Gadd45g, A Novel Antidepressant Target, Mediates Metformin-Induced Neuronal Differentiation of Neural Stem Cells Via DNA Demethylation.

Increased neurogenesis elicits antidepressive-like effects. The antidiabetic drug metformin (Met) reportedly promotes hippocampal neurogenesis, which ameliorates spatial memory deficits and depression-like behaviors. However, the precise molecular mechanisms underpinning Met-induced neuronal differentiation of neural stem cells (NSCs) remain unclear. We showed that Met enhanced neuronal differentiation of NSCs via Gadd45g but not Gadd45a and Gadd45b. We further found that Gadd45g increased demethylation of neurogenic differentiation 1 promoter by regulating the activity of passive and active DNA demethylation enzymes through an adenylate-activated protein kinase -independent mechanism in Met-treated NSCs. Importantly, genetic deficiency of Gadd45g decreased hippocampal neurogenesis, which could contribute to spatial memory decline, and depression-like behaviors in the adult mice, whereas forced expression of Gadd45g alleviated the depressive-like behaviors. Our findings provide a model that Gadd45g-mediated DNA demethylation contributes to Met-induced neuronal genesis and its antidepressant-like effects and propose the concept that targeting Gadd45g regulation of neurogenesis might serve as a novel antidepressant strategy.

Mettl3-mediated m6 A modification of Lrp2 facilitates neurogenesis through Ythdc2 and elicits antidepressant-like effects.

N6 -methyladenosine (m6 A) is the most abundant mRNA modification affecting diverse biological processes. However, the functions and precise mechanisms of m6 A signaling in adult hippocampal neurogenesis and neurogenesis-related depression remain largely enigmatic. We found that depletion of Mettl3 or Mettl14 in neural stem cells (NSCs) dramatically reduced m6 A abundance, proliferation, and neuronal genesis, coupled with enhanced glial differentiation. Conversely, overexpressing Mettl3 promoted proliferation and neuronal differentiation. Mechanistically, the m6 A modification of Lrp2 mRNA by Mettl3 enhanced its stability and translation efficiency relying on the reader protein Ythdc2, which in turn promoted neurogenesis. Importantly, mice lacking Mettl3 manifested reduced hippocampal neurogenesis, which could contribute to spatial memory decline, and depression-like behaviors. We found that these defective behaviors were notably reversed by Lrp2 overexpression. Moreover, Mettl3 overexpression in the hippocampus of depressive mice rescues behavioral defects. Our findings uncover the biological role of m6 A modification in Lrp2-mediated neurogenesis via m6 A-binding protein Ythdc2, and propose a rationale that targeting Mettl3-Ythdc2-Lrp2 axis regulation of neurogenesis might serve as a promising antidepressant strategy.

PPARß mediates mangiferin-induced neuronal differentiation of neural stem cells through DNA demethylation.

Adult hippocampal neurogenesis (AHN) is heavily implicated in the pathogenesis of various neuropsychiatric disorders. The mangiferin (MGF), a bioactive compound of the mango, reportedly produces biological effects on a variety of neuropsychiatric disorders. However, the function and underlying mechanisms of MGF in regulating hippocampal neurogenesis remain unknown. Here we discovered that the transcriptome and methylome of MGF-induced neural stem cells (NSCs) are distinct from the control. RNA-seq analysis revealed that the diferentially expressed genes (DEGs) were signifcantly enriched in the PPARs. Furthermore, we found that MGF enhanced neuronal differentiation and proliferation of neural stem cells (NSCs) via PPARß but not PPARα and PPARγ. The combination of WGBS and RNA-seq analysis showed that the expression of some neurogenesis genes was negatively correlated with the DNA methylation level generally. We further found that PPARß increased demethylation of Mash1 promoter by modulating the expressions of active and passive DNA demethylation enzymes in MGF-treated NSCs. Importantly, genetic deficiency of PPARß decreased hippocampal neurogenesis in the adult mice, whereas the defective neurogenesis was notably rescued by Mash1 overexpression. Our findings uncover a model that PPARß-mediated DNA demethylation of Mash1 contributes to MGF-induced neuronal genesis, and advance the concept that targeting PPARß-TET1/DNMT3a-Mash1 axis regulation of neurogenesis might serve as a novel neurotherapeutic strategy.

Fabrication of functional group-rich monoliths for magnetic field-assisted in-tube solid phase microextraction of inorganic selenium species in water samples followed by online chromatographic determination.

