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Background: APOBEC3G (A3G) and related cytidine deaminases of the APOBEC3 family of proteins are potent inhibitors of many retroviruses, including HIV-1. Formation of infectious HIV-1 requires the suppression of multiple cytidine deaminases by Vif. HIV-1 Vif suppresses various APOBEC3 proteins through the common mechanism of recruiting the Cullin5-ElonginBElonginC E3 ubiquitin ligase to induce target protein polyubiquitination and proteasome-mediated degradation. The domains in Vif and various APOBEC3 proteins required for APOBEC3 recognition and degradation have not been fully characterized. Methods and Findings: In the present study, we have demonstrated that the regions of APOBEC3F (A3F) that are required for its HIV-1-mediated binding and degradation are distinct from those reported for A3G. We found that the C-terminal cytidine deaminase domain (C-CDD) of A3F alone is sufficient for its interaction with HIV-1 Vif and its Vif-mediated degradation. We also observed that the domains of HIV-1 Vif that are uniquely required for its functional interaction with full-length A3F are also required for the degradation of the C-CDD of A3F; in contrast, those Vif domains that are uniquely required for functional interaction with A3G are not required for the degradation of the C-CDD of A3F. Interestingly, the HIV- 1 Vif domains required for the degradation of A3F are also required for the degradation of A3C and A3DE. On the other hand, the Vif domains uniquely required for the degradation of A3G are dispensable for the degradation of cytidine deaminases A3C and A3DE. Conclusions: Our data suggest that distinct regions of A3F and A3G are targeted by HIV-1 Vif molecules. However, HIV-1 Vif suppresses A3F, A3C, and A3DE through similar recognition determinants, which are conserved among Vif molecules from diverse HIV-1 strains. Mapping these determinants may be useful for the design of novel anti-HIV inhibitors. [ABSTRACT FROM AUTHOR]

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 transactivator 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 than a dominant-negative ubiquitin mutant (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, which 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 to Tat downregulation. Thus, in this study, we report a novel role of the deubiquitinase, USP21, in HIV-1 infection. USP21 represents a potentially useful target for the development of novel anti-HIV drugs. [ABSTRACT FROM AUTHOR]

NF-ĸB-interacting long noncoding RNA (NKILA) was recently identified as a negative regulator of NF-ĸB signaling and plays an important role in the development of various cancers. It is well known that NF-ĸB-mediated activation of human immunodeficiency virus type 1 (HIV-1) long terminal repeat (LTR)-driven gene expression is required for HIV-1 transcription and reactivation of latency. However, whether NKILA plays essential roles in HIV-1 replication and latency is unclear. Here, by ectopic expression and silencing experiments, we demonstrate that NKILA potently inhibits HIV-1 replication in an NF-ĸB-dependent manner by suppressing HIV-1 LTR promoter activity. Moreover, NKILA showed broad-spectrum inhibition on the replication of HIV-1 clones with different coreceptor tropisms as well as on LTR activity of various HIV-1 clinical subtypes. Chromatin immunoprecipitation (ChIP) assays revealed that NKILA expression abolishes the recruitment of p65 to the duplicated ĸB binding sites in the HIV-1 LTR. NKILA mutants disrupting NF-ĸB inhibition also lost the ability to inhibit HIV-1 replication. Notably, HIV-1 infection or reactivation significantly downregulated NKILA expression in T cells in order to facilitate viral replication. Downregulated NKILA was mainly due to reduced acetylation of histone K27 on the promoter of NKILA by HIV-1 infection, which blocks NKILA expression. Knockdown of NKILA promoted the reactivation of latent HIV-1 upon phorbol myristate acetate (PMA) stimulation, while ectopic NKILA suppressed the reactivation in a wellestablished clinical model of withdrawal of azidothymidine (AZT) in vitro. These findings improve our understanding of the functional suppression of HIV-1 replication and latency by NKILA through NF-ĸB signaling. [ABSTRACT FROM AUTHOR]

The HIV-1 reservoir is a major obstacle to complete eradication of the virus. Although many proteins and RNAs have been characterized as regulators in HIV-1/AIDS pathogenesis and latency, only a few long noncoding RNAs (lncRNAs) have been shown to be closely associated with HIV-1 replication and latency. In this study, we demonstrated that lncRNA uc002yug.2 plays a key role in HIV-1 replication and latency. uc002yug.2 potentially enhances HIV-1 replication, long terminal repeat (LTR) activity and the activation of latent HIV-1 in both cell lines and CD4+ T cells from patients. Further investigation revealed that uc002yug.2 activates latent HIV-1 through downregulating RUNX1b and -1c and upregulating Tat protein expression. The accumulated evidence supports our model that the Tat protein has the key role in the uc002yug.2-mediated regulatory effect on HIV-1 reactivation. Moreover, uc002yug.2 showed an ability to activate HIV-1 similar to that of suberoylanilide hydroxamic acid or phorbol 12-myristate 13-acetate using latently infected cell models. These findings improve our understanding of lncRNA regulation of HIV-1 replication and latency, providing new insights into potential targeted therapeutic interventions. IMPORTANCE The latent viral reservoir is the primary obstacle to curing HIV-1 disease. To date, only a few lncRNAs, which play major roles in various biological processes, including viral infection, have been identified as regulators in HIV-1 latency. In this study, we demonstrated that lncRNA uc002yug.2 is important for both HIV-1 replication and activation of latent viruses. Moreover, uc002yug.2 was shown to activate latent HIV-1 through regulating alternative splicing of RUNX1 and increasing the expression of Tat protein. These findings highlight the potential merit of targeting lncRNA uc002yug.2 as an activating agent for latent HIV-1. [ABSTRACT FROM AUTHOR]

