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A primary function of HIV-1 Nef is the enhancement of viral infectivity and replication. Whether counteraction of the antiretroviral proteins SERINC3 and SERINC5 is the cause of this positive influence on viral growth-rate and infectivity remains unclear. Here, we utilized CRISPR/Cas9 to knockout SERINC3 and SERINC5 in a leukemic CD4-positive T cell line (CEM) that displays nef-related infectivity and growth-rate phenotypes. Viral replication was attenuated in CEM cells infected with HIV-1 lacking Nef (HIV-1ΔNef). This attenuated growth-rate phenotype was observed regardless of whether the coding regions of the serinc3 or serinc5 genes were intact. Moreover, knockout of serinc5 alone or of both serinc5 and serinc3 together failed to restore the infectivity of HIV1ΔNef virions produced from infected CEM cells. Our results corroborate a similar study using another T-lymphoid cell line (MOLT-3) and indicate that the antagonism of SERINC3 and SERINC5 does not fully explain the virology of HIV-1 lacking Nef.

The lentiviral nef gene encodes several discrete activities aimed at co-opting or antagonizing cellular proteins and pathways to defeat host defenses and maintain persistent infection. Primary functions of Nef include downregulation of CD4 and MHC class-I from the cell surface, disruption or mimicry of T-cell receptor signaling, and enhancement of viral infectivity by counteraction of the host antiretroviral proteins SERINC3 and SERINC5. In the absence of Nef, SERINC3 and SERINC5 incorporate into virions and inhibit viral fusion with target cells, decreasing infectivity. However, whether Nef’s counteraction of SERINC3 and SERINC5 is the cause of its positive influence on viral growth-rate in CD4-positive T cells is unclear. Here, we utilized CRISPR/Cas9 to knockout SERINC3 and SERINC5 in a leukemic CD4-positive T cell line (CEM) that displays robust nef-related infectivity and growth-rate phenotypes. As previously reported, viral replication was severely attenuated in CEM cells infected with HIV-1 lacking Nef (HIV-1ΔNef). This attenuated growth-rate phenotype was observed regardless of whether or not the coding regions of the serinc3 and serinc5 genes were intact. Moreover, knockout of serinc3 and serinc5 failed to restore the infectivity of HIV-1ΔNef virions produced from infected CEM cells in single-cycle replication experiments using CD4-positive HeLa cells as targets. Taken together, our results corroborate a recent study using another T-lymphoid cell line (MOLT-3) and suggest that Nef modulates a still unidentified host protein(s) to enhance viral growth rate and infectivity in CD4-positive T cells. Importance HIV-1 Nef is a major pathogenicity factor in vivo. A well-described activity of Nef is the enhancement of virion-infectivity and viral propagation in vitro. The infectivity-effect has been attributed to Nef’s ability to prevent the cellular, antiretroviral proteins SERINC3 and SERINC5 from incorporating into viral particles. While the activity of the SERINCs as inhibitors of retroviral infectivity has been well-documented, the role these proteins play in controlling HIV-1 replication is less clear. We report here that genetic disruption of SERINC3 and SERINC5 rescues neither viral replication-rate nor the infectivity of cell-free virions produced from CD4-positive T cells of the CEM lymphoblastoid line infected with viruses lacking Nef. This indicates that failure to modulate SERINC3 and SERINC5 is not the cause of the virologic attenuation of nef-negative HIV-1 observed using this system.

The HIV-1 Nef protein suppresses multiple immune surveillance mechanisms to promote viral pathogenesis and is an attractive target for the development of novel therapeutics. A key function of Nef is to remove the CD4 receptor from the cell surface by hijacking clathrin- and adaptor protein complex 2 (AP2)-dependent endocytosis. However, exactly how Nef does this has been elusive. Here, we describe the underlying mechanism as revealed by a 3.0-Å crystal structure of a fusion protein comprising Nef and the cytoplasmic domain of CD4 bound to the tetrameric AP2 complex. An intricate combination of conformational changes occurs in both Nef and AP2 to enable CD4 binding and downregulation. A pocket on Nef previously identified as crucial for recruiting class I MHC is also responsible for recruiting CD4, revealing a potential approach to inhibit two of Nef’s activities and sensitize the virus to immune clearance., https://doi.org/10.1038/s41594-020-0463-z