Efficient separation and enrichment is a crucial step in the analysis of Se(IV) and Se(VI). In the present study, for the first time, online monolith-based magnetic field-assisted in-tube solid phase microextraction (MFA/IT-SPME) was applied to capture inorganic selenium species in water samples. To this aim, porous monoliths mixed with magnetic nanoparticles were synthesized in a silica capillary and employed as a microextraction column (MEC) for MFA/IT-SPME. After that, a magnetic coil utilized to induce variable magnetic fields in adsorption and desorption steps was entwined around the MEC. Se(IV) was coordinated with o-phenylenediamine to form a coordination compound that was infused onto the MEC to be captured. Results evidenced that application of magnetic field during the extraction procedure assisted the capture of the Se(IV)-OPA complex, with an enhancement in the extraction efficiency from 83% to 97%. Under the optimized conditions, MFA/IT-SPME was online combined with HPLC equipped with a diode array detector (DAD) to perform quantification of Se(IV) and Se(VI) in environmental water samples. Total inorganic Se was quantified after pre-reduction of Se(VI) to Se(IV) prior to applying the established approach, and a subtraction method was adopted to calculate the Se(VI) and Se(IV) contents. The limit of detection for Se(IV) was as low as 0.012 µg L-1. The reliability of the suggested method was investigated by assaying Se(IV) and Se(VI) species in real-life water samples with satisfactory recoveries (81.1%-116%) and repeatability (RSDs below 9%).

Electrochemical Enzyme Sensor Based on the Two-Dimensional Metal-Organic Layers Supported Horseradish Peroxidase.

Metal-organic frames (MOFs) have recently been used to support redox enzymes for highly sensitive and selective chemical sensors for small biomolecules such as oxygen (O2), hydrogen peroxide (H2O2), etc. However, most MOFs are insulative and their three-dimensional (3D) porous structures hinder the electron transfer pathway between the current collector and the redox enzyme molecules. In order to facilitate electron transfer, here we adopt two-dimensional (2D) metal-organic layers (MOLs) to support the HRP molecules in the detection of H2O2. The correlation between the current response and the H2O2 concentration presents a linear range from 7.5 µM to 1500 µM with a detection limit of 0.87 µM (S/N = 3). The sensitivity, reproducibility, and stability of the enzyme sensor are promoted due to the facilitated electron transfer.

Unmasking the Critical Role of the Ordering Degree of Bimetallic Nanocatalysts on Oxygen Reduction Reaction by In Situ Raman Spectroscopy.

Precise control and accurate understanding of the ordering degree of bimetallic nanocatalysts (BNs) are challenging yet crucial to acquire advanced materials for the oxygen reduction reaction (ORR). AuCu BNs with various ordering degrees were synthesized to evaluate the influence of ordering degree on the ORR at a molecular level using in situ Raman spectroscopy. The activity of AuCu BNs was improved by over 2 times after a disorder-to-order transition, making the performance of highly ordered AuCu BNs exceed that of benchmark Pt/C. Direct Raman spectroscopic evidence of key intermediate (*OH) demonstrates that the active site is the combination site of Au and Cu. Moreover, two distinct *OH species are observed on the ordered and disordered structure, and the ordered site is more beneficial for ORR due to its lower affinity to *OH. This work deepens the understanding on the important role of ordering degree on BNs and enables the design of improved catalysts.

In Situ Raman Probing of Hot-Electron Transfer at Gold-Graphene Interfaces with Atomic Layer Accuracy.

Plasmonic metals under photoexcitation can generate energetic hot electrons to directly induce chemical reactions. However, the capability and fundamental insights of the transportation of these hot electrons at plasmonic metal-2D material interfaces remain unclear. Herein, hot-electron transfer at Au-graphene interfaces has been in situ studied using surface-enhanced Raman spectroscopy (SERS) with atomic layer accuracy. Combining in situ SERS studies with density functional theory calculations, it is proved that hot electrons can be injected from plasmonic Au nanoparticles to graphene and directly penetrate graphene to trigger photocatalytic reactions. With increasing graphene layers, the transportation of hot electrons decays rapidly and would be completely blocked after five layers of graphene. Moreover, the transfer of hot electrons can be modulated by applying an external electric field, and the hot-electron transfer efficiency under electrochemical conditions is improved by over three times in the presence of a monolayer of graphene.

In Situ Raman Observation of Oxygen Activation and Reaction at Platinum-Ceria Interfaces during CO Oxidation.

