Deubiquitinating Enzyme USP21 Inhibits HIV-1 Replication by Downregulating Tat Expression

Abstract

Ubiquitination plays an important role in human immunodeficiency virus-1 (HIV-1) infection. HIV proteins such as Vif and Vpx mediate the degradation of the host proteins APOBEC3 and SAMHD1, respectively, through the proteasome pathway. However, whether deubiquitylating enzymes play an essential role in HIV-1 infection is largely unknown. Here, we demonstrate that the deubiquitinase, USP21, potently inhibits HIV-1 production by indirectly downregulating the expression of HIV-1 trans-activator of transcription (Tat), which is essential for transcriptional elongation in HIV-1. USP21 deubiquitylates Tat via its deubiquitinase activity, but a stronger ability to reduce Tat expression compared to Ub-KO showed that other mechanisms may contribute to USP21-mediated inhibition of Tat. Further investigation showed that USP21 downregulates cyclin T1 mRNA levels by increasing methylation of histone K9 in the promoter of cyclin T1, a subunit of the positive transcription elongation factor b (P-TEFb) that interacts with Tat and transactivation response element (TAR) and is required for transcription stimulation and Tat stability. Moreover, USP21 had no effect on the function of other HIV-1 accessory proteins, including Vif, Vpr, Vpx, and Vpu, indicating that USP21 was specific to Tat. These findings improve our understanding of USP21-mediated functional suppression of HIV-1 production. Importance Ubiquitination plays an essential role in viral infection. Deubiquitinating enzymes (DUBs) reverse ubiquitination by cleaving ubiquitins from target proteins, thereby affecting viral infection. The role of the members of the USP family, that comprises the largest subfamily of DUBs, is largely unknown in HIV-1 infection. Here, we screened a series of USP members and found that USP21 inhibits HIV-1 production by specifically targeting Tat, but not the other HIV-1 accessory proteins. Further investigations revealed that USP21 reduces Tat expression in two ways. First, USP21 deubiquitinates polyubiquitinated Tat causing Tat instability, and second USP21 reduces the mRNA levels of cyclin T1 (CycT1), an important component of P-TEFb, that leads 56 to Tat downregulation. Thus, in this study, we report a novel role of the deubiquitinase, USP21, in HIV-1 viral infection. USP21 represents a potentially useful target for the development of novel anti-HIV drugs.

The Deubiquitinase USP29 Promotes SARS-CoV-2 Virulence by Preventing Proteasome Degradation of ORF9b

“…USP29-HA C294A was generated from USP29-HA using site-directed mutagenesis. Ub-Flag/Myc ( 10 ), RIG-I(N)-Myc ( 26 ), MAVS, IFN-β-Luc, NF-κB-Luc, and Renilla ( 27 ) have been previously described. The SARS-CoV-2 GFP/ΔN (Wuhan-Hu-1, MN908947, Clade:19A) expression plasmid was constructed by replacing the regions encoding viral N (from nucleotide positions 28274 to 29533) based on the MN908947 genome with the GFP reporter gene.…”

“…HIV-1 is a well-studied pathogen that manipulates the host's ubiquitin network to antagonize host restriction factors ( 5 – 9 ). In contrast, the host also utilizes the ubiquitin-proteasome system (UPS) to destabilize viral proteins such as deubiquitination by the deubiquitinating enzyme (DUB) USP21 of the HIV-1 protein Tat, which leads to Tat instability ( 10 ). Recent reports have shown that the E3 ubiquitin ligases RNF5 and CRL2 (ZYG11B) are recruited by membrane protein and ORF10 protein of SARS-CoV-2, respectively; However, CRL2 is dispensable for SARS-CoV-2 infection ( 11 , 12 ).…”

Insights Into the Properties, Biological Functions, and Regulation of USP21

“…Although two studies found that USP21 maintained the protein level of EZH2, the enzymatic catalytic subunit of the polycomb repressive complex 2 that can modulate gene expression by trimethylating K27 on histone 3 (H3K27), in which the impacts of USP21 on H3K27me3 level were not tested ( Chen et al, 2017b ; Ma et al, 2021 ). Reciprocally, USP21 expression dramatically elevated H3K9 methylation without affecting H3K4 methylation or H3K27 acetylation on the promoter of cyclin T1, which resulted in decreased cyclin T1 expression ( Gao et al, 2021 ). Whether this correlates with the hydrolysis of H2Aub remaines unclear.…”

“…The recent identification of USP21 as the DUB responsible for the stabilization of AIM2 and immediate AIM2 inflammasome activation upon DNA stimulation further complicates the role of USP21 in regulating DNA-mediated innate immunity ( Hong et al, 2021 ). Moreover, another study indicated that USP21 inhibits human immunodeficiency virus 1 (HIV-1) replication in vitro by downregulating the expression of transactivator of transcription (Tat), which is essential for transcriptional elongation in HIV-1 ( Gao et al, 2021 ). Collectively, these studies suggested a dual role for USP21 in anti-viral infections.…”

Deubiquitinase ubiquitin-specific protease 3 (USP3) inhibits HIV-1 replication via promoting APOBEC3G (A3G) expression in both enzyme activity-dependent and -independent manners

“…[35][36][37][38][39][40] HIV-1 is a well-studied pathogen, [41,42] and our studies and those of other groups have discovered several different modes of DUB regulation of HIV-1 replication by targeting either virus or host proteins. [43][44][45][46][47][48] For example, some DUBs have different effects on HIV-1 infectivity by targeting the HIV-1 Tat protein. Ubiquitin-specific protease 7 (USP7) promotes HIV-1 production by stabilizing Tat protein, [49] whereas our recent study showed that USP21 inhibits HIV-1 replication by downregulating Tat expression.…”