The HIV-1 Nef protein suppresses multiple immune surveillance mechanisms to promote viral pathogenesis and is an attractive target for the development of novel therapeutics. A key function of Nef is to remove the CD4 receptor from the cell surface by hijacking clathrin- and adaptor protein complex 2 (AP2)-dependent endocytosis. However, exactly how Nef does this has been elusive. Here, we describe the underlying mechanism as revealed by a 3.0-A crystal structure of a fusion protein comprising Nef and the cytoplasmic domain of CD4 bound to the tetrameric AP2 complex. An intricate combination of conformational changes occurs in both Nef and AP2 to enable CD4 binding and downregulation. A pocket on Nef previously identified as crucial for recruiting class I MHC is also responsible for recruiting CD4, revealing a potential approach to inhibit two of Nef's activities and sensitize the virus to immune clearance.

The cellular protein SERINC5 inhibits the infectivity of diverse retroviruses, and its activity is counteracted by the glycosylated Gag (glycoGag) protein of murine leukemia virus (MLV), the S2 protein of equine infectious anemia virus (EIAV), and the Nef protein of human immunodeficiency virus type 1 (HIV-1). Determining the regions within SERINC5 that provide restrictive activity or Nef sensitivity should inform mechanistic models of the SERINC5/HIV-1 relationship. Here, we report that deletion of the conserved sequence EDTEE, which is located within a cytoplasmic loop of SERINC5 and which is reminiscent of an acidic-cluster membrane trafficking signal, increases the sensitivity of SERINC5 to antagonism by Nef, while it has no effect on the intrinsic activity of the protein as an inhibitor of infectivity. These effects correlated with enhanced removal of the ΔEDTEE mutant relative to that of wild-type SERINC5 from the cell surface and with enhanced exclusion of the mutant protein from virions by Nef. Mutational analysis indicated that the acidic residues, but not the threonine, within the EDTEE motif are important for the relative resistance to Nef. Deletion of the EDTEE sequence did not increase the sensitivity of SERINC5 to antagonism by the glycoGag protein of MLV, suggesting that its virologic role is Nef specific. These results are consistent with the reported mapping of the cytoplasmic loop that contains the EDTEE sequence as a general determinant of Nef responsiveness, but they further indicate that sequences inhibitory to as well as supportive of Nef activity reside in this region. We speculate that the EDTEE motif might have evolved to mediate resistance against retroviruses that use Nef-like proteins to antagonize SERINC5. IMPORTANCE Cellular membrane proteins in the SERINC family, especially SERINC5, inhibit the infectivity of retroviral virions. This inhibition is counteracted by retroviral proteins, specifically, HIV-1 Nef, MLV glycoGag, and EIAV S2. One consequence of such a host-pathogen “arms race” is a compensatory change in the host antiviral protein as it evolves to escape the effects of viral antagonists. This is often reflected in a genetic signature, positive selection, which is conspicuously missing in SERINC5. Here we show that despite this lack of genetic evidence, a sequence in SERINC5 nonetheless provides relative resistance to antagonism by HIV-1 Nef.

