Host factor SMYD3 is recruited by Ebola virus nucleoprotein to facilitate viral mRNA transcription

Abstract

The polymerase complex of Ebola virus (EBOV) is the functional unit for transcription and replication of viral genome. Nucleoprotein (NP) is a multifunctional protein with high RNA binding affinity and recruits other viral proteins to form functional polymerase complex. In our study, we investigated host proteins associated with EBOV polymerase complex using NP as bait in a transcription and replication competent minigenome system by mass spectrometry analysis and identified SET and MYND domain-containing protein 3 (SMYD3) as a novel host protein which was required for the replication of EBOV. SMYD3 specifically interacted with NP and was recruited to EBOV inclusion bodies through NP. The depletion of SMYD3 dramatically suppressed EBOV mRNA production. A mimic of non-phosphorylated VP30, which is a transcription activator, could partially rescue the viral mRNA production downregulated by the depletion of SMYD3. In addition, SMYD3 promoted NP-VP30 interaction in a dose-dependent manner. These results revealed that SMYD3 was a novel host factor recruited by NP to supporting EBOV mRNA transcription through increasing the binding of VP30 to NP. Thus, our study provided a new understanding of mechanism underlying the transcription of EBOV genome, and a novel anti-EBOV drug design strategy by targeting SMYD3.

The ORF8 Protein of SARS-CoV-2 Mediates Immune Evasion through Potently Downregulating MHC-I

“…Immunofluorescence assay was performed as previously described 48 . HEK293T cells were seeded on in μ-slide chambered coverslips (Ibidi; 80826), and transfected as indicated.…”

“…The proteins were then visualized with ProteoSilver Plus Silver Stain Kit (Sigma Aldrich) according to the manufacturer’s instructions. The whole lane was cut into ten slices and prepared for liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis as previously described 48 . Functional pathways representative of each gene signature was analyzed for enrichment in gene categories from the Gene Ontology Biological Processes (GO-BP) database (Gene Ontology Consortium) using DAVID Bioinformatics Resources, observing correlation between two replicate experiments.…”

Reporter Assays for Ebola Virus Nucleoprotein Oligomerization, Virion-Like Particle Budding, and Minigenome Activity Reveal the Importance of Nucleoprotein Amino Acid Position 111

“…Though we did not detect differential interactions between the two NP genotypes, our approach yielded multiple members of the adaptor related protein 1 (AP-1) complex as strong candidate host protein interactors (Supplementary Figure S5A). Several AP-1 members had been identified previously [82,83,85] but were not further confirmed. The AP-1 complex mediates intracellular transport, and therefore could be important for viral transcription and replication, or VLP formation and budding.…”

“…By studying the host interactome of NP, we confirmed a strong interaction between NP and the clathrin adaptor AP-1 complex (Supplementary Figure S5B), which mediates intracellular transport and could facilitate virion assembly. AP-1 members were among the few proteins that co-purified with NP in multiple MS/MS experiments [82,83,85], but the interaction was not specifically validated by a second approach like reciprocal co-IP in those studies. In retroviruses, the Gag protein facilitates budding by hijacking the AP-1 complex [88].…”

“…The binding interfaces of VP30 and VP35 are on the opposite face of NP as the R111 residue ( Figure 2B), suggesting that these viral interactions are likely to be unaffected. Only a handful of host interactome studies have been performed on EBOV NP [82][83][84][85], all utilizing the NP amino acid sequence from the Mayinga isolate of the EBOV Yambuku variant (EBOV/Yam-May), the first EBOV isolated in 1976. To build upon these previous results, we performed co-immunoprecipitation tandem mass spectrometry (co-IP MS/MS) using myc-tagged NP from EBOV/Makona variant bearing either R111 or K109E/K110E/R111E, which had the most dramatic charge-reversal.…”