A Novel Imaging Approach for Single-Cell Real-Time Analysis of Oncolytic Virus Replication and Efficacy in Cancer Cells.

Oncolytic viruses (OVs) are novel cancer gene therapies that are moving toward the forefront of modern medicines. However, their full therapeutic potential is hindered by the lack of convenient and reliable strategies to visualize and quantify OV growth kinetics and therapeutic efficacy in live cells. In this study, we present an innovative imaging approach for single-cell real-time analysis of OV replication and efficacy in cancer cells. We selected SG33 as a prototypic new OV that derives from wild-type Myxoma virus (MYXV). Lausanne Toulouse 1 (T1) was used as control. We equipped SG33 and T1 genomes with the ANCHOR system and infected a panel of cell lines. The ANCHOR system is composed of a fusion protein (OR-GFP) that specifically binds to a short nonrepetitive DNA target sequence (ANCH) and spreads onto neighboring sequences by protein oligomerization. Its accumulation on the tagged viral DNA results in the creation of fluorescent foci. We found that (1) SG33 and T1-ANCHOR DNA can be readily detected and quantified by live imaging, (2) both OVs generate perinuclear replication foci after infection clustering into horse-shoe shape replication centers, and (3) SG33 replicates to higher levels as compared with T1. Lastly, as a translational proof of concept, we benchmarked SG33 replication and oncolytic efficacy in primary cancer cells derived from pancreatic adenocarcinoma (PDAC) both at the population and at the single-cell levels. In vivo, SG33 significantly replicates in experimental tumors to inhibit tumor growth. Collectively, we provide herein for the first time a novel strategy to quantify each step of OV infection in live cells and in real time by tracking viral DNA and provide first evidence of theranostic strategies for PDAC patients. Thus, this approach has the potential to rationalize the use of OVs for the benefit of patients with incurable diseases.

Oncolytic properties of non-vaccinia poxviruses.

Vaccinia virus, a member of the Poxviridae family, has been extensively used as an oncolytic agent and has entered late stage clinical development. In this study, we evaluated the potential oncolytic properties of other members of the Poxviridae family. Numerous tumor cell lines were infected with ten non-vaccinia poxviruses to identify which virus displayed the most potential as an oncolytic agent. Cell viability indicated that tumor cell lines were differentially susceptible to each virus. Raccoonpox virus was the most potent of the tested poxviruses and was highly effective in controlling cell growth in all tumor cell lines. To investigate further the oncolytic capacity of the Raccoonpox virus, we have generated a thymidine kinase (TK)-deleted recombinant Raccoonpox virus expressing the suicide gene FCU1. This TK-deleted Raccoonpox virus was notably attenuated in normal primary cells but replicated efficiently in numerous tumor cell lines. In human colon cancer xenograft model, a single intratumoral inoculation of the recombinant Raccoonpox virus, in combination with 5-fluorocytosine administration, produced relevant tumor growth control. The results demonstrated significant antitumoral activity of this new modified Raccoonpox virus armed with FCU1 and this virus could be considered to be included into the growing armamentarium of oncolytic virotherapy for cancer.

Cowpox Virus: A New and Armed Oncolytic Poxvirus.

Oncolytic virus therapy has recently been recognized as a promising new therapeutic approach for cancer treatment. In this study, we are proposing for the first time to evaluate the in vitro and in vivo oncolytic capacities of the Cowpox virus (CPXV). To improve the tumor selectivity and oncolytic activity, we developed a thymidine kinase (TK)-deleted CPXV expressing the suicide gene FCU1, which converts the non-toxic prodrug 5-fluorocytosine (5-FC) into cytotoxic 5-fluorouracil (5-FU) and 5-fluorouridine-5'-monophosphate (5-FUMP). This TK-deleted virus replicated efficiently in human tumor cell lines; however, it was notably attenuated in normal primary cells, thus displaying a good therapeutic index. Furthermore, this new recombinant poxvirus rendered cells sensitive to 5-FC. In vivo, after systemic injection in mice, the TK-deleted variant caused significantly less mortality than the wild-type strain. A biodistribution study demonstrated high tumor selectivity and low accumulation in normal tissues. In human xenograft models of solid tumors, the recombinant CPXV also displayed high replication, inducing relevant tumor growth inhibition. This anti-tumor effect was improved by 5-FC co-administration. These results demonstrated that CPXV is a promising oncolytic vector capable of expressing functional therapeutic transgenes.

Genome sequence of SG33 strain and recombination between wild-type and vaccine myxoma viruses.

