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Vol. 19, Issue 6, 2544-2552, June 2008

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A Supraphysiological Nuclear Export Signal Is Required for Parvovirus Nuclear Export

Dieuwke Engelsma^*, Noelia Valle^,, Alexander Fish, Nathalie Salomé^||, José M. Almendral, and Maarten Fornerod^*

Departments of *Tumor Biology and Molecular Carcinogenesis, The Netherlands Cancer Institute, 1066 CX Amsterdam, The Netherlands; Centro de Biología Molecular "Severo Ochoa" (Consejo Superior de Investigaciones Científicas-Universidad Autónoma de Madrid), 28049 Cantoblanco, Madrid, Spain; and ^||Infection and Cancer Program, Division F010 and INSERM U701, Deutsches Krebsforschungszentrum, D-69120 Heidelberg, Germany

Submitted January 8, 2008; Revised March 4, 2008; Accepted March 24, 2008
Monitoring Editor: Susan Wente

CRM1 exports proteins that carry a short leucine-rich peptide signal, the nuclear export signal (NES), from the nucleus. Regular NESs must have low affinity for CRM1 to function optimally. We previously generated artificial NESs with higher affinities for CRM1, termed supraphysiological NESs. Here we identify a supraphysiological NES in an endogenous protein, the NS2 protein of parvovirus Minute Virus of Mice (MVM). NS2 interacts with CRM1 without the requirement of RanGTP, whereas addition of RanGTP renders the complex highly stable. Mutation of a single hydrophobic residue that inactivates regular NESs lowers the affinity of the NS2 NES for CRM1 from supraphysiological to regular. Mutant MVM harboring this regular NES is compromised in viral nuclear export and productivity. In virus-infected mouse fibroblasts we observe colocalization of NS2, CRM1 and mature virions, which is dependent on the supraphysiological NS2 NES. We conclude that supraphysiological NESs exist in nature and that the supraphysiological NS2 NES has a critical role in active nuclear export of mature MVM particles before cell lysis.

Present address: Instituto de Investigaciones Biomédicas "Alberto Sols" 28029 Madrid, Spain.

Address correspondence to: Maarten Fornerod (m.fornerod{at}nki.nl).

Abbreviations used: 2Nt, amino-terminus of VP2; GFP, green fluorescent protein; LMB, leptomycin B; MVM, minute virus of mice; NE, nuclear envelope; NES, nuclear export signal; NPC, nuclear pore complex; NS2, non-structural protein 2; SAABs, Smn-associated autonomous parvovirus-associated replication bodies; supraNES, supraphysiological NES; VP, viral protein.