Oncolytic Rodent Protoparvoviruses Evade a TLR- and RLR-Independent Antiviral Response in Transformed Cells.

The oncolytic rodent protoparvoviruses (PVs) minute virus of mice (MVMp) and H-1 parvovirus (H-1PV) are promising cancer viro-immunotherapy candidates capable of both exhibiting direct oncolytic activities and inducing anticancer immune responses (AIRs). Type-I interferon (IFN) production is instrumental for the activation of an efficient AIR. The present study aims at characterizing the molecular mechanisms underlying PV modulation of IFN induction in host cells. MVMp and H-1PV triggered IFN production in semi-permissive normal mouse embryonic fibroblasts (MEFs) and human peripheral blood mononuclear cells (PBMCs), but not in permissive transformed/tumor cells. IFN production triggered by MVMp in primary MEFs required PV replication and was independent of the pattern recognition receptors (PRRs) Toll-like (TLR) and RIG-like (RLR) receptors. PV infection of (semi-)permissive cells, whether transformed or not, led to nuclear translocation of the transcription factors NFĸB and IRF3, hallmarks of PRR signaling activation. Further evidence showed that PV replication in (semi-)permissive cells resulted in nuclear accumulation of dsRNAs capable of activating mitochondrial antiviral signaling (MAVS)-dependent cytosolic RLR signaling upon transfection into naïve cells. This PRR signaling was aborted in PV-infected neoplastic cells, in which no IFN production was detected. Furthermore, MEF immortalization was sufficient to strongly reduce PV-induced IFN production. Pre-infection of transformed/tumor but not of normal cells with MVMp or H-1PV prevented IFN production by classical RLR ligands. Altogether, our data indicate that natural rodent PVs regulate the antiviral innate immune machinery in infected host cells through a complex mechanism. In particular, while rodent PV replication in (semi-)permissive cells engages a TLR-/RLR-independent PRR pathway, in transformed/tumor cells this process is arrested prior to IFN production. This virus-triggered evasion mechanism involves a viral factor(s), which exert(s) an inhibitory action on IFN production, particularly in transformed/tumor cells. These findings pave the way for the development of second-generation PVs that are defective in this evasion mechanism and therefore endowed with increased immunostimulatory potential through their ability to induce IFN production in infected tumor cells.

Design and Characterization of Mutated Variants of the Oncotoxic Parvoviral Protein NS1.

Oncotoxic proteins such as the non-structural protein 1 (NS1), a constituent of the rodent parvovirus H1 (H1-PV), offer a novel approach for treatment of tumors that are refractory to other treatments. In the present study, mutated NS1 variants were designed and tested with respect to their oncotoxic potential in human hepatocellular carcinoma cell lines. We introduced single point mutations of previously described important residues of the wild-type NS1 protein and a deletion of 114 base pairs localized within the N-terminal domain of NS1. Cell-viability screening with HepG2 and Hep3B hepatocarcinoma cells transfected with the constructed NS1-mutants led to identification of the single-amino acid NS1-mutant NS1-T585E, which led to a 30% decrease in cell viability as compared to NS1 wildtype. Using proteomics analysis, we could identify new interaction partners and signaling pathways of NS1. We could thus identify new oncotoxic NS1 variants and gain insight into the modes of action of NS1, which is exclusively toxic to human cancer cells. Our in-vitro studies provide mechanistic explanations for the observed oncolytic effects. Expression of NS1 variants had no effect on cell viability in NS1 unresponsive control HepG2 cells or primary mouse hepatocytes. The availability of new NS1 variants in combination with a better understanding of their modes of action offers new possibilities for the design of innovative cancer treatment strategies.

Generation and Validation of Monoclonal Antibodies Suitable for Detecting and Monitoring Parvovirus Infections.

