myxoma virus eliminates human multiple myeloma cells, we queried the apoptotic pathways which were activated following viral infection using immunoblot and other cell biology approaches. Results Our results indicate that myxoma virus infection initiates apoptosis in multiple myeloma cells through activation of the extrinsic initiator caspase-8. Caspase-8 activation subsequently results in cleavage of Bid and loss of mitochondrial membrane potential causing secondary activation of caspase-9. Activation of caspase-8 appears to be independent of extrinsic death ligands and instead correlates with depletion of cellular inhibitors of apoptosis. We hypothesize that this depletion results from virally mediated host-protein shutoff since a myxoma construct which overexpresses the viral decapping enzymes displays improved oncolytic potential. Conclusion Taken together, these results suggest that myxoma virus eliminates human multiple myeloma cells through a pathway unique to oncolytic poxviruses making it an excellent therapeutic option for the treatment of relapsed or refractory patients.

Section: Discussionmentioning

confidence: 85%

Myxoma Virus Induces Ligand Independent Extrinsic Apoptosis in Human Myeloma Cells

Dunlap

Clinical Lymphoma Myeloma and Leukemia

2016

“…15 MYXV infections were performed by incubating human resting or activated T cells with vMyx-green fluorescent protein (GFP) 16 or vMyx-GFP/ tomato red fluorescent protein (TrFP) 17 (at MOI 5 10) for 1 hour at room temperature. For both binding and infection, mock-treated cells were incubated in complete media containing no virus under the same incubation conditions.…”

Section: Methods Virus Binding and Infection Conditionsmentioning

confidence: 99%

“…Next, the T cells were mixed with human MM cells (U266 cells) that have previously shown susceptibility to MYXV oncolysis. 10,14,15 This mixture of T cells and MM cells was incubated at 37°C for an additional 48 hours. As controls, T cells alone or MM cells alone were subjected to the same MYXV treatment, 6 anti-CD3/CD28 activation and culture conditions.…”

Section: Activated T Lymphocytes Efficiently Transfer Myxv and Kill Smentioning

confidence: 99%

Myxoma virus suppresses proliferation of activated T lymphocytes yet permits oncolytic virus transfer to cancer cells

Villa¹,

Wasserfall²,

Meacham³

et al. 2015

Blood

Self Cite

“…Previously, the only primary human leukocyte type known to be permissive to VACV was activated T cells (35). Among primary human leukocyte subtypes, VACV is able to bind to monocytes, activated T cells, B cells, and neutrophils (35,49,50), but it is able to express only viral genes in monocytes and activated T cells to a significant degree (49,51,52). Primary human monocytes and B cells have been shown to express little or no VACV late gene A56 (35), suggesting that these cell types are abortive.…”

Section: Discussionmentioning

confidence: 99%

Primary Human Macrophages Serve as Vehicles for Vaccinia Virus Replication and Dissemination

Byrd

Shepherd

Lan

et al. 2014

J Virol

Human monocytic and professional antigen-presenting cells have been reported only to exhibit abortive infections with vaccinia virus (VACV). We found that monocyte-derived macrophages (MDMs), including granulocyte macrophage colony-stimulating factor (GM-CSF)-polarized M1 and macrophage colony-stimulating factor (M-CSF)-polarized M2, but not human AB serumderived cells, were permissive to VACV replication. The titers of infectious virions in both cell-free supernatants and cellular lysates of infected M1 and M2 markedly increased in a time-dependent manner. The majority of virions produced in permissive MDMs were extracellular enveloped virions (EEV), a secreted form of VACV associated with long-range virus dissemination, and were mainly found in the culture supernatant. Infected MDMs formed VACV factories, actin tails, virion-associated branching structures, and cell linkages, indicating that MDMs are able to initiate de novo synthesis of viral DNA and promote virus release. VACV replication was sensitive to inhibitors against the Akt and Erk1/2 pathways that can be activated by VACV infection and M-CSF stimulation. Classical activation of MDMs by lipopolysaccharide (LPS) plus gamma interferon (IFN-␥) stimulation caused no effect on VACV replication, while alternative activation of MDMs by interleukin-10 (IL-10) or LPS-plus-IL-1␤ treatment significantly decreased VACV production. The IL-10-mediated suppression of VACV replication was largely due to Stat3 activation, as a Stat3 inhibitor restored virus production to levels observed without IL-10 stimulation. In conclusion, our data demonstrate that primary human macrophages are permissive to VACV replication. After infection, these cells produce EEV for long-range dissemination and also form structures associated with virions which may contribute to cell-cell spread. O rthopoxvirus, a genus of the subfamily Chordopoxvirinae of the family Poxviridae, is composed of large DNA viruses that infect a wide array of mammalian species. The most famous member of the genus is the human-specific variola virus, which causes smallpox. In nature, variola virus has a strict human-specific tropism, and nonhuman reservoirs of the virus have never been found. Variola virus is generally transmitted via inhalation, with subsequent infection and replication in epithelial cells of the oral and respiratory mucosa. The next stage of infection involves viral infiltration of lymphoid organs accompanied by strong viremia and skin lesions. Recent studies using high doses of variola virus to infect Cynomolgus macaques in an attempt to develop an animal model of smallpox have demonstrated that infected animals develop systemic infection and hemorrhagic symptoms (1, 2). These symptoms were correlated with monocyte/macrophage-mediated viremia and dissemination (1, 2). In mice, macrophages are crucial to control the infection of the orthopoxvirus ectromelia virus (ECTV) (3, 4). However, ECTV replicates in macrophages (5) and directly contributes to dissemination within the host (6). Giv...