Ebola virus VP35 interaction with dynein LC8 regulates viral RNA synthesis

Abstract

Ebola virus VP35 inhibits alpha/beta interferon production and functions as a viral polymerase cofactor. Previously, the 8-kDa cytoplasmic dynein light chain (LC8) was demonstrated to interact with VP35, but the functional consequences were unclear. Here we demonstrate that the interaction is direct and of high affinity and that binding stabilizes the VP35 N-terminal oligomerization domain and enhances viral RNA synthesis. Mutational analysis demonstrates that VP35 interaction is required for the functional effects of LC8.

FIG 1

LC8 interacts with EBOV VP35 protein. (A) The N terminus of VP35 is sufficient to interact with LC8. A co-IP experiment was performed with FLAG-tagged WT VP35, VP35-N (containing amino acids [aa] 1 to 218), VP35-C (aa 219 to 340), and HA-tagged LC8 (HA-LC8). VP35 proteins were pulled down by anti-FLAG antibody, and protein expression was detected by Western blotting (immunoblotting [IB]). WCE, whole-cell extracts. (B) VP35 mediates the interaction with LC8 via an LC8 binding consensus motif (K/SXTQT). VP35 mutants (the T73A and Q74A mutants) were utilized in a co-IP experiment with HA-tagged LC8. Protein expression was detected by Western blotting with anti-VP35 and anti-HA antibodies. (C) SDS-PAGE gel of MBP-VP35 50–150 Q74A (lane 1), MBP-VP35 50–150 (lane 2), MBP-LC8 (lane 3), and MBP (lane 4) used in SEC-MALS and ITC experiments. (D) VP35 forms a 1:1 complex with LC8. Elution profiles of MBP-LC8 (blue), MBP-VP35 50–150 (black), MBP-VP35 50–150/MBP-LC8 complex (green), and MBP (inset) from a Superdex 200 column were obtained and analyzed by SEC-MALS. The theoretical monomeric molecular masses for MBP-VP35 50–150 and MBP-LC8 are 55.2 and 54.4 kDa, respectively. (E) ITC data for MBP-LC8 binding to MBP-WT VP35 50–150 (left), mutant MBP-VP35 50–150 Q74A (center), and mutant MBP-VP35 50–150 Q72A Q74A (right). Representative raw data and binding isotherms are shown, and the reported values for K_D and n are averages from at least two independent experiments.

FIG 2

LC8 binding enhances the stability of the N terminus of VP35. (A) SDS-PAGE gel of VP35 50–150 (lane 1) and LC8 (lane 2) used in circular dichroism (CD) experiments. Lane M, molecular mass markers. (B) CD wavelength scans for WT VP35 50–150 (black lines) and the VP35 50–150 Q72A Q74A mutant (red lines) at the temperatures indicated. Mean residue ellipticity is in 10³ degrees · cm² · dmol⁻¹. (C) Thermal denaturation (T_m) of WT VP35 50–150 (black, left axis; T_m = 63.8 ± 0.1°C), the VP35 50–150 Q72A Q74A mutant (red, right axis; T_m = 63.8 ± 1°C), and LC8 (inset, blue; T_m = 66.3 ± 2°C) monitored at 222 nm. (D) Thermal denaturation of the WT VP35 50–150/LC8 complex (black, left axis; T_m = 70.8 ± 1°C) and the VP35 50–150 Q72A Q74A/LC8 mutant complex (red, right axis; T_m = 66.4 ± 2°C) monitored at 222 nm. Reported T_ms are averages of results from at least two independent experiments.

FIG 3

LC8 enhances EBOV RNA synthesis in a dose-dependent manner. (A) Increasing amounts of HA-tagged LC8 plasmid (50, 500 ng) were cotransfected with the plasmids required to reconstitute the EBOV RNA polymerase complex (L, NP, VP35, VP30) along with a plasmid encoding the Renilla luciferase minigenome RNA and a firefly luciferase expression plasmid, which served as a control for transfection efficiency. Relative activity was determined by normalizing Renilla luciferase activity to firefly luciferase activity. The error bars indicate standard deviations from three independent replicates. *, P = 0.04; **, P = 0.03, as determined by Student's t test. The Western blot shows expression of β-tubulin, VP35, and LC8 (anti-HA antibody). (B) A minigenome experiment similar to that described for panel A was performed except that WT VP35 or the indicated VP35 mutants were used, and the LC8 plasmid amount was kept constant at 500 ng. The error bars indicate standard deviations from three independent replicates. The Western blot shows the expression of VP35, LC8, and β-tubulin, detected with anti-VP35, anti-HA, and anti-β-tubulin antibodies, respectively. *, P = 0.01; **, P = 0.006, as determined by Student's t test.

FIG 4

LC8 expression enhances EBOV transcription. (A) A minigenome assay was performed as described for Fig. 2A, except that a replication-deficient minigenome construct was used. *, P = 0.01; **, P = 0.0045, as determined by Student's t test. (B) A minigenome experiment similar to that described for panel A was performed except that WT VP35 or the indicated VP35 mutants were included and the LC8 plasmid amount was kept constant at 500 ng. The error bars indicate standard deviations from three independent replicates. The Western blot shows the expression of VP35 and LC8, detected with anti-VP35 and anti-HA antibodies, respectively. *, P = 0.007; **, P = 0.002, as determined by Student's t test.