For many applications it is necessary to detect target proteins in living cells. This is particularly the case when monitoring viral infections, in which the presence (or absence) of distinct target polypeptides potentially provides vital information about the pathology caused by the agent. To obtain suitable tools with which to monitor parvoviral infections, we thus generated monoclonal antibodies (mAbs) in order to detect the major non-structural protein NS1 in the intracellular environment and tested them for sensitivity and specificity, as well as for cross-reactivity towards related species. Using different immunogens and screening approaches based on indirect immunofluorescence, we describe here a panel of mAbs suitable for monitoring active infections with various parvovirus species by targeting the major non-structural protein NS1. In addition to mAbs detecting the NS1 of parvovirus H-1 (H-1PV) (belonging to the Rodent protoparvovirus 1 species, which is currently under validation as an anti-cancer agent), we generated tools with which to monitor infections by human cutavirus (CuV) and B19 virus (B19V) (belonging to the Primate protoparvovirus 3 and the Primate erythroparvovirus 1 species, respectively, which were both found to persistently infect human tissues). As well as mAbs able to detect NS1 from a broad range of parvoviruses, we obtained entities specific for either (distinct) members of the Rodent protoparvovirus 1 species, human CuV, or human B19V.

Virotherapy in Germany-Recent Activities in Virus Engineering, Preclinical Development, and Clinical Studies.

Virotherapy research involves the development, exploration, and application of oncolytic viruses that combine direct killing of cancer cells by viral infection, replication, and spread (oncolysis) with indirect killing by induction of anti-tumor immune responses. Oncolytic viruses can also be engineered to genetically deliver therapeutic proteins for direct or indirect cancer cell killing. In this review-as part of the special edition on "State-of-the-Art Viral Vector Gene Therapy in Germany"-the German community of virotherapists provides an overview of their recent research activities that cover endeavors from screening and engineering viruses as oncolytic cancer therapeutics to their clinical translation in investigator-initiated and sponsored multi-center trials. Preclinical research explores multiple viral platforms, including new isolates, serotypes, or fitness mutants, and pursues unique approaches to engineer them towards increased safety, shielded or targeted delivery, selective or enhanced replication, improved immune activation, delivery of therapeutic proteins or RNA, and redirecting antiviral immunity for cancer cell killing. Moreover, several oncolytic virus-based combination therapies are under investigation. Clinical trials in Germany explore the safety and potency of virotherapeutics based on parvo-, vaccinia, herpes, measles, reo-, adeno-, vesicular stomatitis, and coxsackie viruses, including viruses encoding therapeutic proteins or combinations with immune checkpoint inhibitors. These research advances represent exciting vantage points for future endeavors of the German virotherapy community collectively aimed at the implementation of effective virotherapeutics in clinical oncology.

Human Retrotransposons and the Global Shutdown of Homeostatic Innate Immunity by Oncolytic Parvovirus H-1PV in Pancreatic Cancer.

Although the oncolytic parvovirus H-1PV has entered clinical trials, predicting therapeutic success remains challenging. We investigated whether the antiviral state in tumor cells determines the parvoviral oncolytic efficacy. The interferon/interferon-stimulated genes (IFN/ISG)-circuit and its major configurator, human endogenous retroviruses (HERVs), were evaluated using qRT-PCR, ELISA, Western blot, and RNA-Seq techniques. In pancreatic cancer cell lines, H-1PV caused a late global shutdown of innate immunity, whereby the concomitant inhibition of HERVs and IFN/ISGs was co-regulatory rather than causative. The growth-inhibitory IC50 doses correlated with the power of suppression but not with absolute ISG levels. Moreover, H-1PV was not sensitive to exogenous IFN despite upregulated antiviral ISGs. Such resistance questioned the biological necessity of the oncotropic ISG-shutdown, which instead might represent a surrogate marker for personalized oncolytic efficacy. The disabled antiviral homeostasis may modify the activity of other viruses, as demonstrated by the reemergence of endogenous AluY-retrotransposons. This way of suppression may compromise the interferogenicity of drugs having gemcitabine-like mechanisms of action. This shortcoming in immunogenic cell death induction is however amendable by immune cells which release IFN in response to H-1PV.

Non-viral gene delivery of the oncotoxic protein NS1 for treatment of hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is related to increasing incidence rates and poor clinical outcomes due to lack of efficient treatment options and emerging resistance mechanisms. The aim of the present study is to exploit a non-viral gene therapy enabling the expression of the parvovirus-derived oncotoxic protein NS1 in HCC. This anticancer protein interacts with different cellular kinases mediating a multimodal host-cell death. Lipoplexes (LPX) designed to deliver a DNA expression plasmid encoding NS1 are characterized using a comprehensive set of in vitro assays. The mechanisms of cell death induction are assessed and phosphoinositide-dependent kinase 1 (PDK1) is identified as a potential predictive biomarker for a NS1-LPX-based gene therapy. In an HCC xenograft mouse model, NS1-LPX therapeutic approach results in a significant reduction in tumor growth and extended survival. Data provide convincing evidence for future studies using a targeted NS1 gene therapy for PDK1 overexpressing HCC.

