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)/CKIIα, 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. Rodent parvoviruses are non-enveloped lytic viruses that are thought excellent tools for a virotherapy of cancer because of their strong natural oncolytic potential and low pathogenicity in humans. Egress of non-enveloped lytic viruses is commonly thought to occur as a virus burst after cell disintegration. Indeed, we showed in the past that autonomous parvoviruses induce severe cytopathic effects to the host cell, manifested in restructuring and degradation of cytoskeletal filaments, thereby supporting such mode of virus spread. Here, we focus on the impact of virus-induced actin degradation, and particularly the functioning of the actin-severing protein gelsolin. Although not required for DNA replication or progeny particle production, gelsolin appears to facilitate a regulated virus egress from the nucleus to the cell periphery via (virus modified) lysosomal/late endosomal vesicles. These results challenge the current view that lytic virus egress is just a passive process at the end of infection and suggests that these pathogens are endowed with the ability to efficiently spread from cell to cell potentially in solid (tumor) tissue.

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 CKIIα 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 CKIIα or tropomyosin. The cytotoxic adaptor function of NS1 was confirmed with fusion peptides, where the tropomyosin-binding domain of NS1 and CKIIα are physically linked. These adaptor peptides were able to mimic NS1 in its ability to induce death of transformed MVM-permissive cells.

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. Previously, it was thought that non-enveloped lytic parvoviruses were released through a lytic burst of cells at the end of infection. However, recent work demonstrated that these small non-enveloped single-stranded DNA viruses are actively transported through vesicles from the nucleus, the site of replication and assembly, to the cell periphery. The current investigation demonstrates that progeny particles become engulfed into COPII-vesicles in the endoplasmic reticulum (ER) and are transported through the Golgi to the plasma membrane (PM). ERM family proteins radixin and moesin appear to play an essential role in this cellular secretion pathway. While passing through ER and Golgi cisternae, PVs maturate through post-assembly modifications, which significantly increase the infectivity of progeny virions. Finally, the vesicular transport of parvoviral particles was shown to regulate virus-induced cytolysis, thereby accelerating the further release and spread of progeny virions. As rodent PVs are currently viewed as oncolytic agents for cancer virotherapy, it is important to further investigate the mechanism of PV egress — not only to improve the spreading of these agents through the tumor mass, but also to optimize the induction of an anti-tumor immune response upon virus — induced cytolysis.

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.