Previous studies showed that full-length P protein and at least four additional shorter products P2 (PΔN19), P3(PΔN52), P4(PΔN68), and P5 were detected in RABV-infected cells, viral gene P transfected cells, and purified RABV virions [13]. BECN1 plays an interacting partner role for the mammalian phosphatidylinositol 3kinase catalytic subunit type 3(PIK3C3) involving macroautophagy, in which it is an essential chaperone or adaptor [17–19]. However, in the relationship between the virus and autophagy, although it has been reported that BECN1 interacts with a virus protein to regulate autophagy, the specific domain responsible for the BECN1 interaction is not clear[20, 21]. The present study showed that BECN1 exists in a ring-like structure, and identified that among five truncated P proteins (PΔC75, PΔN19, PΔN52, PΔN68, and P5), residues 173–222 induced autophagy by interacting with N-terminal residues 1–139 of BECN1. Meanwhile, only the full-length P protein and P5 were visibly colocalized with the BECN1 ring-like structure (Fig. 4). Notably, in co-IP experiments, P5 showed stronger binding than full length P protein. Therefore, we concluded that RABV small phosphoprotein P5 is responsible for binding to the BECN1 ringlike structure.
As an essential cofactor of RABV RNA polymerase, P may participate in additional physiological processes [22]. Our previous research reported incomplete autophagy induced by the RABV phosphoprotein [9]. In this study, we demonstrated that the P proteins with, but not without, amino acid segment 173–222 are involved in increasing the level of endogenous lipidated LC3-II. In particular, the LC3-II was surrounded by the P5 protein (Figs. 1 and 2). However, P5 did not change the levels of autophagy associated proteins ATG5, ATG7, ULK1, BECN1, and P62, markedly. In addition, the P5-induced autophagosome did not colocalize with lysosomes (Figs. 1 and 3). Nonetheless, we observed that P5 upregulated the phosphorylation of AMPK, MAPK (P38, ERK), AKT, and MTOR, and decreased BECN1-dependent CASP2 levels (Fig. 6). Collectively, our data demonstrated that amino acid residues 173–222 of the viral P protein are responsible for inducing incomplete autophagy, and the binding of P5 to the BECN1 ring-like structure induced this incomplete autophagy by activating the CASP2 signaling pathway.
Autophagy can remove intracellular pathogens, including bacteria and viruses, by activating various cellular defense responses, including direct digestion of intracytoplasmic virions [20, 23], recognition of pathogen-associated molecular patterns through the delivery of viral genomes to endosomal toll-like receptors [24], activation of innate immune signaling [25], and regulation of the inflammatory response [26–30]. However, many viruses also subvert the autophagic machinery to enhance viral replication [31–36]. In this study, we demonstrated that P5 overexpression increased the level of viral N protein, viral N mRNA, viral anti-genomic RNA, and infectious RABV progeny, and these indexes were significantly inhibited in presence of Flag-P5 and knockdown of Becn1 (Fig. 5). In addition, we also demonstrated that the P5 still increased the RABV replication when autophagy was inhibited (Fig. 5). These results suggest that RABV replication is regulated by the binding of P5 to the BECN1 ring-like structure.