The cellular protein SERINC5 inhibits the infectivity of diverse retroviruses and is counteracted by the glycoGag protein of MLV, the S2 protein of EIAV, and the Nef protein of HIV-1. Determining regions within SERINC5 that provide restrictive activity or Nef-sensitivity should inform mechanistic models of the SERINC5/HIV-1 relationship. Here, we report that deletion of the highly conserved sequence EDTEE, which is located within a cytoplasmic loop of SERINC5 and is reminiscent of an acidic cluster membrane trafficking signal, increases the sensitivity of SERINC5 to antagonism by Nef while having no effect on the intrinsic activity of the protein as an inhibitor of infectivity. The effects on infectivity correlated with enhanced removal of the ΔEDTEE mutant relative to wild type SERINC5 from the cell surface and with enhanced exclusion of the mutant protein from virions by Nef. Mutational analysis revealed that the acidic residues, but not the threonine, within the EDTEE motif are important for the relative resistance to Nef. Deletion of the EDTEE sequence did not increase the sensitivity of SERINC5 to antagonism by the glycoGag protein of MLV, suggesting that its virologic role is Nef-specific. These results are consistent with the reported mapping of the cytoplasmic loop that contains the EDTEE sequence as a general determinant of Nef-responsiveness, but they further indicate that sequences inhibitory to as well as supportive of Nef-activity reside in this region. We speculate that the EDTEE motif might have evolved to mediate resistance against retroviruses that use Nef-like proteins to antagonize SERINC5.ImportanceCellular membrane proteins in the SERINC family, especially SERINC5, inhibit the infectivity of retroviral virions. This inhibition is counteracted by retroviral proteins, specifically HIV-1 Nef, MLV glycoGag, and EIAV S2. One consequence of such a host-pathogen “arms race” is compensatory change in the host antiviral protein as it evolves to escape the effects of the viral antagonist. This is often reflected in a genetic signature, positive selection, which is conspicuously missing inSERINC5. Here we show that despite this lack of genetic evidence, a sequence in SERINC5 nonetheless provides relative resistance to antagonism by HIV-1 Nef.

The design of a new clinical candidate histamine-H(3) receptor antagonist for the potential treatment of excessive daytime sleepiness (EDS) is described. Phenethyl-R-2-methylpyrrolidine containing biphenylsulfonamide compounds were modified by replacement of the sulfonamide linkage with a sulfone. One compound from this series, 2j (APD916) increased wakefulness in rodents as measured by polysomnography with a duration of effect consistent with its pharmacokinetic properties. The identification of a suitable salt form of 2j allowed it to be selected for further development.

Antagonism of the histamine-H(3) receptor is one tactic being explored to increase wakefulness for the treatment of disorders such as excessive daytime sleepiness (EDS) as well as other sleep or cognitive disorders. Phenethyl-R-2-methylpyrrolidine containing biphenylsulfonamide compounds were shown to be potent and selective antagonists of the H(3) receptor. Several of these compounds demonstrated in vivo activity in a rat model of (R)-alpha-methyl histamine (RAMH) induced dipsogenia, and one compound (4e) provided an increase in wakefulness in rats as measured by polysomnographic methods. However, more detailed analysis of the PK/PD relationship suggested the presence of a common active metabolite which may preclude this series of compounds from further development.

A new series of H 3 antagonists derived from the natural product Conessine are presented. Several compounds from these new series retain the potency and selectivity of earlier diamine based analogs while exhibiting improved PK characteristics. One compound ( 3u ) demonstrated functional antagonism of the H 3 receptor in an in vivo pharmacological model.

BST2/tetherin, an antiviral restriction factor, inhibits the release of enveloped viruses from the cell surface. Human immunodeficiency virus-1 (HIV-1) antagonizes BST2 through viral protein u (Vpu), which downregulates BST2 from the cell surface. We report the crystal structure of a protein complex containing Vpu and BST2 cytoplasmic domains and the core of the clathrin adaptor protein complex 1 (AP1). This, together with our biochemical and functional validations, reveals how Vpu hijacks the AP1-dependent membrane trafficking pathways to mistraffick BST2. Vpu mimics a canonical acidic dileucine-sorting motif to bind AP1 in the cytosol, while simultaneously interacting with BST2 in the membrane. These interactions enable Vpu to build on an intrinsic interaction between BST2 and AP1, presumably causing the observed retention of BST2 in juxtanuclear endosomes and stimulating its degradation in lysosomes. The ability of Vpu to hijack AP-dependent trafficking pathways suggests a potential common theme for Vpu-mediated downregulation of host proteins. DOI: http://dx.doi.org/10.7554/eLife.02362.001, eLife digest HIV is a retrovirus that attacks the immune system, making the body increasingly susceptible to opportunistic infections and disease and eventually leading to AIDS. While antiretroviral drugs have allowed people with AIDS to live longer, there is no cure or vaccine for HIV. Two types of HIV exist, with HIV-1 being much more common and pathogenic than HIV-2. Like other ‘complex’ retroviruses, the HIV-1 genome contains genes that encode various proteins that allow the virus to disrupt the immune response of the host it is attacking. Viral protein u is a protein encoded by HIV-1 (but not HIV-2) that counteracts an antiviral protein called BST2 in the host. BST2, which is part of the host's innate immune response, prevents newly formed viruses from leaving the surface of infected cells. By counteracting BST2, viral protein u allows the virus to spread in the host more efficiently. Like many proteins, newly produced BST2 is packaged inside structures called vesicles in a part of the cell called the trans-Golgi network, and then sent to its destination. Complexes formed by various proteins make sure that the vesicles take their cargo to their correct destinations within the cell. Two adaptor protein complexes—known as AP1 and AP2—are thought to be involved the transport of BST2. However, it is not known how viral protein u stops BST2 from reaching the cell surface, or how it decreases the amount of BST2 in the cell as a whole. Jia et al. show how viral protein u and BST2 jointly interact with AP1. This interaction leads to the mistrafficking and degradation of BST2 and the counteraction of its antiviral activity. DOI: http://dx.doi.org/10.7554/eLife.02362.002