Tumor Selectivity of Oncolytic Parvoviruses: From in vitro and Animal Models to Cancer Patients.

Oncolytic virotherapy of cancer is among the innovative modalities being under development and especially promising for targeting tumors, which are resistant to conventional treatments. Presently, at least a dozen of viruses, belonging to nine different virus families, are being tested within the frames of various clinical studies in cancer patients. Continuously growing preclinical evidence showing that the autonomous rat parvovirus H-1 (H-1PV) is able to kill tumor cells that resist conventional treatments and to achieve a complete cure of various human tumors in animal models argues for its inclusion in the arsenal of oncolytic viruses with an especially promising bench to bedside translation potential. Oncolytic parvovirus safe administration to humans relies on the intrinsic preference of these agents for quickly proliferating, metabolically, and biochemically disturbed tumor versus normal cells (tumor selectivity or oncotropism). The present review summarizes and discusses (i) preclinical evidence of H-1PV innocuousness for normal cells and healthy tissues in vitro and in animals, respectively, (ii) toxicological assessments of H-1PV mono- or combined therapy in tumor-bearing virus-permissive animal models, as well as (iii) historical results of experimental infection of human cancer patients with H-1PV. Altogether, these data argue against a risk of H-1PV inducing significant toxic effects in human patients. This highly favorable safety profile allowed the translation of H-1PV preclinical research into a Phase I/IIa clinical trial being currently in progress.

Double-faceted mechanism of parvoviral oncosuppression.

The H-1 parvovirus (H-1PV) exerts oncosuppressive action that has two components: oncotoxicity and immunostimulation. While many human tumor cells, including conventional drug-resistant ones, can be killed by H-1PV, some fail to support progeny virus production, necessary for infection propagation in neoplastic tissues. This limitation can be overcome through forced selection of H-1PV variants capable of enhanced multiplication and spreading in human tumor cells. In the context of further developing H-1PV for use in cancer therapy, arming it with immunostimulatory CpG motifs under conditions preserving replication and oncolysis enhances its action as an anticancer vaccine adjuvant. A first clinical study of H-1PV treatment in glioma patients has yielded evidence of intratumoral synthesis of the viral oncotoxic protein NS1 and immune cell infiltration.

PKCη/Rdx-driven phosphorylation of PDK1: a novel mechanism promoting cancer cell survival and permissiveness for parvovirus-induced lysis.

The intrinsic oncotropism and oncosuppressive activities of rodent protoparvoviruses (PVs) are opening new prospects for cancer virotherapy. Virus propagation, cytolytic activity, and spread are tightly connected to activation of the PDK1 signaling cascade, which delays stress-induced cell death and sustains functioning of the parvoviral protein NS1 through PKC(η)-driven modifications. Here we reveal a new PV-induced intracellular loop-back mechanism whereby PKCη/Rdx phosphorylates mouse PDK1:S138 and activates it independently of PI3-kinase signaling. The corresponding human PDK1phosphoS135 appears as a hallmark of highly aggressive brain tumors and may contribute to the very effective targeting of human gliomas by H-1PV. Strikingly, although H-1PV does not trigger PDK1 activation in normal human cells, such cells show enhanced viral DNA amplification and NS1-induced death upon expression of a constitutively active PDK1 mimicking PDK1phosphoS135. This modification thus appears as a marker of human glioma malignant progression and sensitivity to H-1PV-induced tumor cell killing.

Tumor suppressing properties of rodent parvovirus NS1 proteins and their derivatives.

Cancer chemotherapy with monospecific agents is often hampered by the rapid development of tumor resistance to the drug used. Therefore, combination treatments aiming at several different targets are sought. Viral regulatory proteins, modified or not, appear ideal for this purpose because of their multimodal killing action against neoplastically transformed cells. The large nonstructural protein NS1 of rodent parvoviruses is an excellent candidate for an anticancer agent, shown to interfere specifically with cancer cell growth and survival. The present review describes the structure, functions, and regulation of the multifunctional protein NS1, its specific interference with cell processes and cell protein activities, and what is known so far about the mechanisms underlying NS1 interference with cancer growth. It further outlines prospects for the development of new, multimodal cancer toxins and their potential applications.

