Structures of minute virus of mice replication initiator protein N-terminal domain: Insights into DNA nicking and origin binding.

Members of the Parvoviridae family all encode a non-structural protein 1 (NS1) that directs replication of single-stranded viral DNA, packages viral DNA into capsid, and serves as a potent transcriptional activator. Here we report the X-ray structure of the minute virus of mice (MVM) NS1 N-terminal domain at 1.45Å resolution, showing that sites for dsDNA binding, ssDNA binding and cleavage, nuclear localization, and other functions are integrated on a canonical fold of the histidine-hydrophobic-histidine superfamily of nucleases, including elements specific for this Protoparvovirus but distinct from its Bocaparvovirus or Dependoparvovirus orthologs. High resolution structural analysis reveals a nickase active site with an architecture that allows highly versatile metal ligand binding. The structures support a unified mechanism of replication origin recognition for homotelomeric and heterotelomeric parvoviruses, mediated by a basic-residue-rich hairpin and an adjacent helix in the initiator proteins and by tandem tetranucleotide motifs in the replication origins.

Parvoviral virions deploy a capsid-tethered lipolytic enzyme to breach the endosomal membrane during cell entry.

Enveloped viruses deliver their virions into the host cell by fusion with the cellular plasma or endosomal membrane, thus creating topological continuity between the cytosol and the inside of the viral envelope. Nonenveloped viruses are, by their very nature, denied this strategy and must employ alternative methods to breach their host cell's delimiting membrane. We show here that the compact icosahedral parvoviral virion gains entry by deploying a lipolytic enzyme, phospholipase A(2) (PLA(2)), that is expressed at the N terminus of VP1, the minor coat protein. This region of VP1 is normally sequestered within the viral shell but is extruded during the entry process as a capsid-tethered domain. A single amino acid substitution in the active site of the VP1 PLA(2) inactivates enzymatic activity and abrogates infectivity. We have used transencapsidation of a vector expressing green fluorescent protein to show that infection by this PLA(2)-defective mutant can be complemented by coinfection with wild-type or mutant full virions, provided they can express a functional PLA(2). Even though wild-type empty capsids contain an active form of the enzyme, it is not externalized under physiological conditions, and such capsids are not able to complement the PLA(2) mutant. Significantly, highly efficient rescue can be achieved by polyethyleneimine-induced endosome rupture or by coinfection with adenovirus as long as uptake of the two viruses is simultaneous and the adenovirus is capable of deploying pVI, a capsid protein with endosomolytic activity. Together, these results demonstrate a previously unrecognized enzymatic mechanism for nonenveloped virus penetration.

Cyclin A activates the DNA polymerase delta -dependent elongation machinery in vitro: A parvovirus DNA replication model.

Replication of the single-stranded linear DNA genome of parvovirus minute virus of mice (MVM) starts with complementary strand synthesis from the 3'-terminal snap-back telomere, which serves as a primer for the formation of double-stranded replicative form (RF) DNA. This DNA elongation reaction, designated conversion, is exclusively dependent on cellular factors. In cell extracts, we found that complementary strand synthesis was inhibited by the cyclin-dependent kinase inhibitor p21(WAF1/CIP1) and rescued by the addition of proliferating cell nuclear antigen, arguing for the involvement of DNA polymerase (Pol) delta in the conversion reaction. In vivo time course analyses using synchronized MVM-infected A9 cells allowed initial detection of MVM RF DNA at the G(1)/S phase transition, coinciding with the onset of cyclin A expression and cyclin A-associated kinase activity. Under in vitro conditions, formation of RF DNA was efficiently supported by A9 S cell extracts, but only marginally by G(1) cell extracts. Addition of recombinant cyclin A stimulated DNA conversion in G(1) cell extracts, and correlated with a concomitant increase in cyclin A-associated kinase activity. Conversely, a specific antibody neutralizing cyclin A-dependent kinase activity, abolished the capacity of S cell extracts for DNA conversion. We found no evidence for the involvement of cyclin E in the regulation of the conversion reaction. We conclude that cyclin A is necessary for activation of complementary strand synthesis, which we propose as a model reaction to study the cell cycle regulation of the Pol delta-dependent elongation machinery.

DNA unwinding functions of minute virus of mice NS1 protein are modulated specifically by the lambda isoform of protein kinase C.