Understanding the fundamental insights of oxygen activation and reaction at metal-oxide interfaces is of significant importance yet remains a major challenge due to the difficulty in in situ characterization of active oxygen species. Herein, the activation and reaction of molecular oxygen during CO oxidation at platinum-ceria interfaces has been in situ explored using surface-enhanced Raman spectroscopy (SERS) via a borrowing strategy, and different active oxygen species and their evolution during CO oxidation at platinum-ceria interfaces have been directly observed. In situ Raman spectroscopic evidence with isotopic exchange experiments demonstrate that oxygen is efficiently dissociated to chemisorbed O on Pt and lattice Ce-O species simultaneously at interfacial Ce3+ defect sites under CO oxidation, leading to a much higher activity at platinum-ceria interfaces compared to that at Pt alone. Further in situ time-resolved SERS studies and density functional theory simulations reveal a more efficient molecular pathway through the reaction between adsorbed CO and chemisorbed Pt-O species transferred from the interfaces. This work deepens the fundamental understandings on oxygen activation and CO oxidation at metal-oxide interfaces and offers a sensitive technique for the in situ characterization of oxygen species under working conditions.

SERINC3 and SERINC5 restrict HIV-1 infectivity and are counteracted by Nef.

HIV-1 Nef and the unrelated mouse leukaemia virus glycosylated Gag (glycoGag) strongly enhance the infectivity of HIV-1 virions produced in certain cell types in a clathrin-dependent manner. Here we show that Nef and glycoGag prevent the incorporation of the multipass transmembrane proteins serine incorporator 3 (SERINC3) and SERINC5 into HIV-1 virions to an extent that correlates with infectivity enhancement. Silencing of both SERINC3 and SERINC5 precisely phenocopied the effects of Nef and glycoGag on HIV-1 infectivity. The infectivity of nef-deficient virions increased more than 100-fold when produced in double-knockout human CD4(+) T cells that lack both SERINC3 and SERINC5, and re-expression experiments confirmed that the absence of SERINC3 and SERINC5 accounted for the infectivity enhancement. Furthermore, SERINC3 and SERINC5 together restricted HIV-1 replication, and this restriction was evaded by Nef. SERINC3 and SERINC5 are highly expressed in primary human HIV-1 target cells, and inhibiting their downregulation by Nef is a potential strategy to combat HIV/AIDS.

HIV-1 gag recruits PACSIN2 to promote virus spreading.

The p2b domain of Rous sarcoma virus (RSV) Gag and the p6 domain of HIV-1 Gag contain late assembly (L) domains that engage the ESCRT membrane fission machinery and are essential for virus release. We now show that the PPXY-type RSV L domain specifically recruits the BAR domain protein PACSIN2 into virus-like particles (VLP), in addition to the NEDD4-like ubiquitin ligase ITCH and ESCRT pathway components such as TSG101. PACSIN2, which has been implicated in the remodeling of cellular membranes and the actin cytoskeleton, is also recruited by HIV-1 p6 independent of its ability to engage the ESCRT factors TSG101 or ALIX. Moreover, PACSIN2 is robustly recruited by NEDD4-2s, a NEDD4-like ubiquitin ligase capable of rescuing HIV-1 budding defects. The NEDD4-2s-induced incorporation of PACSIN2 into VLP correlated with the formation of Gag-ubiquitin conjugates, indicating that PACSIN2 binds ubiquitin. Although PACSIN2 was not required for a single cycle of HIV-1 replication after infection with cell-free virus, HIV-1 spreading was nevertheless severely impaired in T cell lines and primary human peripheral blood mononuclear cells depleted of PACSIN2. HIV-1 spreading could be restored by reintroduction of wild-type PACSIN2, but not of a SH3 domain mutant unable to interact with the actin polymerization regulators WASP and N-WASP. Overall, our observations indicate that PACSIN2 promotes the cell-to-cell spreading of HIV-1 by connecting Gag to the actin cytoskeleton.

Human CRM1 augments production of infectious human and feline immunodeficiency viruses from murine cells.

Productive replication of human immunodeficiency virus type 1 (HIV-1) occurs efficiently only in humans. The posttranscriptional stages of the HIV-1 life cycle proceed poorly in mouse cells, with a resulting defect in viral assembly and release. Previous work has shown that the presence of human chromosome 2 increases HIV-1 production in mouse cells. Recent studies have shown that human chromosome region maintenance 1 (hCRM1) stimulates Gag release from rodent cells. Here we report that expressions of hCRM1 in murine cells resulted in marked increases in the production of infectious HIV-1 and feline immunodeficiency virus (FIV). HIV-1 production was also increased by hSRp40, and a combination of hCRM1 and hSRp40 resulted in a more-than-additive effect on HIV-1 release. In contrast, the overexpression of mouse CRM1 (mCRM1) minimally affected HIV-1 and FIV production and did not antagonize hCRM1. In the presence of hCRM1 there were large increases in the amounts of released capsid, which paralleled the increases in the infectious titers. Consistent with this finding, the ratios of unspliced to spliced HIV-1 mRNAs in mouse cells expressing hCRM1 and SRp40 became similar to those of human cells. Furthermore, imaging of intron-containing FIV RNA showed that hCRM1 increased RNA export to the cytoplasm.By testing chimeras between mCRM1 and hCRM1 and comparing those sequences to feline CRM1, we mapped the functional domain to HEAT (Huntingtin, elongation factor 3, protein phosphatase 2A, and the yeast kinase TOR1) repeats 4A to 9A and a triple point mutant in repeat 9A, which showed a loss of function. Structural analysis suggested that this region of hCRM1 may serve as a binding site for viral or cellular factors to facilitate lentiviral RNA nuclear export.

