Properties of two EBV Mta nuclear export signal sequences

Area of Science:

• Molecular Biology
• Virology
• Cell Biology

Background:

• The Epstein-Barr virus (EBV) Mta protein is a key regulator of viral lytic gene expression.
• Mta influences RNA splicing and transport, mediating the cytoplasmic accumulation of unspliced viral transcripts.
• Mta shuttles between the nucleus and cytoplasm, a process critical for its function.

Purpose of the Study:

• To investigate the nuclear export signals (NES) within the EBV Mta protein.
• To elucidate the role of these NES motifs in Mta's nucleocytoplasmic shuttling and its effect on viral gene expression.
• To explore the potential link between Mta's export function and its interaction with cellular splicing factors.

Main Methods:

• Deletion and mutation analyses of putative NES motifs within Mta.
• Heterokaryon assays to assess Mta's nucleocytoplasmic shuttling.
• Fusion of Mta-derived sequences to a reporter protein (GFP-NLS-pyruvate kinase) to confer export properties.
• Leptomycin B treatment to confirm Crm-1-dependent export.
• Co-immunoprecipitation and immunofluorescence assays to study Mta's interaction with the splicing factor SC35.

Main Results:

• Mta contains at least two functional NES motifs, one leucine-rich (aa 227-236) and an adjacent hydrophobic one (aa 218-227).
• Both motifs mediate Crm-1-dependent nuclear export, as evidenced by leptomycin B sensitivity.
• Mutations in these NES sequences abolish Mta's ability to accumulate BALF2 transcripts in the cytoplasm.
• NES mutant Mta proteins exhibit altered interactions with the splicing factor SC35, binding more tightly.
• Mta co-localizes with SC35 and modifies its nuclear distribution.

Conclusions:

• The EBV Mta protein utilizes two distinct NES motifs for Crm-1-dependent nucleocytoplasmic export.
• These export functions are essential for Mta's role in regulating the cytoplasmic accumulation of viral replication transcripts.
• Mta's export activity is intrinsically linked to its interaction with cellular splicing factors, suggesting a coordinated regulation of splicing and export.