Novel PKCηIs Required To Activate Replicative Functions of the Major Nonstructural Protein NS1 of Minute Virus of Mice

Abstract

The multifunctional protein NS1 of minute virus of mice (MVMp) is posttranslationally modified and at least in part regulated by phosphorylation. The atypical lambda isoform of protein kinase C (PKC) phosphorylates residues T435 and S473 in vitro and in vivo, leading directly to an activation of NS1 helicase function, but it is insufficient to activate NS1 for rolling circle replication. The present study identifies an additional cellular protein kinase phosphorylating and regulating NS1 activities. We show in vitro that the recombinant novel PKC phosphorylates NS1 and in consequence is able to activate the viral polypeptide in concert with PKC for rolling circle replication. Moreover, this role of PKC was confirmed in vivo. We thereby created stably transfected A9 mouse fibroblasts, a typical MVMp-permissive host cell line with Flag-tagged constitutively active or inactive PKC mutants, in order to alter the activity of the NS1 regulating kinase. Indeed, tryptic phosphopeptide analyses of metabolically 32 P-labeled NS1 expressed in the presence of a dominant-negative mutant, PKCDN, showed a lack of distinct NS1 phosphorylation events. This correlates with impaired synthesis of viral DNA replication intermediates, as detected by Southern blotting at the level of the whole cell population and by BrdU incorporation at the single-cell level. Remarkably, MVM infection triggers an accumulation of endogenous PKC in the nuclear periphery, suggesting that besides being a target for PKC, parvovirus infections may also affect the regulation of this NS1 regulating kinase. Altogether, our results underline the tight interconnection between PKC-mediated signaling and the parvoviral life cycle.The regulation not only of cellular proteins but also of viral proteins by phosphorylation has attracted research interest for many years. Besides characterization of proteins which become phosphorylated and the identification of their regulatory kinases, much effort has been spent on the analysis of the signaling pathways involved and their functional consequences. We are particularly interested in understanding how the multifunctional nonstructural protein NS1 of the autonomous parvovirus minute virus of mice (MVMp) is regulated. MVM consists of a small icosahedral capsid with a linear singlestranded DNA of negative polarity as a genome. The DNA of MVMp codes for the nonstructural proteins NS1 and NS2, of which the latter exists in three different isoforms, differing in their unique C termini only, as well as two capsid proteins, VP1 and VP2. NS1 is endowed with numerous biochemical activities, such as ATP binding and hydrolysis (12, 62), helicase (44, 62), site-specific binding to the cognate recognition motif [ACCA] 2-3 which is scattered throughout the viral genome (13, 19), and site-and strand-specific endonuclease (11,18,44). Furthermore, NS1 takes part in protein-protein interactions to form homo-oligomers (40, 52) or complexes with cellular partner proteins like the transcription factor SP1 (35), the cochaperone SGT (20, 58), or...

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

“…So far, we have identified two members of the protein kinase C (PKC) family, PKC and PKC , as being able to activate NS1 for viral DNA amplification (10,18,26,30). Interestingly, mutagenesis at consensus PKC phosphorylation sites dissociated NS1 functions necessary for virus amplifications, interactions with cellular proteins, and induction of host cell perturbations, death, and lysis (6,9,10,26).…”

“…Stable transfectants were generated with pP38-Flag ERM (using the above-mentioned mutants of Ez, Rdx, and Moe) and the selection plasmid pSV2neo at a molar ratio of 25:1 (18 (18,26). The presence of PKC and PKC in low-affinity (HA-1) and high-affinity (HA-2) fractions from HA chromatography, respectively, is indicated.…”

“…The purification and identification of NS1-activating PKC and PKC from HeLa cellular extracts by consecutive column chromatography (phosphocellulose, DE52, protamine chloride, and hydroxyl apatite [HA]) have been described previously (18,26). The presence of ERM proteins at the final purification step (i.e., in the PKC -containing, NS1-activating fractions from HA-2) was determined by analyzing HA-bound proteins eluting at 116 mM (Fig.…”

“…After 24 h, the treated cells were infected with 30 PFU/cell CsCl-purified MVM and analyzed for the presence of Ez, Rdx, Moe, and NS1 by immunofluorescence. Viral DNA replication was measured upon addition of 1 M bromodeoxyuridine (BrdU) for 2 h, followed by immunofluorescence determination of BrdU incorporation using specific antibodies (18). Statistical evaluation was performed based on three individual experiments, each analyzing Ͼ200 cells.…”

“…To test this possibility, functional knockouts of Ez, Rdx, and Moe were designed and tested for their effects on minute virus of mice 3Ј; Rdxdl[P], 5Ј-TAAAAACTGTGATGTCTGCGdlGTCATCCTCCAACGGAG AA-3Ј and 5Ј-TTCTCCGTTGGAGGATAGACdlCGCAGACATCACAGTTTTT A-3Ј, where mutated codons are indicated by italics, with the changed nucleotides in boldface; deletions are indicated by dl; ellipses indicate variable numbers of wildtype nucleotides which are not shown in the sequence. The Flag-tagged mutants were subcloned into pCR2.1, sequenced, and transferred as PmeI/NotI restriction fragments into HpaI/NotI-cleaved pP38 (18), yielding pP38-FlagEzT566A, pP38-FlagEzT566E, pP38-Flag-RdxT564A, pP38-FlagRdxT564E, pP38FlagRdxY146F, pP38-FlagRdxdl[P], pP38-FlagMoeT547A, and pP38-FlagMoeT547E, respectively.…”