Zekun Yang, Haixia Feng, Qiongjie Wang, Chengwei Xiang, Yin Han, Fei Xu, Mengyao Jing, Hongmei Li, Jianhua Liang, Xianming He, Ruiai Chen, Ting Xiong
Regulation of Duck Tembusu virus (DTMUV) non-structural protein 1 (NS1) remains unclear. Exogenous and endogenous co-immunoprecipitation (Co-IP) assays demonstrated an association between the T-complex protein 1 subunit 2 (CCT2) and NS1. Further experiments mapped the NS1 region required for assembly of this complex to amino acid residues 172-352 of NS1.DTMUV increased CCT2 expression; CCT2 overexpression increased NS1 accumulation, whereas CCT2 knockdown delayed NS1 and envelope (E) protein accumulation. Although NS1 also associated with chaperonin-containing T-complex protein 1 subunit 6 (CCT6), CCT6 knockdown did not significantly alter NS1 messenger RNA (mRNA) or clearly change NS1 protein abundance. NS1 underwent lysine 48 (K48)- and lysine 63 (K63)-linked polyubiquitination. Increasing tripartite motif-containing protein 21 (TRIM21) reduced NS1 abundance and enhanced NS1-associated ubiquitin signals; the proteasome inhibitor MG132 promoted NS1 accumulation, which was highest with CCT2 co-expression, supporting proteasome-related turnover and CCT2-associated stabilization without establishing direct TRIM21 ubiquitination. CCT2 interacted with microtubule-associated protein 1 light chain 3C (LC3C), and DTMUV infection was associated with reduced late-stage lipidated LC3 (LC3-II) abundance and SQSTM1 mRNA expression. In dose-gradient Co-IP experiments, increasing NS1 expression was associated with a dose-associated reduction in CCT2 recovery in LC3C immunoprecipitates; the concomitant reduction in immunoprecipitated LC3C across the NS1 dose gradient precludes conclusions regarding direct competition or a binding capacity-dependent mechanism. These findings indicate that CCT2 is a DTMUV-associated host factor capable of stabilizing intracellular NS1 protein and promoting the production of infectious DTMUV progeny. CCT2 contributes to the homeostasis of NS1 protein.