BST2 (HM1.24; CD317; tetherin) is an interferon-inducible transmembrane protein that restricts the release of several enveloped viruses, including HIV, from infected cells. Before its activity as an antiviral factor was described, BST2 was identified as an inducer of NF-κB activity. Here we show that human BST2 induces NF-κB in a dose-dependent manner. This activity is separable from the restriction of virus release: a YxY sequence in the cytoplasmic domain of BST2 is required for the induction of NF-κB but is dispensable for restriction, whereas the glycosylphosphatidylinositol (GPI) addition site in the protein's ectodomain is required for restriction but is largely dispensable for the induction of NF-κB. Mutations predicted to disrupt the coiled-coil structure of the BST2 ectodomain impaired both signaling and restriction, but disruption of the tetramerization interface differentially affected signaling. The induction of NF-κB by BST2 was impaired by inhibition of transforming growth factor β (TGF-β)-activated kinase 1 (TAK1) or by calcium chelation, suggesting potential linkage to the mitogen-activated protein kinase and endoplasmic reticulum (ER) stress response pathways. Consistent with a role for TAK1, BST2 coimmunoprecipitated with TAK1 and the TAK1-associated pseudophosphatase TAB1; these interactions required the YxY sequence in BST2. Moreover, signaling by BST2 was blocked by expression of an IκB-mutant that inhibits the canonical pathway of NF-κB activation. The expression of HIV-1 Vpu inhibited the induction of NF-κB by BST2; this inhibition required Vpu's ability to bind the cellular β-TrCP-E3-ubiquitin ligase complex. The expression of HIV-1 lacking vpu augmented the induction of NF-κB activity by BST2, suggesting that BST2 can act as a virus sensor. This augmentation was also inhibited by Vpu in a β-TrCP-dependent manner. The role of BST2 in the host-pathogen relationship is apparently multifaceted: signaling during the innate immune response, sensing of viral gene expression, and direct restriction of virus release. HIV-1 Vpu counteracts each of these functions.

The design of a new clinical candidate histamine-H(3) receptor antagonist for the potential treatment of excessive daytime sleepiness (EDS) is described. Phenethyl-R-2-methylpyrrolidine containing biphenylsulfonamide compounds were modified by replacement of the sulfonamide linkage with a sulfone. One compound from this series, 2j (APD916) increased wakefulness in rodents as measured by polysomnography with a duration of effect consistent with its pharmacokinetic properties. The identification of a suitable salt form of 2j allowed it to be selected for further development.

A new family of Histamine H3 receptor antagonists (5a–t) has been prepared based on the structure of the natural product Conessine, a known H3 antagonist. Several members of the new series are highly potent and selective binders of rat and human H3 receptors and display inverse agonism at the human H3 receptor. Compound 5n exhibited promising rat pharmacokinetic properties and demonstrated functional antagonism of the H3 receptor in an in-vivo pharmacological model.