Determinants of viral assembly and infectivity within the Ebola virus VP24 protein

About This Grant

Summary Ebola virus (EBOV), a filovirus, causes periodic outbreaks with high fatality rates. To understand the basis for viral pathogenesis, emergence, and to devise control strategies, it is important to define the mechanisms that underlie EBOV assembly and release of infectious virus. The VP24 protein, produced by one of seven viral genes, is a 24 kDa multifunctional protein unique to the filovirus family. EBOV VP24 blocks cellular responses to interferons (IFN) by preventing nuclear translocation of tyrosine phosphorylated STAT1 (pSTAT1), a transcription factor central to antiviral IFN signaling. VP24 accomplishes this by interacting with importin α (IMPA) nuclear transport proteins, preventing binding and nuclear import of STAT1, a key transcription factor needed for IFN responses. That VP24 interacts with IMPA nuclear import factors suggests that VP24 can traffic into the nucleus. In addition, transfection studies identified a nuclear export signal (NES) at the C-terminus of EBOV VP24, further supporting nuclear its trafficking. However, whether EBOV VP24 undergoes nucleocytoplasmic trafficking and the functional significance of this trafficking remain to be determined. VP24 also plays a critical but incompletely understood role in viral genome packaging and production of infectious viral particles. Most notably, VP24, the EBOV nucleoprotein and EBOV VP35 together form filamentous nucleocapsid structures, called nucleocapsid- like structures (NCLS) that are morphologically indistinguishable from the nucleocapsids present in EBOV virions. The presence of VP24 also renders NCLS competent for actin-dependent transport. Fundamental questions remain including how VP24 facilitates actin-dependent transport and the specific molecular features required for NCLS formation and trafficking. Our Preliminary Data indicates that VP24 can traffic into and out of the nucleus and that disruption of IMPA binding significantly impairs virus growth at a late stage in the replication cycle. Disruption of nuclear export function completely abrogates infectious particle production. Consistent with these observations, an NES mutant VP24 recombinant virus could not be recovered. Providing mechanistic insight, initial studies on the NES mutant VP24 suggest that it is significantly impaired for actin-dependent trafficking. Finally, proteomic analysis suggests that VP24 recruits components of the Wave regulatory complex (WRC), a regulator of Arp2/3 dependent actin polymerization via its NES sequence. These data support roles for VP24 IMPA binding and NES sequences in virus production and suggest that the NES may recruit the WRC to promote NCLS trafficking. The proposed studies will determine how VP24 NES sequences recruits the WRC to promote actin-dependent trafficking of viral nucleocapsids and incorporation into viral particles, and it will define the IFN-dependent and IFN-independent consequences of VP24-IMPA interaction.