BST-2/tetherin blocks the release of various enveloped viruses including HIV-1 with a "physical tethering" model. The detailed contribution of N-linked glycosylation to this model is controversial. Here, we confirmed that mutation of glycosylation sites exerted an effect of post-translational mis-trafficking, leading to an accumulation of BST-2 at intracellular CD63-positive vesicles. BST-2 with this phenotype potently inhibited the release of multivesicular body-targeted HIV-1 and hepatitis B virus, without affecting the co-localization of BST-2 with EEA1 and LAMP1. These results suggest that N-linked glycosylation of human BST-2 is dispensable for intracellular virion retention and imply that this recently discovered intracellular tethering function may be evolutionarily distinguished from the canonical antiviral function of BST-2 by tethering nascent virions at the cell surface. [ABSTRACT FROM AUTHOR]

The human immunodeficiency virus type 1 (HIV-1)-encoded virion infectivity factor (Vif) is required to inactivate the host restriction factor APOBEC3 by engaging Cullin 5 (Cul5)-RING ubiquitin ligase (CRL5). Core binding factor beta (CBF-β) is a novel regulator of Vif-CRL5 function; as yet, its mechanism of regulation remains unclear. In the present study, we demonstrate that CBF-β promotion of Vif-CRL5 assembly is independent of its influence on Vif stability and is also a conserved feature of primate lentiviral Vif proteins. Furthermore, CBF-β is critical for the formation of the Vif-ElonginB/ElonginC-Cul5 core E3 ubiquitin ligase complex in vitro. CBF-β from diverse vertebrate species supported HIV-1 Vif function, indicating the conserved nature of Vif-CBF-β interfaces. Considering the importance of the interaction between Vif and CBF-β in viral CRL5 function, disrupting this interaction represents an attractive pharmacological intervention against HIV-1. [ABSTRACT FROM AUTHOR]

The human immunodeficiency virus type 1 (HIV-1)-encoded virion infectivity factor (Vif) is required to inactivate the host restriction factor APOBEC3 by engaging Cullin 5 (Cul5)-RING ubiquitin ligase (CRL5). Core binding factor beta (CBF-β) is a novel regulator of Vif-CRL5 function; as yet, its mechanism of regulation remains unclear. In the present study, we demonstrate that CBF-β promotion of Vif-CRL5 assembly is independent of its influence on Vif stability and is also a conserved feature of primate lentiviral Vif proteins. Furthermore, CBF-β is critical for the formation of the Vif-ElonginB/ElonginC-Cul5 core E3 ubiquitin ligase complex in vitro. CBF-β from diverse vertebrate species supported HIV-1 Vif function, indicating the conserved nature of Vif-CBF-β interfaces. Considering the importance of the interaction between Vif and CBF-β in viral CRL5 function, disrupting this interaction represents an attractive pharmacological intervention against HIV-1. IMPORTANCE HIV-1 encodes virion infectivity factor (Vif) to inactivate its host's antiviral APOBEC3 proteins. Vif triggers APOBEC3 degradation by forming Vif-Cullin 5 (Cul5)-RING ubiquitin ligase (CRL5). Core binding factor beta (CBF-β) is a novel regulator of Vif- CRL5 function whose mechanism of regulation remains poorly defined. In the present study, we demonstrate that the promotion of Vif-CRL5 assembly by CBF-β can be separated from its influence on Vif stability. The promotion of Vif-CRL5 assembly, but not the influence on Vif stability, is conserved among primate lentiviral Vif proteins: we found that CBF-β from diverse vertebrate species supported HIV-1 Vif function. Considering the importance of the interaction between Vif and CBF-β in viral CRL5 function and HIV-1 replication, disrupting this interaction is an attractive strategy against HIV-1. [ABSTRACT FROM AUTHOR]

Core binding factor beta (CBFß), a transcription regulator through RUNX binding, was recently reported critical for Vif function. Here, we mapped the primary functional domain important for Vif function to amino acids 15 to 126 of CBFß. We also revealed that different lengths and regions are required for CBFß to assist Vif or RUNX. The important interaction domains that are uniquely required for Vif but not RUNX function represent novel targets for the development of HIV inhibitors. [ABSTRACT FROM AUTHOR]