Host cells impose a broad range of obstacles to the replication of retroviruses. Tetherin (also known as CD317, BST-2 or HM1.24) impedes viral release by retaining newly budded HIV-1 virions on the surface of cells. HIV-1 Vpu efficiently counteracts this restriction. Here, we show that HIV-1 Vpu induces the depletion of tetherin from cells. We demonstrate that this phenomenon correlates with the ability of Vpu to counteract the antiviral activity of both overexpressed and interferon-induced endogenous tetherin. In addition, we show that Vpu co-immunoprecipitates with tetherin and β-TrCP in a tri-molecular complex. This interaction leads to Vpu-mediated proteasomal degradation of tetherin in a β-TrCP2-dependent manner. Accordingly, in conditions where Vpu-β-TrCP2-tetherin interplay was not operative, including cells stably knocked down for β-TrCP2 expression or cells expressing a dominant negative form of β-TrCP, the ability of Vpu to antagonize the antiviral activity of tetherin was severely impaired. Nevertheless, tetherin degradation did not account for the totality of Vpu-mediated counteraction against the antiviral factor, as binding of Vpu to tetherin was sufficient for a partial relief of the restriction. Finally, we show that the mechanism used by Vpu to induce tetherin depletion implicates the cellular ER-associated degradation (ERAD) pathway, which mediates the dislocation of ER membrane proteins into the cytosol for subsequent proteasomal degradation. In conclusion, we show that Vpu interacts with tetherin to direct its β-TrCP2-dependent proteasomal degradation, thereby alleviating the blockade to the release of infectious virions. Identification of tetherin binding to Vpu provides a potential novel target for the development of drugs aimed at inhibiting HIV-1 replication.

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Background Humanized mice generate a lymphoid system of human origin subsequent to transplantation of human CD34+ cells and thus are highly susceptible to HIV infection. Here we examined the efficacy of antiretroviral treatment (ART) when added to food pellets, and of long-acting (LA) antiretroviral compounds, either as monotherapy or in combination. These studies shall be inspiring for establishing a gold standard of ART, which is easy to administer and well supported by the mice, and for subsequent studies such as latency. Furthermore, they should disclose whether viral breakthrough and emergence of resistance occurs similar as in HIV-infected patients when ART is insufficient. Methods/Principal Findings NOD/shi-scid/γ c null (NOG) mice were used in all experimentations. We first performed pharmacokinetic studies of the drugs used, either added to food pellets (AZT, TDF, 3TC, RTV) or in a LA formulation that permitted once weekly subcutaneous administration (TMC278: non-nucleoside reverse transcriptase inhibitor, TMC181: protease inhibitor). A combination of 3TC, TDF and TMC278-LA or 3TC, TDF, TMC278-LA and TMC181-LA suppressed the viral load to undetectable levels in 15/19 (79%) and 14/14 (100%) mice, respectively. In successfully treated mice, subsequent monotherapy with TMC278-LA resulted in viral breakthrough; in contrast, the two LA compounds together prevented viral breakthrough. Resistance mutations matched the mutations most commonly observed in HIV patients failing therapy. Importantly, viral rebound after interruption of ART, presence of HIV DNA in successfully treated mice and in vitro reactivation of early HIV transcripts point to an existing latent HIV reservoir. Conclusions/Significance This report is a unique description of multiple aspects of HIV infection in humanized mice that comprised efficacy testing of various treatment regimens, including LA compounds, resistance mutation analysis as well as viral rebound after treatment interruption. Humanized mice will be highly valuable for exploring the antiviral potency of new compounds or compounds targeting the latent HIV reservoir.

BackgroundHumanized mice (hu mice) are based on the transplantation of hematopoietic stem and progenitor cells into immunodeficient mice and have become important pre-clinical models for biomedical research. However, data about their hematopoiesis over time are scarce. We therefore characterized leukocyte reconstitution in NSG mice, which were sublethally irradiated and transplanted with human cord blood-derived CD34+ cells at newborn age, longitudinally in peripheral blood and, for more detailed analyses, cross-sectionally in peripheral blood, spleen and bone marrow at different time points.ResultsHuman cell chimerism and absolute human cell count decreased between week 16 and 24 in the peripheral blood of hu mice, but were stable thereafter as assessed up to 32 weeks. Human cell chimerism in spleen and bone marrow was maintained over time. Notably, human cell chimerism in peripheral blood and spleen as well as bone marrow positively correlated with each other. Percentage of B cells decreased between week 16 and 24, whereas percentage of T cells increased; subsequently, they levelled off with T cells clearly predominating at week 32. Natural killer cells, monocytes and plasmacytoid dendritic cells (DCs) as well as CD1c + and CD141+ myeloid DCs were all present in hu mice. Proliferative responses of splenic T cells to stimulation were preserved over time. Importantly, the percentage of more primitive hematopoietic stem cells (HSCs) in bone marrow was maintained over time.ConclusionsOverall, leukocyte reconstitution was maintained up to 32 weeks post-transplantation in our hu NSG model, possibly explained by the maintenance of HSCs in the bone marrow. Notably, we observed great variation in multi-lineage hematopoietic reconstitution in hu mice that needs to be taken into account for the experimental design with hu mice.Electronic supplementary materialThe online version of this article (doi:10.1186/s12865-017-0209-9) contains supplementary material, which is available to authorized users.