The parvovirus minute virus of mice NS1 protein is a multifunctional protein involved in a variety of processes during virus propagation, ranging from viral DNA replication to promoter regulation and cytotoxic action to the host cell. Since NS1 becomes phosphorylated during infection, it was proposed that the different tasks of this protein might be regulated in a coordinated manner by phosphorylation. Indeed, comparing biochemical functions of native NS1 with its dephosphorylated counterpart showed that site-specific nicking of the origin and the helicase and ATPase activities are remarkably reduced upon NS1 dephosphorylation while site-specific affinity of the protein to the origin became enhanced. As a consequence, the dephosphorylated polypeptide is deficient for initiation of DNA replication. By adding fractionated cell extracts to a kinase-free in vitro replication system, the combination of two protein components containing members of the protein kinase C (PKC) family was found to rescue the replication activity of the dephosphorylated NS1 protein upon addition of PKC cofactors. One of these components, termed HA-1, also stimulated NS1 helicase function in response to acidic lipids but not phorbol esters, indicating the involvement of atypical PKC isoforms in the modulation of this NS1 function (J. P. F. Nüesch, S. Dettwiler, R. Corbau, and J. Rommelaere, J. Virol. 72:9966-9977, 1998). The present study led to the identification of atypical PKClambda/iota as the active component of HA-1 responsible for the regulation of NS1 DNA unwinding and replicative functions. Moreover, a target PKClambda phosphorylation site was localized at S473 of NS1. By site-directed mutagenesis, we showed that this residue is essential for NS1 helicase activity but not promoter regulation, suggesting a possible modulation of NS1 functions by PKClambda phosphorylation at residue S473.

Mutations in the NTP-binding motif of minute virus of mice (MVM) NS-1 protein uncouple ATPase and DNA helicase functions.

The NS-1 protein of minute virus of mice (MVM) is required for viral DNA replication and transcriptional regulation. To define the domain structure of NS-1, we have generated point mutations in its putative NTP-binding/ATPase domain. We show that all mutants were unable to support replication of MVM DNA in a transient DNA replication assay. Furthermore, all mutants, except for the K405S substitution, were able to transactivate the P38 promoter in transient transfection experiments. NS-1 proteins bearing COOH-terminal deletions of 29 and 33 amino acid residues were also transcriptionally inert. Biochemical analysis of recombinant NS-1 expressed in insect cells shows that mutations in the putative NTP-binding/ATPase domain severely reduced helicase activity in vitro. However, affinity labeling experiments indicate that none of these mutations, except for K469T, impaired NTP-binding activity. Finally, all point mutants retained significant levels of ATPase activity, except for the E444Q mutant (1%). These findings suggest that the replication and transcription activities of NS-1 reside in separate functional domains. In addition, NS-1 proteins with mutations in the putative nucleotide binding fold have lost helicase activity, whereas most retain nucleotide binding and ATPase functions, suggesting that the mutations have uncoupled the ATPase and helicase activities.

Minute virus of mice-induced modification of the murine DNA polymerase alpha-primase complex permits the salt-induced dissociation of 12S DNA primase and 10S DNA polymerase alpha components.

DNA polymerase alpha-primase complexes in extracts of MVM-infected murine cells were dissociated in the presence of 0.3M KCl to generate a 12S DNA primase and a 10S DNA polymerase alpha that were readily separated by sedimentation in glycerol gradients. A 12S DNA polymerase alpha-primase complex refractory to dissociation in 0.3M KCl was identified in extracts of MVM-infected HeLa cells. In extracts of mock-infected murine and HeLa cells DNA primase and DNA polymerase alpha were not dissociated from each other in 0.3M KCl but remained in a stable complex that sedimented at 10S. We propose that a novel 12S DNA polymerase alpha-primase complex prone to disruption by salt is induced by MVM infection and that the DNA primase component of the complex is modified.

Expression of minute virus of mice major nonstructural protein in insect cells: purification and identification of ATPase and helicase activities.

The gene encoding the major nonstructural (NS-1) protein of minute virus of mice (MVM) has been expressed in insect cells using a baculovirus expression system. This 83-kDa polypeptide was found to be localized in the soluble (cytosolic) fraction in insect cells, in contrast with the nuclear localization of NS-1 expressed in MVM-infected mouse LA-9 cells. The protein was purified by immunoaffinity chromatography using a monoclonal antibody (MAb) prepared to an NS-1 fusion peptide [(Yeung et al., Virology 185, 35-45 (1991)]. Recombinant NS-1 was eluted using either low pH or a synthetic peptide corresponding to the epitope of the MAb. The peptide-eluted material is greater than 95% pure and biologically active in that it has ATPase activity and ATP-dependent helicase activity as determined by a strand displacement assay.