Vesicular transport of progeny parvovirus particles through ER and Golgi regulates maturation and cytolysis.

Progeny particles of non-enveloped lytic parvoviruses were previously shown to be actively transported to the cell periphery through vesicles in a gelsolin-dependent manner. This process involves rearrangement and destruction of actin filaments, while microtubules become protected throughout the infection. Here the focus is on the intracellular egress pathway, as well as its impact on the properties and release of progeny virions. By colocalization with cellular marker proteins and specific modulation of the pathways through over-expression of variant effector genes transduced by recombinant adeno-associated virus vectors, we show that progeny PV particles become engulfed into COPII-vesicles in the endoplasmic reticulum (ER) and are transported through the Golgi to the plasma membrane. Besides known factors like sar1, sec24, rab1, the ERM family proteins, radixin and moesin play (an) essential role(s) in the formation/loading and targeting of virus-containing COPII-vesicles. These proteins also contribute to the transport through ER and Golgi of the well described analogue of cellular proteins, the secreted Gaussia luciferase in absence of virus infection. It is therefore likely that radixin and moesin also serve for a more general function in cellular exocytosis. Finally, parvovirus egress via ER and Golgi appears to be necessary for virions to gain full infectivity through post-assembly modifications (e.g. phosphorylation). While not being absolutely required for cytolysis and progeny virus release, vesicular transport of parvoviruses through ER and Golgi significantly accelerates these processes pointing to a regulatory role of this transport pathway.

Molecular pathways: rodent parvoviruses--mechanisms of oncolysis and prospects for clinical cancer treatment.

Rodent parvoviruses (PV) are recognized for their intrinsic oncotropism and oncolytic activity, which contribute to their natural oncosuppressive effects. Although PV uptake occurs in most host cells, some of the subsequent steps leading to expression and amplification of the viral genome and production of progeny particles are upregulated in malignantly transformed cells. By usurping cellular processes such as DNA replication, DNA damage response, and gene expression, and/or by interfering with cellular signaling cascades involved in cytoskeleton dynamics, vesicular integrity, cell survival, and death, PVs can induce cytostasis and cytotoxicity. Although productive PV infections normally culminate in cytolysis, virus spread to neighboring cells and secondary rounds of infection, even abortive infection or the sole expression of the PV nonstructural protein NS1, is sufficient to cause significant tumor cell death, either directly or indirectly (through activation of host immune responses). This review highlights the molecular pathways involved in tumor cell targeting by PVs and in PV-induced cell death. It concludes with a discussion of the relevance of these pathways to the application of PVs in cancer therapy, linking basic knowledge of PV-host cell interactions to preclinical assessment of PV oncosuppression.

Ezrin-radixin-moesin family proteins are involved in parvovirus replication and spreading.

The propagation of autonomous parvoviruses is strongly dependent on the phosphorylation of the major nonstructural protein NS1 by members of the protein kinase C (PKC) family. Minute virus of mice (MVM) replication is accompanied by changes in the overall phosphorylation pattern of NS1, which is newly modified at consensus PKC sites. These changes result, at least in part, from the ability of MVM to modulate the PDK-1/PKC pathway, leading to activation and redistribution of both PDK-1 and PKCeta. We show that proteins of the ezrin-radixin-moesin (ERM) family are essential for virus propagation and spreading through their functions as adaptors for PKCeta. MVM infection led to redistribution of radixin and moesin in the cell, resulting in increased colocalization of these proteins with PKCeta. Radixin was found to control the PKCeta-driven phosphorylation of NS1 and newly synthesized capsids in vivo. Conversely, radixin phosphorylation and activation were driven by the NS1/CKIIalpha complex. Altogether, these data argue for ERM proteins being both targets and modulators of parvovirus infection.

Vesicular egress of non-enveloped lytic parvoviruses depends on gelsolin functioning.

