Apple necrotic mosiac virus (ApNMV) is a newly identified causal agent that is highly associated with the occurrence of apple mosaic disease in China. However, resistance gene against this virus has not been identified yet. We reported here that nitrate treatment destablized viral protein 1a via the ubiquitin-proteasome pathways to restrain ApNMV genomic RNA accumulation. A nitrate-responsive BTB/TAZ domain-containing protein MdBT2 was identified in a yeast-two-hybrid screening of apple cDNA library using viral protein 1a as bait, and 1a was confirmed to interact with MdBT2 both in vivo and in vitro. MdBT2 was further verified to promote the ubiquitination and degradation of viral protein 1a through the proteasome pathways in a MdCUL3A-scaffold protein in E3 ligase complex-independent manner. Viral genomic RNA accumulation was decreased in MdBT2 overexpression transgenic apple leaves but enhanced in MdBT2 antisense leaves compared to that in wild type. Moreover, MdBT2 was found to interfere with the interactions between viral replication proteins 1a and 2apol by competing with the latter. Taken together, our work demonstrated that nitrate-inducible MdBT2 functioned as a limiting factor in ApNMV viral RNA accumulation by promoting the ubiquitination and degradation of viral protein 1a and interfering with the interactions between viral replication proteins.