Apple mosaic disease is one of the most widely distributed and destructive diseases in apple cultivation worldwide, especially in China, whose apple yields account for more than 50% of the global total. Apple necrotic mosaic virus (ApNMV) is a newly identified ilarvirus that is closely associated with apple mosaic disease in China; however, basic viral protein interactions that play key roles in virus replication and the viral life cycle have not been determined in ApNMV. Here, we first identify an ApNMV-Lw isolate that belongs to subgroup 3 in the genus Ilarvirus. ApNMV-Lw was used to investigate interactions among viral components. ApNMV 1a and 2a pol , encoded by RNA1 and RNA2, respectively, were co-localized in plant cell cytoplasm. ApNMV 1a interacted with itself at both the inter-and intramolecular levels, and its N-terminal portion played a key role in these interactions. 1a also interacted with 2a pol , and 1a's C-terminal, together with 2a pol 's N-terminal, was required for this interaction. Moreover, the first 115 amino acids of 2a pol were sufficient for permitting the 1a-2a pol interaction. This study provides insight into the protein interactions among viral replication components of ApNMV, facilitating future investigations on its pathogenicity, as well as the development of strategies to control the virus and disease.Viruses 2020, 12, 474 2 of 15 caused by apple mosaic disease, as previous work has shown that the net photosynthetic rate of leaves with mosaic symptoms is decreased to from approximately 3% to more than 45% [8].In a long period, apple mosaic virus (ApMV), belonging to the genus Ilarvirus in the family Bromoviridae [9], was traditionally believed to be the sole causal agent of apple mosaic disease until prunus necrotic ringspot virus (PNRSV) [10,11], cucumber mosaic virus (CMV) [12], and apple necrotic mosaic virus (ApNMV) [13,14] were also reported to be associated with this disease. Specifically, 90% of the detected apple tree leaves (111 samples collected from different regions of China) with mosaic symptoms were infected with PNRSV, while ApMV was detected in none of them [11]. Furthermore, ApNMV was detected in 92.1% (268 out of 291 apple trees from different regions of China) of symptomatic trees, while ApMV was detected in none of them [14]. Moreover, the distribution of ApNMV was correlated with the symptomatic leaves, and these leaves were unevenly distributed in diseased apple trees [13]. In addition, ApNMV has also been reported to be isolated from crabapple (Malus spp.) leaves with mosaic symptoms [15]. Thus, in China, ApMV is not the only agent that can induce mosaic symptoms, as PNRSV and especially ApNMV, are also associated with apple mosaic disease. However, the induction of mosaic symptoms by ApNMV has not been verified via Koch's Postulates [16], classical criteria for directly linking a specific pathogen with its corresponding symptom.ApNMV was first reported to be associated with the apple mosaic disease by a Japanese group [13]. ApNMV, along with ApMV and PNRSV, ...