The Vpu accessory protein promotes HIV-1 release by counteracting Tetherin/BST-2, an interferon-regulated restriction factor, which retains virions at the cell-surface. Recent reports proposed β-TrCP-dependent proteasomal and/or endo-lysosomal degradation of Tetherin as potential mechanisms by which Vpu could down-regulate Tetherin cell-surface expression and antagonize this restriction. In all of these studies, Tetherin degradation did not, however, entirely account for Vpu anti-Tetherin activity. Here, we show that Vpu can promote HIV-1 release without detectably affecting Tetherin steady-state levels or turnover, suggesting that Tetherin degradation may not be necessary and/or sufficient for Vpu anti-Tetherin activity. Even though Vpu did not enhance Tetherin internalization from the plasma membrane (PM), it did significantly slow-down the overall transport of the protein towards the cell-surface. Accordingly, Vpu expression caused a specific removal of cell-surface Tetherin and a re-localization of the residual pool of Tetherin in a perinuclear compartment that co-stained with the TGN marker TGN46 and Vpu itself. This re-localization of Tetherin was also observed with a Vpu mutant unable to recruit β-TrCP, suggesting that this activity is taking place independently from β-TrCP-mediated trafficking and/or degradation processes. We also show that Vpu co-immunoprecipitates with Tetherin and that this interaction involves the transmembrane domains of the two proteins. Importantly, this association was found to be critical for reducing cell-surface Tetherin expression, re-localizing the restriction factor in the TGN and promoting HIV-1 release. Overall, our results suggest that association of Vpu to Tetherin affects the outward trafficking and/or recycling of the restriction factor from the TGN and as a result promotes its sequestration away from the PM where productive HIV-1 assembly takes place. This mechanism of antagonism that results in TGN trapping is likely to be augmented by β-TrCP-dependent degradation, underlining the need for complementary and perhaps synergistic strategies to effectively counteract the powerful restrictive effects of human Tetherin. Restriction factors are cellular proteins that interfere with the multiplication and transmission of viruses and are therefore important components of natural immunity. Tetherin (also known as BST-2) is a recently identified restriction factor that traps viruses at the cell-surface, preventing their release and thus infection of other cells. Viruses have, however, developed means to counteract this restriction factor. Viral protein U (Vpu) is an accessory protein encoded by HIV-1, the causative agent of AIDS. Vpu antagonizes Tetherin and consequently promotes the release of HIV-1 particles. A series of recent reports proposed that Vpu would induce the degradation of this restriction factor in order to overcome its anti-viral activity. Here, we report that Vpu is able to enhance HIV-1 release in absence of Tetherin degradation. Instead, we found that Vpu interacts with Tetherin and interferes with the transport of the restriction factor towards the cell-surface. This would lead to re-localization of Tetherin in an intracellular organelle called the trans-Golgi network, resulting in insufficient levels of Tetherin at the cell-surface to trap progeny viruses. This mechanism of antagonism that results in TGN trapping could be augmented by the induction of degradation to effectively counteract the powerful restrictive effects of human Tetherin. Further characterization of this mechanism will improve our understanding of host antiviral defenses as well as provide new targets for the development of novel anti-HIV drugs.

Human immunodeficiency virus type 1 (HIV-1) viral protein R (Vpr) has been shown to cause G2 cell cycle arrest in human cells by inducing ATR-mediated inactivation of p34cdc2, but factors directly engaged in this process remain unknown. We used tandem affinity purification to isolate native Vpr complexes. We found that damaged DNA binding protein 1 (DDB1), viral protein R binding protein (VPRBP), and cullin 4A (CUL4A)—components of a CUL4A E3 ubiquitin ligase complex, DDB1-CUL4A^VPRBP—were able to associate with Vpr. Depletion of VPRBP by small interfering RNA impaired Vpr-mediated induction of G2 arrest. Importantly, VPRBP knockdown alone did not affect normal cell cycle progression or activation of ATR checkpoints, suggesting that the involvement of VPRBP in G2 arrest was specific to Vpr. Moreover, leucine/isoleucine-rich domain Vpr mutants impaired in their ability to interact with VPRBP and DDB1 also produced strongly attenuated G2 arrest. In contrast, G2 arrest–defective C-terminal Vpr mutants were found to maintain their ability to associate with these proteins, suggesting that the interaction of Vpr with the DDB1-VPRBP complex is necessary but not sufficient to block cell cycle progression. Overall, these results point toward a model in which Vpr could act as a connector between the DDB1-CUL4A^VPRBP E3 ubiquitin ligase complex and an unknown cellular factor whose proteolysis or modulation of activity through ubiquitination would activate ATR-mediated checkpoint signaling and induce G2 arrest. Human immunodeficiency virus type 1 (HIV-1), the causative agent of AIDS, encodes several proteins termed accessory, which play a critical role in viral pathogenesis. Collectively, HIV-1 accessory proteins Vif, Vpr, Vpu, and Nef have been shown to manipulate host cell biology to promote viral replication, immune evasion, and AIDS progression. In this context, Vpr has been found to block normal cell growth. This interruption of cell division by Vpr is thought to increase viral replication and induce programmed cell death. However, how Vpr is able to block cell division remains unknown. To understand this mechanism, we have studied the interaction between Vpr and novel cellular proteins. We identified a novel complex comprising Vpr and cellular proteins involved in a process called ubiquitination. Ubiquitination is a mechanism by which a small protein, ubiquitin, is conjugated to cellular proteins to modulate their activity or induce their degradation. We demonstrated that association of Vpr with this ubiquitinating complex might be responsible for the defect in cell growth. Further characterization of this protein complex and the elucidation of the mechanism by which it affects cell growth might lead to the development of new anti-HIV drugs and an improved understanding of the basic cellular processes controlling cell division.

