NS1 of Duck Tembusu Virus Interacts with CCT2: Reduced CCT2 Recovery in LC3C Co-Immunoprecipitation In Vitro and the Proviral Role of CCT2 in DTMUV Infection.

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.