Nuclear Targeting of the Parvoviral Replicator Molecule NS1: Evidence for Self-Association Prior to Nuclear Transport

Virus-induced apoptosis of infected cells can limit both the time and the cellular machinery available for virus replication. Hence, many viruses have evolved strategies to specifically inhibit apoptosis. However, Aleutian mink disease parvovirus (ADV) is the first example of a DNA virus that not only induces apoptosis but also utilizes caspase activity to facilitate virus replication. To determine the function of caspase activity during ADV replication, virus-infected cell lysates or purified ADV proteins were incubated with various purified caspases. Caspases cleaved the major nonstructural protein of ADV (NS1) at two caspase recognition sequences, whereas ADV structural proteins could not be cleaved. Importantly, the NS1 products could be identified in ADV-infected cells but were not present in infected cells pretreated with caspase inhibitors. By mutating putative caspase cleavage sites (D to E), we mapped the two cleavage sites to amino acid residues NS1:227 (INTD2S) and NS1:285 (DQTD2S). Replication of ADV containing either of these mutations was reduced 10 3 -to 10 4 -fold compared to that of wild-type virus, and a construct containing both mutations was replication defective. Immunofluorescent studies revealed that cleavage was required for nuclear localization of NS1. The requirement for caspase activity during permissive replication suggests that limitation of caspase activation and apoptosis in vivo may be a novel approach to restricting virus replication.

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Caspase Cleavage of the Nonstructural Protein NS1 Mediates Replication of Aleutian Mink Disease Parvovirus

Best¹,

Shelton²,

Pompey³

et al. 2003

“…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 heterogeneous nuclear ribonucleoproteins (28). NS1 plays a key role in many processes necessary for progeny virus production.…”

mentioning

confidence: 99%

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

Lachmann

Rommeleare

Nüesch

2003

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...

“…The large nonstructural protein, NS1, is a multifunctional protein with site-specific DNA binding, nickase, ATPase, and helicase activities that have been mapped to specific regions of the NS1 protein and are critical for MVM replication (8,17,19,20). The small nonstructural protein, NS2, is also required for MVM replication in a host-cell-specific manner; in murine cells NS2-null mutants (18), as well as a number of other characterized NS2 mutants (5), generate little double-stranded replicative intermediates and little to no progeny single-stranded DNA (ssDNA) is produced.…”

mentioning

confidence: 99%

Interaction between Parvovirus NS2 Protein and Nuclear Export Factor Crm1 Is Important for Viral Egress from the Nucleus of Murine Cells

Miller

Pintel

2002

A mutation that disrupts the interaction between the NS2 protein of minute virus of mice and the nuclear export factor Crm1 results in a block to egress of mutant-generated full virions from the nucleus of infected murine cells. These mutants produce wild-type levels of monomer and dimer replicative DNA forms but are impaired in their ability to generate progeny single-stranded DNA in restrictive murine cells in the first round of infection. The NS2-Crm1 interaction mutant can be distinguished phenotypically from an NS2-null mutant and reveals a role for the Crm1-mediated export pathway at a late step in viral infection.The autonomous parvovirus minute virus of mice (MVM) produces two nonstructural proteins that play critical roles in the replication of the virus (6)(7)(8)18). The large nonstructural protein, NS1, is a multifunctional protein with site-specific DNA binding, nickase, ATPase, and helicase activities that have been mapped to specific regions of the NS1 protein and are critical for MVM replication (8,17,19,20). The small nonstructural protein, NS2, is also required for MVM replication in a host-cell-specific manner; in murine cells NS2-null mutants (18), as well as a number of other characterized NS2 mutants (5), generate little double-stranded replicative intermediates and little to no progeny single-stranded DNA (ssDNA) is produced. Cells from a variety of other species support MVM replication at near-wild-type levels in the absence of NS2 (18).The role(s) NS2 plays in MVM replication remains unclear. In the absence of NS2, wild-type levels of the VP1 and VP2 structural proteins are synthesized at early times postinfection; however, they do not efficiently assemble into MVM capsids (5). Although the mechanisms are not well understood, the production of MVM progeny ssDNA is tightly connected to the availability of assembled MVM capsids (8), and so it is likely that, at least to some extent, the lack of progeny ssDNA produced during NS2 mutant infection is a consequence of the lack of proper capsid assembly. The lack of replication of monomer replicative DNA forms (mRF) and dimer replicative DNA forms (dRF) in NS2-null mutant infection is more difficult to explain, as this process is known to proceed efficiently in the absence of capsid production (23).NS2 was recently shown to interact with two members of the 14-3-3 family of signaling proteins (3) and the nuclear export factor Crm1 (2, 21). Crm1 is a sequence-specific nuclear export receptor that binds leucine-rich sequences within proteins in cooperation with RanGTP and actively transports these proteins from the nucleus to the cytoplasm (9, 24). The NS2 interaction with Crm1 was previously shown to be important in proper localization of NS2 within the cell and was mapped to NS2 amino acids (aa) 81 to 103 by peptide analysis (N. Salome, personal communication) and glutathione S-transferase (GST) pull-down assays (21). The role of the NS2-Crm1 interaction during MVM infection, however, has not been determined.We have investigated the consequence o...