The ubiquitin system

The ubiquitin system. replication. Previous study showed that avibirnavirus particles could be engulfed into the autophagosome and degradation of virus particles took apart. Selective autophagy is a highly specific and regulated degradation pathway for the clearance of damaged or unwanted cytosolic components and superfluous organelles as well as invading microbes. However, whether and how selective autophagy removes avibirnavirus Fmoc-PEA capsids is largely unknown. Here, we have shown that selective autophagy specifically clears ubiquitinated avibirnavirus protein VP2 by p62 recognition and that p62 is an inhibitor of avibirnavirus replication, highlighting the role of p62 as a potential drug target for mediating the removal of ubiquitinated virus components from cells. family (22, 23) and causes a highly contagious and immunosuppressive disease in young chickens. IBDV is a nonenveloped virus and contains two segments of double-stranded RNAs, including segment A and segment B (24, 25). The smaller segment B encodes VP1, the RNA-dependent RNA polymerase (26, 27). The larger segment A encodes VP5 and the polyprotein pVP2-VP4-VP3, which is autoproteolytically processed to generate precursor VP2 (pVP2), the viral protease VP4, and a major structural protein, VP3, through the proteolytic activity of VP4 (28, 29). Subsequently, pVP2 is further cleaved at its C-terminal end to the mature VP2 along with four short peptides (30, 31). The IBDV capsid is a single-layer capsid composed of VP2 on the outer surface and VP3 on the inner Fmoc-PEA surface (24, 32). VP2, as an avibirnavirus capsid, plays a key role in virus adsorption, virus invasion, and the induction of host immune response (33). Critical amino acid mutations in VP2 result in immune escape from vaccination against avibirnavirus (34,C36). The first link between autophagy and avibirnavirus was discovered by electron microscopy visualization in 1976, when the degradation of IBDV within autophagic vacuoles was observed (37). Since then, increasing evidence has demonstrated that avibirnavirus protein VP2 induces autophagy upon virus entry (33). However, Plau whether autophagic induction degrades VP2 is still unclear. Accordingly, in the present study we demonstrate that the avibirnavirus capsid protein VP2 is efficiently ubiquitinated at lysine residue 411 (K411). Thereafter, p62 recognizes the K411-ubiquitinated VP2 and delivers it to autophagosomes for degradation. Thus, our study highlights the role of p62 in mediating selective autophagy as a means to remove cytoplasmic pathogens. RESULTS The avibirnavirus capsid protein VP2 undergoes autophagic degradation. A previous study demonstrated that the degradation of IBDV particles in autophagic Fmoc-PEA vacuoles and the binding of viral protein VP2 to HSP90AA1 activate autophagy by the AKT-MTOR pathway in the early stage of infection (33). Accordingly, we investigated whether VP2, the major capsid protein of avibirnavirus, is subject to autophagic degradation via autophagic induction. The gene was inserted into lentivirus vector PCDH-CMV-MCS-EF1 to construct a DF-1 cell line that stably expresses VP2. Immunoblot assay and immunofluorescence assay (IFA) using anti-VP2 monoclonal antibody revealed that viral protein VP2 is successfully expressed in the VP2 stably expressed DF-1 cells (Fig. 1A and ?andB).B). Cell viability assays showed that the activity of the VP2 stably expressed cells is not influenced significantly compared to that of wild-type (WT) DF-1 cells (Fig. 1C). Subsequently, we tested the levels of VP2 in VP2 stably expressed DF-1 cells and 293T cells transiently transfected with Flag-VP2 in the presence or absence of an autophagy inducer or inhibitor. Western blot assays (WB) showed that the autophagy inhibitors wortmannin (Wort) and chloroquine (CQ) significantly increase the level of VP2, while the autophagy activators rapamycin (Rapa) and Earles balanced salt solution (EBSS) markedly decrease the level of VP2 (Fig. 1D to ?toG).G). Similarly, the level of VP2 was Fmoc-PEA also measured in avibirnavirus-infected cells treated with Fmoc-PEA autophagy inducer or inhibitor. The results showed that CQ or Wort treatment inhibited the.