SARS-CoV-2, the etiological agent of COVID-19, has so far resulted in >6.1 million deaths worldwide. The spike protein (S) of the virus directs infection of the lungs and other tissues by binding the angiotensin-converting enzyme 2 (ACE2) receptor.

HIV up-regulates cell-surface expression of specific ligands for the activating NKG2D receptor, including ULBP-1, -2, and -3, but not MICA or MICB, in infected cells both in vitro and in vivo. However, the viral factor(s) involved in NKG2D ligand expression still remains undefined. HIV-1 Vpr activates the DNA damage/stress-sensing ATR kinase and promotes G2 cell-cycle arrest, conditions known to up-regulate NKG2D ligands. We report here that HIV-1 selectively induces cell-surface expression of ULBP-2 in primary CD4+ T lymphocytes by a process that is Vpr dependent. Importantly, Vpr enhanced the susceptibility of HIV-1–infected cells to NK cell–mediated killing. Strikingly, Vpr alone was sufficient to up-regulate expression of all NKG2D ligands and thus promoted efficient NKG2D-dependent NK cell–mediated killing. Delivery of virion-associated Vpr via defective HIV-1 particles induced analogous biologic effects in noninfected target cells, suggesting that Vpr may act similarly beyond infected cells. All these activities relied on Vpr ability to activate the ATR-mediated DNA damage/stress checkpoint. Overall, these results indicate that Vpr is a key determinant responsible for HIV-1–induced up-regulation of NKG2D ligands and further suggest an immunomodulatory role for Vpr that may not only contribute to HIV-1–induced CD4+ T-lymphocyte depletion but may also take part in HIV-1–induced NK-cell dysfunction.

Vpu promotes the efficient release of human immunodeficiency virus type 1 (HIV-1) by overcoming the activity of tetherin, a host cell restriction factor that retains assembled virions at the cell surface. In this study, we analyzed the intracellular localization and trafficking of subtype B Vpu in HIV-1-producing human cells. We found that mutations of conserved positively charged residues (R30 and K31) within the putative overlapping tyrosine- and dileucine-based sorting motifs of the Vpu hinge region affected both the accumulation of the protein in the trans -Golgi network (TGN) and its efficient delivery to late endosomal degradative compartments. A functional characterization of this mutant revealed that the mislocalization of Vpu from the TGN correlated with an attenuation of HIV-1 release. Interestingly, clathrin light chain small interfering RNA-directed disruption of Vpu trafficking from the TGN to the endosomal system slightly stimulated Vpu-mediated HIV-1 release and completely restored the activity of the Vpu R30A,K31A mutant. An analysis of the C-terminal deletion mutants of Vpu identified an additional determinant in the second helical structure of the protein, which regulated TGN retention/localization, and further revealed the functional importance of Vpu localization in the TGN. Finally, we show that a large fraction of Vpu colocalizes with tetherin in the TGN and provide evidence that the degree of Vpu colocalization with tetherin in the TGN is important for efficient HIV-1 release. Taken together, our results reveal that Vpu traffics between the TGN and the endosomal system and suggest that the proper distribution of Vpu in the TGN is critical to overcome the restricting activity of tetherin on HIV-1 release.

HIV proteins Nef and Vpu down-modulate various host factors to evade immune defenses. Indeed, the CD4 receptor is down-regulated by Nef and Vpu, whereas virion-tethering BST2 is depleted by Vpu. Antibody-dependent cell-mediated cytotoxicity (ADCC) is increasingly recognized as a potentially powerful anti-HIV response. Given that epitopes which are specific for ADCC-competent anti-HIV antibodies are transitionally exposed upon CD4-mediated HIV entry, we investigated whether by depleting CD4 and BST2, HIV could negatively affect ADCC function.