The host cell ubiquitin ligase protein CHIP is a potent suppressor of HIV-1 replication.

HIV-1 Tat is degraded in the host cell both by proteasomal and lysosomal pathways, but the specific molecules that engage with Tat from these pathways are not known. Since E3 ubiquitin ligases are the primary determinants of substrate specificity within the ubiquitin dependent proteasomal degradation of proteins, we first sought to identify the E3 ligase associated with Tat degradation. Based on the intrinsic disordered nature of Tat protein we focused our attention on host cell E3 ubiquitin ligase CHIP (carboxy terminus of Hsp70-binding protein). Co-transfection of Tat with a CHIP expressing plasmid decreased the levels of Tat protein in a dose-dependent manner, without affecting the corresponding mRNA levels. Additionally, the rate of Tat protein degradation as measured by cycloheximide (CHX) chase assay was increased in presence of CHIP. A CHIP mutant lacking the U-box domain which is responsible for protein ubiquitination (CHIP∆U-box) was unable to degrade Tat protein. Furthermore, CHIP promoted ubiquitination of Tat by both wild type as well as lysine-K48 ubiquitin which has only a single lysine residue at 48 position. CHIP transfection in HIV-1 reporter TZM-bl cells resulted in decreased Tat-dependent HIV-1 long terminal repeat (LTR) promoter transactivation as well as HIV-1 virion production. CHIP knockdown in HEK-293T cells using CRISPR-Cas9 led to higher virion production and enhanced Tat-mediated HIV-1 LTR promoter transactivation, along with stabilization of Tat protein. Together, these results suggest a novel role of host cell E3 ubiquitin ligase protein CHIP in regulating HIV-1 replication through ubiquitin dependent degradation of its regulatory protein Tat.