The VSV M protein interacts with Ndufaf4
To identify potential cellular proteins that interact with the VSV M protein, yeast two-hybrid screening was performed using the VSV M protein as a bait against a normalized HeLa cDNA library. Putative positive clones were isolated and sequenced. Among these clones, Ndufaf4 was identified as a potential cellular partner of VSV M. The M-Ndufaf4 interaction was confirmed by yeast cotransformation assay. All the yeasts cotransformed with the indicated constructs grew well on SD/–2 medium, but only BD-M/AD-Ndufaf4 and BD-p53/AD-T (the positive control) grew on SD/–4 medium (Fig. 1A). Therefore, Ndufaf4 may be a binding partner of VSV M.
To further confirm the M-Ndufaf4 interaction, a GST pulldown assay was performed with purified GST-tagged M protein expressed in E. coli and Flag-tagged Ndufaf4 protein expressed in BSR-T7/5 cells. The presence of GST-M, but not GST alone, resulted in an interaction with Flag-Ndufaf4 (Fig. 1 B). Purified GST-Ndufaf4 protein expressed in E. coli and Flag-M expressed in BSR-T7/5 cells were also used in the pulldown assay. The results showed that GST-Ndufaf4, but not GST, interacted with Flag-M (Fig. 1C). Overall, these results indicated that M interacts with Ndufaf4.
Identification of critical amino acids in the M protein involved in the M-Ndufaf4 interaction
To determine the critical regions within the M protein that mediate its interaction with the Ndufaf4 protein, the carboxy-terminal globular domain and amino-terminal flexible domain of M were individually fused to the DNA-binding domain of GAL4, and the ability of the fusion proteins to interact with the Ndufaf4 protein was assessed using the Y2H system. Yeast transformed with BD-M/AD-Ndufaf4 and BD-M2/AD-Ndufaf4 grew well on SD/–2 and SD/–4 media; however, yeast transformed with BD-M1/AD-Ndufaf4 did not grow on SD/–4 medium (Fig. 2 A). The above results showed that the carboxy-terminal globular domain of M participates in the M-Ndufaf4 interaction.
To further identify the critical residues in the M protein involved in the M-Ndufaf4 interaction, all amino acids at the surface of the globular domain of the M protein were chosen for mutagenesis analysis [18, 19]. As shown by the results, all cells expressing the mutants grew as well as cells expressing wild-type M on SD/–4 medium, except for cells expressing E156A, E156A/H157A, or D180A/E181A M (Fig. 2 B). These results indicate that the E156/H157 and D180/E181 amino acids in M are critical for the M-Ndufaf4 interaction.
Ndufaf4 inhibits VSV replication
To investigate the role of Ndufaf4 in VSV propagation, HeLa cells were transfected with pCMV-Flag-Ndufaf4 or pCMV-Flag-N empty vector and then infected with VSV at an MOI of 0.1. VSV G protein levels were detected by Western blotting, and the VSV titer was determined by TCID50 assay at 4, 8 and 12 hours postinfection. As shown by the results, overexpression of Ndufaf4 decreased expression of the VSV G protein at 12 hours postinfection and the VSV titer at 8 and 12 hours postinfection (Fig. 3 A and B). These results show that overexpression of Ndufaf4 inhibits the growth of VSV.
To further analyze the effect of Ndufaf4 knockdown on VSV replication, HeLa cells were transfected with plasmid to deliver short hairpin RNA (shRNA) targeting the Ndufaf4 gene. Western blot analyses demonstrated that the specific shRNA efficiently decreased the expression level of Ndufaf4 (Fig. 3 C). Furthermore, transfection of HeLa cells with the plasmid resulted in the efficient knockdown of Ndufaf4 expression, which promoted expression of the G protein and the VSV titer (Fig. 3 D and E). Collectively, these results suggest that Ndufaf4 is a cellular antiviral protein against VSV replication.
The anti-VSV effect of Ndufaf4 is independent of the type I IFN response
To further explore the mechanism exploited by Ndufaf4 to affect VSV replication, we detected the effect of Ndufaf4 overexpression or knockdown on the type I IFN response. HeLa cells were transfected with Ndufaf4-overexpression or Ndufaf4-knockdown plasmid and then infected with VSV at an MOI of 5. The mRNA levels of IFN-βand the IFN-stimulated genes ISG56, IFITM3 and MX1 were detected by by real-time RT-PCR at 4, 8, 12 hours post-infection. Compared with their transcription in the control group, neither the overexpression nor knockdown of Ndufaf4 affected the transcription of these genes (Fig. 4 A and B). These results indicated that Ndufaf4 inhibits VSV replication independent of the type I IFN response.