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

Abstract

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...

Replication of Minute Virus of Mice DNA Is Critically Dependent on Accumulated Levels of NS2

“…Why the NS2 protein generated by K96E/L103P migrates more slowly than the other NS2 proteins is not known, but this result was also given in the original report of its isolation (14). during infection, including a block to the egress of assembled capsids from the nucleus and a deficiency in the accumulation of progeny ssDNA (9,17). How the NS2-CRM1 interaction might affect viral replication is not known; however, it may be that this interaction somehow influences the status of assembled capsids required for ssDNA production.…”

“…The 5Ј end of the NS2-specific exon, adjacent to the large-intron 3Ј splice site, contains both an amino acid motif important for the NS2 interaction with Crm1 (5) and a nucleotide motif that functions as an exon splicing enhancer that plays a critical role in upstream large-intron excision (13). The six-amino-acid NS2-CRM1 interaction mutant we previously characterized, which is deficient in ssDNA production (17), shows no decrease in excision of the large intron and makes at least wild-type levels of mutant NS2 (data not shown), and so the phenotype of this mutant is related to the loss of NS2 function rather than a decrease in protein abundance. However, as might be predicted, a single nucleotide mutation in the exon splicing enhancer immediately upstream in this region that inhibits excision of the upstream MVMp large intron in fibroblasts shows, similarly to MVMi, decreased ability to make ssDNA (18).…”

“…Murine A9 and EL4 cells and human NB324K cells were propagated as previously described (17). All virus was made directly from transfection of NB324K cells without further propagation, and the titers of the virus were determined by plaque assay on NB324K (6).…”

“…Southern analysis and Western blot analysis were performed exactly as previously described (17,24). The complete homologous genomic clones were used as probes for Southern analysis, and the antibody for NS1/NS2 Western analysis was a polyclonal rabbit antibody raised to the NH2-terminal common exon of NS1 and NS2.…”

“…Transfection of A9 cells was as previously described (17). Transfections of EL4 cells were performed with an Amaxa nucleofector device (Amaxa, Inc.) by using program B24.…”