Tanapoxvirus: From discovery towards oncolytic immunovirotherapy.

Insufficiency of standard cancer therapeutic agents and a high degree of toxicity associated with chemotherapy and radiotherapy have created a dearth of therapeutic options for metastatic cancers. Oncolytic viruses (OVs) are an emerging therapeutic option for the treatment of various human cancers. Several OVs, including poxviruses, are currently in preclinical and clinical studies and have shown to be effective in treating metastatic cancer types. Tanapoxvirus (TANV), a member of the Poxviridae family, is being developed as an OV for different human cancers due to its desirable safety and efficacy features. TANV causes a mild self-limiting febrile disease in humans, does not spread human to human, and its large genome makes it a relatively safer OV for use in humans. TANV is relatively well characterized at both molecular and clinical levels. Some of the TANV-encoded proteins that are a part of the virus' immune evasion strategy are also characterized. TANV replicates considerably slower than vaccinia virus. TANV has been shown to replicate in different human cancer cells in vitro and regresses human tumors in a nude mouse model. TANV is currently being developed as a therapeutic option for several human cancers including breast cancer, ovarian cancer, colorectal cancer, pancreatic cancer, retinoblastoma, and melanoma. This review provides a comprehensive summary from the discovery to the development of TANV as an OV candidate for a wide array of human cancers.

Structural insight into tanapoxvirus-mediated inhibition of apoptosis.

Premature programmed cell death or apoptosis of cells is a strategy utilized by multicellular organisms to counter microbial threats. Tanapoxvirus (TANV) is a large double-stranded DNA virus belonging to the poxviridae that causes mild monkeypox-like infections in humans and primates. TANV encodes for a putative apoptosis inhibitory protein 16L. We show that TANV16L is able to bind to a range of peptides spanning the BH3 motif of human proapoptotic Bcl-2 proteins and is able to counter growth arrest of yeast induced by human Bak and Bax. We then determined the crystal structures of TANV16L bound to three identified interactors, Bax, Bim and Puma BH3. TANV16L adopts a globular Bcl-2 fold comprising 7 α-helices and utilizes the canonical Bcl-2 binding groove to engage proapoptotic host cell Bcl-2 proteins. Unexpectedly, TANV16L is able to adopt both a monomeric and a domain-swapped dimeric topology where the α1 helix from one protomer is swapped into a neighbouring unit. Despite adopting two different oligomeric forms, the canonical ligand binding groove in TANV16L remains unchanged from monomer to domain-swapped dimer. Our results provide a structural and mechanistic basis for tanapoxvirus-mediated inhibition of host cell apoptosis and reveal the capacity of Bcl-2 proteins to adopt differential oligomeric states whilst maintaining the canonical ligand binding groove in an unchanged state. DATABASE: Structural data are available in the Protein Data Bank (PDB) under the accession numbers 6TPQ, 6TQQ and 6TRR.

Neutralization of matrix metalloproteinase-9 potentially enhances oncolytic efficacy of tanapox virus for melanoma therapy.

Matrix metalloproteinases (MMPs), which are involved in degradation of extracellular matrix, are critical regulators in tumor progression, metastasis and angiogenesis. Although induction of MMPs is frequently observed during the viral infection, the effect of MMPs on viral replication varies between viruses. MMP-9, for instance, is upregulated and promotes the replication of some viruses, such as herpes simplex virus, but inhibits the replication of others. Here, we report that infection with tanapox virus (TPV) promotes the expression of MMP-9 in the melanoma cells. In addition, we show that MMP-9 exerts an anti-viral effect on TPV replication and plays a protective role in TPV-infected melanoma cells in vitro. Moreover, the neutralization of MMP-9 in melanoma cells remarkably enhances the TPV infection and leads to a significant reduction in cell survival. In summary, this study contributes to understanding of the role played by MMP-9 in TPV infectivity and provides more insights for using TPV as cancer virotherapy in future studies. Since TPV has shown substantial oncolytic efficacy in promoting melanoma tumor regression in animal models, identifying mechanisms that suppress MMP-9 expression upon TPV infection can potentially improve its use as a melanoma virotherapy.

The tanapoxvirus 15L protein is a virus-encoded neuregulin that promotes viral replication in human endothelial cells.

