The NS2 Proteins of Parvovirus Minute Virus of Mice Are Required for Efficient Nuclear Egress of Progeny Virions in Mouse Cells

Abstract

The small nonstructural NS2 proteins of parvovirus minute virus of mice (MVMp) were previously shown to interact with the nuclear export receptor Crm1. We report here the analysis of two MVM mutant genomic clones generating NS2 proteins that are unable to interact with Crm1 as a result of amino acid substitutions within their nuclear export signal (NES) sequences. Upon transfection of human and mouse cells, the MVM-NES21 and MVM-NES22 mutant genomic clones were proficient in synthesis of the four virus-encoded proteins. While the MVM-NES22 clone was further able to produce infectious mutant virions, no virus could be recovered from cells transfected with the MVM-NES21 clone. Whereas the defect of MVM-NES21 appeared to be complex, the phenotype of MVM-NES22 could be traced back to a novel distinct NS2 function. Infection of mouse cells with the MVM-NES22 mutant led to stronger nuclear retention not only of the NS2 proteins but also of infectious progeny MVM particles. This nuclear sequestration correlated with a severe delay in the release of mutant virions in the medium and with prolonged survival of the infected cell populations compared with wild-type virus-treated cultures. This defect could explain, at least in part, the small size of the plaques generated by the MVM-NES22 mutant when assayed on mouse indicator cells. Altogether, our data indicate that the interaction of MVMp NS2 proteins with the nuclear export receptor Crm1 plays a critical role at a late stage of the parvovirus life cycle involved in release of progeny viruses.The minute virus of mice prototype strain (MVMp) is an autonomously replicating parvovirus that encodes two types of nonstructural proteins, NS1 and NS2, which are required at various steps of the parvovirus replication cycle. The 83-kDa nuclear phosphoprotein NS1 is a multifunctional protein that exhibits site-specific DNA binding, ATPase, helicase, and nickase activities. These activities account for the role played by NS1 during viral DNA replication (for a review, see reference 22). NS1 also controls transcription in trans from both parvovirus promoters P4 and P38, which drive the expression of nonstructural and capsid proteins, respectively, and from at least one cellular promoter (21,28,29,86). Furthermore, NS1 is described as the major effector of parvovirus-induced cytotoxicity (10, 13, 50; for a review, see reference 87).The small nonstructural NS2 proteins of MVMp consist of three isoforms, NS2-P, -Y, and -L, that differ at their carboxy termini as a result of alternative splicing events (19, 59). They have a molecular mass of about 25 kDa, and all three isoforms share a common amino-terminal domain with NS1 which comprises the first 85 amino acids (aa) of each protein (21,23,43).

Nuclear Export of the Nonenveloped Parvovirus Virion Is Directed by an Unordered Protein Signal Exposed on the Capsid Surface

“…2 to 6). In summary, our data are consistent with previous reports of an effect on nuclear release of the MVM capsid induced by the NS2-CRM1 interaction (15,40), which may also benefit the distinct 2Nt-mediated active transport of mature virions, although the role of CRM1 in both processes must be through an indirect phenomenon in which other pleiotropic properties of this cellular transporter may be involved (17,75), and this phenomenon deserves further research.…”

“…Mutations in the CRM1 binding domain of NS2 that were naturally selected in mice showed, in synchronously infected 324K cells, that an NS2-CRM1 cytoplasmic interaction occurred several hours prior to a general increase in the nuclear release of viral antigens, mainly the NS1 major viral nonstructural protein lacking a recognizable conventional NES, and also of the empty capsid (35). In addition, the release of the capsid into the cytoplasm late in the infection is relatively slow, since it can be stained with an antibody (15,35,40), whereas the export of mature viruses and heated capsids from the nucleus to the cell surface proceeded rapidly, with no cytoplasmic accumulation by IF (Fig. 2 to 6).…”

“…These results may suggest the concourse of a virally induced protein adapter allowing access of the MVM capsid to the CRM1 export route, similar to the transport of some subviral nucleic acid-protein complexes coupled to CRM1 (e.g., see references 16 and 47). Interestingly, efficient MVM capsid release and viral spreading in mouse A9 cells, and to some extent in 324K cells, required the CRM1 interactive domain of the nonstructural NS2 protein (15,40), and thus NS2 could presumably be the MVM-encoded factor that adapts the capsid and 2Nt to the CRM1 pathway in infected cells. However, extensive immunoprecipitation analyses failed to demonstrate any consistent interaction of the intact MVM capsid or mature virions with either CRM1 or NS2 (data not shown), suggesting an indirect involvement of CRM1 in the transport process.…”

“…2A). Later in the infection cycle, the cytoplasm of most infected cells was stained with the capsid MAb antibody (not shown), a phenomenon that was indirectly related to the activity of the NS2 nonstructural protein (15,40), which is described elsewhere (35). The distinct subcellular distribution of the newly formed empty capsids and DNA-filled virions in the MVM infection was further examined by equilibrium centrifugation of 35 S-labeled particles harvested from synchronized cells (Fig.…”

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.…”