Myxomatosis in Europe is the result of the release of a South America strain of myxoma virus in 1952. Several attenuated strains with origins in South America or California have since been used as vaccines in the rabbit industry. We sequenced the genome of the SG33 myxoma virus vaccine strain and compared it with those of other myxoma virus strains. We show that SG33 genome carries a large deletion in its right end. Furthermore, our data strongly suggest that the virus isolate from which SG33 is derived results from an in vivo recombination between a wild-type South America (Lausanne) strain and a California MSD-derived strain. These findings raise questions about the use of insufficiently attenuated virus in vaccination.

MNF, an ankyrin repeat protein of myxoma virus, is part of a native cellular SCF complex during viral infection.

Myxoma virus (MYXV), a member of the Poxviridae family, is the agent responsible for myxomatosis, a fatal disease in the European rabbit (Oryctolagus cuniculus). Like all poxviruses, MYXV is known for encoding multiple proteins that regulate cellular signaling pathways. Among them, four proteins share the same ANK/PRANC structure: M148R, M149R, MNF (Myxoma Nuclear factor) and M-T5, all of them described as virulence factors. This family of poxvirus proteins, recently identified, has drawn considerable attention for its potential role in modulating the host ubiquitin-proteasome system during viral infection. To date, many members of this novel protein family have been shown to interact with SCF components, in vitro. Here, we focus on MNF gene, which has been shown to express a nuclear protein presenting nine ANK repeats, one of which has been identified as a nuclear localization signal. In transfection, MNF has been shown to colocalise with the transcription factor NF-kappaB in the nucleus of TNFalpha-stimulated cells. Functionally, MNF is a critical virulence factor since its deletion generates an almost apathogenic virus. In this study, to pursue the investigation of proteins interacting with MNF and of its mechanism of action, we engineered a recombinant MYXV expressing a GFP-linked MNF under the control of MNF native promoter. Infection of rabbits with MYXV-GFPMNF recombinant virus provided the evidence that the GFP fusion does not disturb the main function of MNF. Hence, cells were infected with MYXV-GFPMNF and immunoprecipitation of the GFPMNF fusion protein was performed to identify MNF's partners. For the first time, endogenous components of SCF (Cullin-1 and Skp1) were co-precipitated with an ANK myxoma virus protein, expressed in an infectious context, and without over-expression of any protein.

M148R and M149R are two virulence factors for myxoma virus pathogenesis in the European rabbit.

Myxoma virus (MYXV), a member of the Poxviridae family, is the agent responsible for myxomatosis, a fatal disease in the European rabbit (Oryctolagus cuniculus). MYXV has a linear double-stranded DNA genome that encodes several factors important for evasion from the host immune system. Among them, four ankyrin (ANK) repeat proteins were identified: M148R, M149R, M150R and M-T5. To date, only M150R and M-T5 were studied and characterized as critical virulence factors. This article presents the first characterization of M148R and M149R. Green Fluorescent Protein (GFP) fusions allowed us to localize them in a viral context. Whereas M149R is only cytoplasmic, interestingly, M148R is in part located in the nucleolus, a unique feature for an ANK repeat poxviral protein. In order to evaluate their implication in viral pathogenicity, targeted M148R, M149R, or both deletions were constructed in the wild type T1 strain of myxoma virus. In vitro infection of rabbit and primate cultured cells as well as primary rabbit cells allowed us to conclude that M148R and M149R are not likely to be implicated in cell tropism or host range functions. However, in vivo experiments revealed that they are virulence factors since after infection of European rabbits with mutant viruses, a delay in the onset of clinical signs, an increase of survival time and a dramatic decrease in mortality rate were observed. Moreover, histological analysis suggests that M148R plays a role in the subversion of host inflammatory response by MYXV.

A virulence factor of myxoma virus colocalizes with NF-kappaB in the nucleus and interferes with inflammation.

NF-kappaB is one of the most important elements that coordinate stress-induced, immune, and inflammatory responses. Myxoma virus, a member of the Poxviridae family responsible for rabbit myxomatosis, codes for several factors that help its survival in the host. In this study, we focused on the product of the M150R gene. We show that the protein has nine ankyrin repeats (ANKs), with the eighth having a close similarity with the nuclear localization signal-containing ANK of I-kappaBalpha, which regulates NF-kappaB activity by sequestering it in the cytosol. Because the viral protein is targeted to the nucleus, it was named MNF, for myxoma nuclear factor. This localization was lost when the eighth ANK was removed. In tumor necrosis factor alpha-treated cells, MNF and NF-kappaB colocalized as dotted spots in the nucleus. In vivo experiments with a knockout virus showed that MNF is a critical virulence factor, with its deletion generating an almost apathogenic virus. Detailed histological examinations revealed an increase in the inflammatory process in the absence of MNF, consistent with the interference of MNF with the NF-kappaB-induced proinflammatory pathway. Because MNF has homologs in other poxviruses, such as vaccinia, cowpox, and variola viruses, this protein is probably part of a key mechanism that contributes to the immunogenic and pathogenic properties of these viruses.