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.

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.

Myxoma virus (MYXV) is a leporipoxvirus that causes lethal disease in Leporids. Hares and rabbits belong to the Leporidae family and are believed to have had a common ancestor 12 million years ago. After seventy years of contact with European hares without causing mortalities or disease manifestation, a recombinant MYXV infected Iberian hares (Lepus granatensis) causing high mortalities. Like all poxviruses, MYXV encodes a wealth of immune modulators required for successful virulence that also mediate host species jumping, for example, into hares. Here, we summarize the data of known MYXV immune modulators, their cellular functions, and their effects on European rabbits. Additionally, we suggest that the critical restrictions MYXV would encounter in colonizing a potentially new host species stem from their interactions with the host’s innate immune environment. Lastly, we synthesize our understanding of some poxvirus genome architectural features that might have facilitated the host species jump of MYXV into hares from rabbits.

Poxviruses encode a plethora of proteins adapted to diverse cellular responses against viruses. The poxvirus-encoded E3-like proteins are multifunctional, regulating diverse cellular antiviral responses. The canonical Vaccinia E3-like proteins have two domains: an N-terminal Z-form nucleic acid-binding domain (Zα-BD) and a C-terminal double-stranded RNA-binding domain (dsRNA-BD). Using protein sequence and structural homology modeling, we identified dsRNA-BD-fold-containing proteins in all the poxviruses except avipoxviruses, salmon poxvirus, and entomopoxviruses. Furthermore, we show that the acquisition of these proteins likely happened under three distinct events and can be classified into three categories: (i) the E3-like proteins with dsRNA-BD with the presence or absence of the N-terminal domain; (ii) unconventional/putative dsRNA-BD-fold-like proteins, present in macropoxvirus and molluscipoxvirus, and (iii) the dsRNA-BD-containing protein present in crocodile poxvirus. Members of leporipoxvirus, waddenpoxvirus, cetaceanpoxvirus, and selected members of orthopoxvirus contain E3-like proteins missing the N-terminal Zα-BD required for necroptosis inhibition. Using AlphaFold, we also show that the Zα-BD of chordopoxviruses, E3-like proteins, is structurally more variable than the dsRNA-binding domain. Compared to orthopoxviruses that inhibit necroptosis and contain an N-terminus Zα-BD, our results show that leporipoxviruses induce receptor-interacting protein kinase (RIPK) RIPK1- and RIPK3-mediated necroptosis in human and mouse necroptosis-competent cell lines. These data suggest that leporipoxviruses lack countermeasures against necroptosis, unlike orthopoxviruses, which encode multiple key regulators of necroptosis, possibly due to a lack of selective pressure in their lagomorph host species. IMPORTANCE: The evolutionary arms race between viruses and their hosts has led to the viral acquisition of genes that antagonize the host’s antiviral defenses. However, related viral and host proteins have evolved under positive, purifying, and neutral selection. Poxvirus-encoded E3-like proteins play a pivotal role in regulating the host’s cellular antiviral and apoptotic responses. The two domains of canonical E3-like proteins, an N-terminal Z-form nucleic acid-binding domain (Zα-BD) and a C-terminal double-stranded RNA-binding domain (dsRNA-BD), have evolved under varying host immune selection pressures. Here, we demonstrate that the dsRNA-BD is structurally conserved among E3 orthologs, whereas the Zα-BD, involved in regulating necroptosis, exhibits structural diversity and is absent in some poxviruses. Leporipoxviruses, which lack Zα-BD-containing proteins and evolved in species without functional necroptosis, do activate necroptosis in necroptotic-competent cells. Our study thus highlights that a lack of selective pressure from the host can shape viral countermeasures and viral divergence.

Tumor burden of individual mouse received Colo205 cells.

Tumor burden of individual mouse received PANC-1 cells.