Deubiquitinases (DUBs) are key regulators of complex cellular processes. HIV-1 Tat is synthesized early after infection and is mainly responsible for enhancing viral production. Here, we report that one of the DUBs, USP7, stabilized the HIV-1 Tat protein through its deubiquitination. Treatment with either a general DUB inhibitor (PR-619) or USP7-specific inhibitor (P5091) resulted in Tat protein degradation. The USP7-specific inhibitor reduced virus production in a latently infected T-lymphocytic cell line J1.1, which produces large amounts of HIV-1 upon stimulation. A potent increase in Tat-mediated HIV-1 production was observed with USP7 in a dose-dependent manner. As expected, deletion of the USP7 gene using the CRISPR-Cas9 method reduced the Tat protein and supported less virus production. Interestingly, the levels of endogenous USP7 increased after HIV-1 infection in human T-cells (MOLT-3) and in mammalian cells transfected with HIV-1 proviral DNA. Thus, HIV-1 Tat is stabilized by the host cell deubiquitinase USP7, leading to enhanced viral production, and HIV-1 in turn up-regulates the USP7 protein level.

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Edited by Charles E. Samuel Human immunodeficiency virus-1 (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. Because 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 (C terminus of HSP70-binding protein). Co-transfection of Tat with a CHIPexpressing plasmid decreased the levels of Tat protein in a dosedependent manner, without affecting the corresponding mRNA levels. Additionally, the rate of Tat protein degradation as measured by cycloheximide (CHX) chase assay was increased in the 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 WT as well as Lys-48ubiquitin, which has only a single lysine residue at position 48. 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.

Hepatitis B virus (HBV) genomic mutations A1762T, G1764A and AG1762/1764TA cause production of HBV X protein (HBx) mutants, namely K130M, V131I and KV130/131MI. These mutations are important biomarkers for the development of cirrhosis and hepatocellular carcinoma (HCC) in chronic HBV patients. This study comparatively analyses the impact of intracellular expression of HBx mutants on HCC cell line Huh7. It was found that expression of KV130/131MI induced: cell proliferation, altered expression of cell cycle regulatory genes in favour of cell proliferation, intracellular reactive oxygen species (ROS) production and mitochondrial depolarization. KV130/131MI may be directly involved in host cell proliferation and hepatocarcinogenesis via altering expression of cell cycle regulatory genes. KV130/131MI may also play pivotal roles in fibrosis and cirrhosis via inducing ROS production and mitochondrial depolarization. Furthermore, these might be the possible reasons for higher occurrence of AG1762/1764TA as compared to A1762T and G1764A in cirrhosis and HCC patients.