Identification of interaction between the host protein CCT5 and rabies virus M protein and the effects on viral replication

【Objective】 This study aimed to clarify the effects of the interaction between the host protein CCT5 and rabies virus （RABV） M protein and overexpressed CCT5 gene on RABV replication， so as to provide a theoretical basis for the function and mechanism of CCT5 protein in RABV replication.【Method】 The relative expressions of CCT5， M， N， P， and G genes and their proteins after RABV infection of BHK-21 and N2a cells were detected by real-time fluorescence quantitative PCR and Western blotting， respectively. The CCT5 gene was amplified by PCR and cloned into the pcDNA3.0 vector to construct a eukaryotic expression vector. After transfection into HEK-293T cells， the expression of CCT5 protein was detected， and the optimal transfection time was selected. The interaction between CCT5 protein and RABV M protein was verified by GST-pull down， Co-IP， and cellular colocalization. The effect of CCT5 gene over-expression on RABV replication.【Result】 After RABV infection of BHK-21 cells， the relative expressions of N， P， M， and G genes generally increased， while the relative expression of CCT5 gene showed an inobvious change. RABV infection of N2a cells did not result in an obvious change in the relative expression of CCT5 protein. Transfection of HEK-293T cells with eukaryotic expression vector pcDNA3.0-CCT5^-Flag resulted in the highest CCT5 relative protein expression at 36 h post-transfection. GST pull-down validation showed that CCT5 protein interacted with RABV M protein in vitro. Co-IP validation showed that CCT5 protein and RABV M protein interact intracellularly. Intracellular colocalization validation showed that CCT5 protein and RABV M protein had extensive colocalization in the cytoplasm. Overexpression of the CCT5 gene could promote RABV replication at multiple levels， including transcription， translation， and progeny virus production.【Conclusion】 The host chaperone protein CCT5 interacts with the RABV M protein both in vivo and in vitro， and the two are colocalized in the cytoplasm. RABV does not regulate the expression of the CCT5 gene and its protein in different cell lines， but the overexpressed CCT5 gene can promote the transcription and translation of the RABV gene and the production of progeny viruses， thus having a positive regulatory effect on RABV replication.