Studies on large double-stranded DNA (dsDNA) viruses such as poxviruses have been helpful in identifying a number of viral and cellular growth factors that contribute to our broad understanding of virus-host interaction. Orthopoxviruses and leporipoxviruses are among the most studied viruses in this aspect. However, tanapoxvirus (TPV), a member of the genus Yatapoxvirus, still remains largely unexplored, as the only known hosts for this virus are humans and monkeys. Here, we describe the initial characterization of an epidermal growth factor (EGF)-like growth factor mimicking human neuregulin from TPV, expressed by the TPV-15L gene. Assays using a baculovirus-expressed and tagged TPV-15L protein demonstrated the ability to phosphorylate neuregulin receptors. Neuregulins represent a large family of EGF-like growth factors that play important roles in embryonic endocardium development, Schwann and oligodendrocyte survival and differentiation, localized acetylcholine receptor expression at the neuromuscular junction, and epithelial morphogenesis. Interestingly, certain neuregulin molecules are able to target specific tissues through interactions with heparin sulfate proteoglycans via an immunoglobulin (Ig)-like domain. Analyses of TPV-15L revealed no Ig-like domain, but it retains the ability to bind heparin and phosphorylate neuregulin receptors, providing compelling evidence that TPV-15L is a functional mimetic of neuregulin. TPV-15L knockout virus experiments demonstrate that the virus replicates in human umbilical vein endothelial cells less efficiently than wild-type TPV-Kenya, indicating that this is a nonessential protein for virus viability but can serve a stimulatory role for replication in some cultured cells. However, the precise role of this protein in host-virus interaction still remains to be deduced.

Tropism of Tanapox virus infection in primary human cells.

Tanapox virus (TPV) belongs to the genus Yatapoxvirus and causes a relatively benign zoonotic disease in man, with symptoms that resemble a mild version of human monkeypox. In order to investigate the underlying mechanisms of TPV pathogenesis, the tropism and replication characteristics of TPV were examined in a variety of primary human cells. A GFP expressing TPV (TPV-GFP) was constructed and used to infect primary human dermal fibroblasts (pHDFs) and peripheral blood mononuclear cells (PBMCs), both of which are believed to be major in vivo targets of poxvirus infection. pHDFs fully supported productive replication and cell-cell spread of TPV-GFP. However, induction of cell cycle arrest in pHDFs by contact mediated inhibition or rapamycin treatment eliminated the ability of TPV to fully stimulate cell cycle progression and dramatically reduced viral replication. TPV-GFP-infected human PBMCs were screened for permissiveness by FACS analysis. CD14+ cells (monocytes) were the primary cellular target for TPV infection. A small proportion of CD3+ cells (T cells) were positive for GFP expression, yet TPV was not able to replicate and spread in cultured peripheral blood lymphocytes, regardless of their state of activation. Primary human monocytes, however, demonstrated robust TPV replication, yet these cells no longer supported replication of TPV once they differentiated into macrophages. This unique ex vivo tropism of TPV gives key insights into the basis for the self-limiting pathogenicity of TPV in man.

A secreted high-affinity inhibitor of human TNF from Tanapox virus.

A class of secreted poxvirus tumor necrosis factor (TNF)-binding proteins has been isolated from Tanapox-infected cell supernatants. The inhibitor bound to a TNF-affinity column and was identified as the product of the 2L gene. Sequence analysis of 2L family members from other yatapoxviruses and swinepox virus yielded no sequence homology to any known cellular gene. The expressed Tanapox virus 2L protein bound to human TNF with high affinity (K(d) = 43 pM) and exhibits an unusually slow off-rate. However, 2L is unable to bind to a wide range of human TNF family members. The 2L protein can inhibit human TNF from binding to TNF receptors I and II as well as block TNF-induced cytolysis. Thus, Tanapox virus 2L represents an inhibitor of human TNF and offers a unique strategy with which to modulate TNF activity.

The replication cycle of tanapox virus in owl monkey kidney cells.

The growth kinetics of tanapox virus in owl monkey kidney cells was elucidated by single-step growth curves at multiplicities of 10, 1.0, and 0.1 plaque forming units (pfu) per cell at 37 and 33 degrees C. Virus replicated equally well at both temperatures and produced a cytopathic effect that was characterized by densely packed rounded cells with retrogressed monolayer and granular vacuolated cytoplasm. Single-step growth curves revealed that the eclipse period varied from 24 h postinfection (hpi) at a multiplicity of infection of 10 pfu/cell to 48 hpi at 0.1 pfu/cell. The length of the latent period also varied from 36 hpi at 10 pfu/cell to 48 hpi at 0.1 pfu/cell. The intracellular virus, extracellular virus, and total virus titers reached their maximums relatively early at 10 pfu/cell as compared with 0.1 pfu/cell. About 78% of the mature progeny virion is retained intracellularly at 10 pfu/cell at 96 hpi. We conclude that tanapox virus replication is similar to other poxviruses, but the replication cycle is longer when compared with vaccinia virus.