The autonomous parvovirus Minute Virus of Mice (MVM) induces specific changes in the cytoskeleton filaments of infected permissive cells, causing in particular the degradation of actin fibers and the generation of "actin patches." This is attributed to a virus-induced imbalance between the polymerization factor N-WASP (Wiscott-Aldrich syndrome protein) and gelsolin, a multifunctional protein cleaving actin filaments. Here, the focus is on the involvement of gelsolin in parvovirus propagation and virus-induced actin processing. Gelsolin activity was knocked-down, and consequences thereof were determined for virus replication and egress and for actin network integrity. Though not required for virus replication or progeny particle assembly, gelsolin was found to control MVM (and related H1-PV) transport from the nucleus to the cell periphery and release into the culture medium. Gelsolin-dependent actin degradation and progeny virus release were both controlled by (NS1)/CKIIalpha, a recently identified complex between a cellular protein kinase and a MVM non-structural protein. Furthermore, the export of newly synthesized virions through the cytoplasm appeared to be mediated by (virus-modified) lysomal/late endosomal vesicles. By showing that MVM release, like entry, is guided by the cytoskeleton and mediated by vesicles, these results challenge the current view that egress of non-enveloped lytic viruses is a passive process.

Parvovirus interference with intracellular signalling: mechanism of PKCeta activation in MVM-infected A9 fibroblasts.

Autonomous parvoviruses are strongly dependent on the phosphorylation of the major non-structural protein NS1 by members of the protein kinase C (PKC) family. Besides being accompanied with changes in the overall phosphorylation pattern of NS1 and acquiring new modifications at consensus PKC sites, ongoing minute virus of mice (MVM) infections lead to the appearance of new phosphorylated cellular protein species. This prompted us to investigate whether MVM actively interferes with phosphoinositol-dependent kinase (PDK)/PKC signalling. The activity, subcellular localization and phosphorylation status of the protein kinases PDK1, PKCeta and PKClambda were measured in A9 cells in the presence or absence of MVM infection. Parvovirus infection was found to result in activation of both PDK1 and PKCeta, as evidenced by changes in their subcellular distribution and overall (auto)phosphorylation. We show evidence that activation of PKCeta by PDK1 is driven by atypical PKClambda. By modifying the hydrophobic motif of PKCeta, PKClambda appeared to control docking and consecutive phosphorylation of PKCeta's activation-loop by PDK1, a process that was inhibited in vivo in the presence of a dominant-negative PKClambda mutant.

A viral adaptor protein modulating casein kinase II activity induces cytopathic effects in permissive cells.

Autonomous parvoviruses induce severe morphological and physiological alterations in permissive host cells, eventually leading to cell lysis and release of progeny virions. Viral cytopathic effects (CPE) result from specific rearrangements and destruction of cytoskeletal micro- and intermediate filaments. We recently reported that inhibition of endogenous casein kinase II (CKII) protects target cells from parvovirus minute virus of mice (MVM)-induced CPE, pointing to this kinase as an effector of MVM toxicity. The present work shows that the parvoviral NS1 protein mediates CKII-dependent cytoskeletal alterations and cell death. NS1 can act as an adaptor molecule, linking the cellular protein kinase CKIIalpha to tropomyosin and thus modulating the substrate specificity of the kinase. This action results in an altered tropomyosin phosphorylation pattern both in vitro and in living cells. The capacity of NS1 to induce CPE was impaired by mutations abolishing binding with either CKIIalpha or tropomyosin. The cytotoxic adaptor function of NS1 was confirmed with fusion peptides, where the tropomyosin-binding domain of NS1 and CKIIalpha are physically linked. These adaptor peptides were able to mimic NS1 in its ability to induce death of transformed MVM-permissive cells.

NS1 interaction with CKII alpha: novel protein complex mediating parvovirus-induced cytotoxicity.

