Phosphorylated VP30 of marburg virus is a repressor of transcription
The filoviruses Marburg virus (MARV) and Ebola virus (EBOV) cause hemorrhagic fever in humans and nonhuman primates, with high case fatality rates. MARV VP30 is known to be phosphorylated and to interact with nucleoprotein (NP), but its role in regulation of viral transcription is disputed. Here, we analyzed phosphorylation of VP30 by mass spectrometry, which resulted in identification of multiple phosphorylated amino acids. Modeling the full-length three-dimensional structure of VP30 and mapping the identified phosphorylation sites showed that all sites lie in disordered regions, mostly in the N-terminal domain of the protein. Minigenome analysis of the identified phosphorylation sites demonstrated that phosphorylation of a cluster of amino acids at positions 46 through 53 inhibits transcription. To test the effect of VP30 phosphorylation on its interaction with other MARV proteins, coimmunoprecipitation analyses were performed. They demonstrated the involvement of VP30 phosphorylation in interaction with two other proteins of the MARV ribonucleoprotein complex, NP and VP35. To identify the role of protein phosphatase 1 (PP1) in the identified effects, a small molecule, 1E7-03, targeting a noncatalytic site of the enzyme that previously was shown to increase EBOV VP30 phosphorylation was used. Treatment of cells with 1E7-03 increased phosphorylation of VP30 at a cluster of phosphorylated amino acids from Ser-46 to Thr-53, reduced transcription of MARV minigenome, enhanced binding to NP and VP35, and dramatically reduced replication of infectious MARV particles. Thus, MARV VP30 phosphorylation can be targeted for development of future antivirals such as PP1-targeting compounds. IMPORTANCE The largest outbreak of MARV occurred in Angola in 2004 to 2005 and had a 90% case fatality rate. There are no approved treatments available for MARV. Development of antivirals as therapeutics requires a fundamental understanding of the viral life cycle. Because of the close similarity of MARV to another member of Filoviridae family, EBOV, it was assumed that the two viruses have similar mechanisms of regulation of transcription and replication. Here, characterization of the role of VP30 and its phosphorylation sites in transcription of the MARV genome demonstrated differences from those of EBOV. The identified phosphorylation sites appeared to inhibit transcription and appeared to be involved in interaction with both NP and VP35 ribonucleoproteins. A small molecule targeting PP1 inhibited transcription of the MARV genome, effectively suppressing replication of the viral particles. These data demonstrate the possibility developing antivirals based on compounds targeting PP1.
Tigabu, Bersabeh; Ramanathan, Palaniappan; Ivanov, Andrey; Lin, Xionghao; Ilinykh, Philipp A.; Parry, Christian S.; Freiberg, Alexander N.; Nekhai, Sergei; and Bukreyev, Alexander, "Phosphorylated VP30 of marburg virus is a repressor of transcription" (2018). The Center For Sickle Cell Disease Faculty Publications. 20.