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

Abstract

SummaryAutonomous 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, PKCh and PKCl 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 PKCh, as evidenced by changes in their subcellular distribution and overall (auto)phosphorylation. We show evidence that activation of PKCh by PDK1 is driven by atypical PKCl. By modifying the hydrophobic motif of PKCh, PKCl appeared to control docking and consecutive phosphorylation of PKCh's activation-loop by PDK1, a process that was inhibited in vivo in the presence of a dominantnegative PKCl mutant.

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

“…This was substantiated by the present study, which showed that Rdx is a substrate of cellular kinases in MVM-infected cells, resulting in Rdx (and consequently PKC [17]) activation. Moreover, our data indicate that the main phosphorylation of Rdx at later stages of infection depends on newly synthesized parvoviral proteins, particularly arguing for the involvement of the recently described NS1/CKII␣ complex (32).…”

“…Few changes in Ez localization were observed between mock-treated and MVM-infected A9 cells. In contrast, as previously reported for PKC (17), MVM induced redistribution of Rdx and Moe, which accumulated in the perinuclear area, showing a strong colocalization with PKC . As previously reported (35), and modulated through the expression of dominant-negative versus constitutively active Rdx mutants (Fig.…”

“…Upon MVM infection, Rdx not only is modified through phosphorylation, but also colocalizes with PKC , correlating with the PKC -driven phosphorylation of both NS1 and progeny virions. Considering that ERM proteins function as mediators between the (actin) cytoskeleton and membrane structures (35), this finding is not surprising, since activation of PKCs is associated with changes in their affinity from cellular scaffold to membrane association (17). NS1 was shown to be regulated in its various functions during productive virus infection by selected phosphorylation events (6,10,27).…”

“…In order to promote viral DNA amplification, NS1 requires additional modifications driven by the novel PKC (18). Early during infection, active PKC is found in limited amounts in the nucleus (17) to drive the initial replication steps. As reflected by the lag period observed in the beginning of the MVM infection cycle, this PKC -driven phosphorylation of NS1 might, however, be rather inefficient.…”

“…During activation, PKCs undergo a variety of conformational changes that allow their interaction with small ligands or regulatory proteins and facilitate translocation of the kinase to specific compartments in which substrates become available. In the course of this sequence of activation steps, PKCs undergo changes in their affinities for cellular scaffold and membrane structures (17). ERM (ezrin [Ez], radixin [Rdx], moesin [Moe]) family proteins are known to be mediators between cellular scaffold (actin) and membrane structures (14) and by analogy have also been proposed to serve as anchoring proteins for PKC (15).…”