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

Abstract

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 CKII␣ 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 CKII␣ or tropomyosin. The cytotoxic adaptor function of NS1 was confirmed with fusion peptides, where the tropomyosinbinding domain of NS1 and CKII␣ are physically linked. These adaptor peptides were able to mimic NS1 in its ability to induce death of transformed MVM-permissive cells.parvovirus minute virus of mice ͉ protein-protein interactions ͉ tropomyosin ͉ protein kinase specificity R odent parvoviruses (PVs) are promising candidates for the virotherapy of cancer because they exert oncolytic action without showing pathogenicity (1). The PV minute virus of mice (MVM) consists of a small, icosahedral nonenveloped capsid containing a single-stranded 5.1-kb linear DNA. During productive infection, MVM induces dramatic morphological and physiological changes in transformed mouse fibroblasts, culminating in cell death and lysis. The cytotoxicity of MVM is attributed mainly to the large nonstructural protein NS1. This 83-kDa multifunctional viral product is endowed with enzymatic and nonenzymatic properties, enabling it to control various processes necessary for progeny particle production and spreading (for a review, see ref.2). Independently of its functions directly involved in particle production, NS1 exerts specific activities that jeopardize the integrity and survival of infected cells (3, 4). The ability of NS1 to interact physically with host proteins is assumed to play an important role in its cytotoxicity. The interaction of NS1 with proteins involved in DNA replication and transcription suggests that scavenging mechanisms may contribute to NS1-triggered perturbation of host cells, but recent data indicate that NS1 could act in a more specific manner, e.g., by modulating the activity of some partner proteins. The cytotoxicity of NS1 correlates with its ability to bind casein kinase II␣ (CKII␣), the catalytic subunit of the cellular casein kinase II, and suppression of CKII activity during infection protects cells from PV-induced cytopathic effects (CPE) (5). This correlation suggests that NS1-mediated modulation of cellular protein kinase...

Molecular Pathways: Rodent Parvoviruses—Mechanisms of Oncolysis and Prospects for Clinical Cancer Treatment

“…These alterations are likely to be especially deleterious to cancer cells, where tropomyosin 1 is underrepresented (40). Of interest, artificial polypeptides combining the targeting domain of NS1 with CKIIa (or a binding site thereof) were found to cause the death of transformed cells (39). This led us to propose that the toxic activity of the NS1 protein depends at least partly on its ability to act as a cytosolic adaptor, allowing a cellular kinase or kinases to phosphorylate illegitimate substrates.…”

“…Filamentous tropomyosin is also a direct target of the NS1/CKIIa complex. Differential NS1/CKIIadriven phosphorylation of tropomyosins 2 and 5 leads to perinuclear restructuring and decay of these filaments (39). These alterations are likely to be especially deleterious to cancer cells, where tropomyosin 1 is underrepresented (40).…”

Ezrin-Radixin-Moesin Family Proteins Are Involved in Parvovirus Replication and Spreading

“…In vitro kinase reactions and tryptic phosphopeptide analyses were performed as described previously (4) using recombinant CKII␣␤ (Roche) and, when indicated, purified glutathione S-transferase-tagged wild-type NS1 protein (33). Rdx used as substrates was produced in bacteria and purified as described previously (32).…”

“…In addition to these enzymatic functions, NS1 serves as a transcription factor for the regulation of viral, as well as cellular, promoters (19). NS1 also interferes with the host cell physiology and morphology through its ability to directly interact with and/or induce posttranslational modifications in specific host cell proteins (3,(31)(32)(33). This involvement of NS1 in multiple facets of the parvoviral life cycle, in conjunction with host cell factories/ proteins, implies that the various functions of the viral proteins are tightly regulated, in particular, through phosphorylation and subcellular (re)distribution (25).…”

“…It was recently shown that NS1 is able to physically interact with CKII␣ (32), thereby modulating the substrate specificity of the cellular kinase (33). To investigate whether this complex of a parvoviral protein with a cellular protein kinase could mediate the activation of ERM proteins To this end, MVM-infected A9 cells and derivatives containing P38-CKIImATP or P38-CKII-E81A, were analyzed at the indicated times p.i.…”