During a productive infection, the prototype strain of the parvovirus minute virus of mice (MVMp) induces dramatic morphological alterations in permissive A9 fibroblasts, culminating in cell lysis at the end of infection. These cytopathic effects (CPE) result from rearrangements and destruction of the cytoskeletal micro- and intermediate filaments, while other structures such as the nuclear lamina and particularly the microtubule network remain protected throughout the infection (J. P. F. Nüesch et al., Virology 331:159-174, 2005). In order to unravel the mechanism(s) by which parvoviruses trigger CPE, we searched for NS1 interaction partners by differential affinity chromatography, using distinct NS1 mutants debilitated specifically for this function. Thereby, we isolated an NS1 partner polypeptide, whose interaction with NS1 correlated with the competence of the viral product for CPE induction, and further identified it by tandem mass spectrometry and Western blotting analyses to consist of the catalytic subunit of casein kinase II, CKIIalpha. This interaction of NS1 with CKIIalpha suggested interference by the viral protein with intracellular signaling. Using permanent cell lines expressing dominant-negative CKIIalpha mutants, we were able to show that this kinase activity was indeed specifically involved in parvoviral CPE and progeny particle release. Furthermore, the NS1/CKIIalpha complex proved to be able to specifically phosphorylate viral capsids, indicating a mediator function of NS1 for CKII activity and specificity, at least in vitro. Altogether our data suggest that parvovirus-induced CPE is mediated by NS1 interference with intracellular CKII signaling.

Tumor suppressive activity of a variant isoform of manganese superoxide dismutase released by a human liposarcoma cell line.

A cell line derived from a pleiomorphic liposarcoma, named LSA, was previously reported to secrete (a) factor(s) exhibiting oncotoxic properties. The present article describes the isolation, purification and sequence analysis of a protein released by LSA cells into conditioned culture medium. This protein proved to be a variant isoform of manganese superoxide dismutase (MnSOD), hence its designation as LSA-type-MnSOD. This LSA-type-SOD differed from conventional SODs in its secretion by producer cells, contrasting with the normal localization of SODs in the mitochondrial matrix. Interestingly, during the protein purification process, LSA-type-SOD cosegregated with a cytotoxic activity directed against a number of tumor cell lines, as determined under in vitro conditions. This cytopathic effect was most likely due to LSA-type-SOD, since it could be fully reproduced using recombinant SOD that was expressed from cDNA clones isolated from LSA cells mRNA preparations and henceforth designated L-rSOD. In addition to its manifestation in cell lines kept in tissue culture, the oncotoxicity of LSA-type-SOD was further reflected in a remarkable capacity of this protein for suppression of mammary tumors in Balb-C-FR(III) mice. Animals subcutaneously injected with L-rSOD in the tumor area showed a complete disruption of established mammary carcinomas, as monitored by nuclear magnetic resonance (NMR) scanning. Moreover, metastatic spreading, which was readily detected in the control group, was suppressed in the treated animals. Altogether these data suggest that LSA-type-SOD interferes with survival and spreading of neoplastically transformed cells and deserves to be future validated as a therapeutic agent against cancer, either alone or in combination with conventional treatments.

Selective alterations of the host cell architecture upon infection with parvovirus minute virus of mice.

During a productive infection, the prototype strain of parvovirus minute virus of mice (MVMp) induces dramatic morphological alterations to the fibroblast host cell A9, resulting in cell lysis and progeny virus release. In order to understand the mechanisms underlying these changes, we characterized the fate of various cytoskeletal filaments and investigated the nuclear/cytoplasmic compartmentalization of infected cells. While most pronounced effects could be seen on micro- and intermediate filaments, manifest in dramatic rearrangements and degradation of filamentous (F-)actin and vimentin structures, only little impact could be seen on microtubules or the nuclear envelope during the entire monitored time of infection. To further analyze the disruption of the cytoskeletal structures, we investigated the viral impact on selective regulatory pathways. Thereby, we found a correlation between microtubule stability and MVM-induced phosphorylation of alpha/beta tubulin. In contrast, disassembly of actin filaments late in infection could be traced back to the disregulation of two F-actin associated proteins gelsolin and Wiscott-Aldrich Syndrome Protein (WASP). Thereby, an increase in the amount of gelsolin, an F-actin severing protein was observed during infection, accounting for the disruption of stress fibers upon infection. Concomitantly, the actin polymerization activity also diminished due to a loss of WASP, the activator protein of the actin polymerization machinery the Arp2/3 complex. No effects could be seen in amount and distribution of other F-actin regulatory factors such as cortactin, cofilin, and profilin. In summary, the selective attack of MVM towards distinct host cell cytoskeletal structures argues for a regulatory feature during infection, rather than a collapse of the host cell as a mere side effect of virus production.