Properties of two EBV Mta nuclear export signal sequences.

The Epstein-Barr virus (EBV) Mta protein is a posttranscriptional regulator of EBV lytic gene expression that affects RNA splicing and transport. Mta mediates cytoplasmic accumulation of unspliced EBV replication gene transcripts and shuttles between the nucleus and cytoplasm. Mta contains a recognized leucine-rich, putative nuclear export signal (NES) between aa 227 and 236. Deletion of this signal sequence eliminated shuttling, while mutation of the core LXL motif in the putative NES diminished but did not abolish the ability of Mta to shuttle from donor to recipient cells in a heterokaryon assay. A double mutation of the LXL motif plus an upstream VTL motif eliminated shuttling, suggesting that Mta may have two NES motifs. In confirmation of this, transfer of either the sequence encoding the leucine-rich aa 227-236 motif or that encoding the adjacent hydrophobic aa 218-227 sequence to a GFP-NLS-pyruvate kinase reporter protein conferred the property of cytoplasmic accumulation onto the heterologous protein. Cytoplasmic accumulation of both the aa 225-237 and 218-227 containing reporters was minimal in the presence of the inhibitor leptomycin B, indicating that both motifs mediated Crm-1-dependent export. Mutations in the NES signal sequences abolished the ability of Mta to mediate cytoplasmic accumulation of BALF2 replication gene transcripts. This included mutation of the LXL motif which still showed cytoplasmic shuttling, suggesting that the NES mutations might have additional effects on Mta function. Wild-type Mta co-immunoprecipitated with the splicing factor SC35 and colocalized with SC35 in transfected cells, modifying endogenous SC35 distribution within the nucleus to give more intense, rounded spots. Interestingly, the NES mutant proteins appeared to have altered interactions with the splicing complex, binding more tightly to SC35 in co-immunoprecipitation assays. These observations suggest a linkage between the splicing and export functions of Mta. Copyright 2001 Academic Press.