AP-2 Adaptor Complex-Dependent Enhancement of HIV-1 Replication by Nef in the Absence of the Nef/AP-2 Targets SERINC5 and CD4.

Human immunodeficiency virus type 1 (HIV-1) Nef hijacks the clathrin adaptor complex 2 (AP-2) to downregulate the viral receptor CD4 and the antiviral multipass transmembrane proteins SERINC3 and SERINC5, which inhibit the infectivity of progeny virions when incorporated. In Jurkat Tag T lymphoid cells lacking SERINC3 and SERINC5, Nef is no longer required for full progeny virus infectivity and for efficient viral replication. However, in MOLT-3 T lymphoid cells, HIV-1 replication remains highly dependent on Nef even in the absence of SERINC3 and SERINC5. Using a knockout (KO) approach, we now show that the Nef-mediated enhancement of HIV-1 replication in MOLT-3 cells does not depend on the Nef-interacting kinases LCK and PAK2. Furthermore, Nef substantially enhanced HIV-1 replication even in triple-KO MOLT-3 cells that simultaneously lacked the three Nef/AP-2 targets, SERINC3, SERINC5, and CD4, and were reconstituted with a Nef-resistant CD4 to permit HIV-1 entry. Nevertheless, the ability of Nef mutants to promote HIV-1 replication in the triple-KO cells correlated strictly with the ability to bind AP-2. In addition, knockdown and reconstitution experiments confirmed the involvement of AP-2. These observations raise the possibility that MOLT-3 cells express a novel antiviral factor that is downregulated by Nef in an AP-2-dependent manner. IMPORTANCE The HIV-1 Nef protein hijacks a component of the cellular endocytic machinery called AP-2 to downregulate the viral receptor CD4 and the antiviral cellular membrane proteins SERINC3 and SERINC5. In the absence of Nef, SERINC3 and SERINC5 are taken up into viral particles, which reduces their infectivity. Surprisingly, in a T cell line called MOLT-3, Nef remains crucial for HIV-1 spreading in the absence of SERINC3 and SERINC5. We now show that this effect of Nef also does not depend on the cellular signaling molecules and Nef interaction partners LCK and PAK2. Nef was required for efficient HIV-1 spreading even in triple-knockout cells that completely lacked Nef/AP-2-sensitive CD4, in addition to the Nef/AP-2 targets SERINC3 and SERINC5. Nevertheless, our results indicate that the enhancement of HIV-1 spreading by Nef in the triple-knockout cells remained AP-2 dependent, which suggests the presence of an unknown antiviral factor that is sensitive to Nef/AP-2-mediated downregulation.

Regulation of heterogeneous electron transfer reactivity by defect engineering through electrochemically induced brominating addition.

Enhancing the electrochemical activity of graphene holds great significance for expanding its applications in various electrochemistry fields. In this study, we have demonstrated a facile and quantitative approach for modulating the defect density of single-layer graphene (SLG) via an electrochemically induced bromination process facilitated by cyclic voltammetry. This controlled defect engineering directly impacts the heterogeneous electron transfer (HET) rate of SLG. By utilizing Raman spectroscopy and scanning electrochemical microscopy (SECM), we have established a correlation between the HET kinetics and both the defect density (nD) and mean distance between defects (LD) of SLG. The variation of the HET rate (k0) with the defect density manifested a distinctive three-stage behavior. Initially, k0 increased slightly with the increasing nD, and then it experienced a rapid increase as nD further increased. However, once the defect density surpassed a critical value of about 1.8 × 1012 cm-2 (LD < 4.2 nm), k0 decreased rapidly. Notably, the results revealed a remarkable 35-fold enhancement of k0 under the optimal defect density conditions compared to pristine SLG. This research paves the way for controllable defect engineering as a powerful strategy to enhance the electrochemical activity of graphene, opening up new possibilities for its utilization in a wide range of electrochemical applications.

Electrochemical regulation of the band gap of single layer graphene: from semimetal to semiconductor.

As a semimetal with a zero band gap and single-atom-scale thickness, single layer graphene (SLG) has excellent electron conductivity on its basal plane. If the band gap could be opened and regulated controllably, SLG would behave as a semiconductor. That means electronic elements or even electronic circuits with single-atom thickness could be expected to be printed on a wafer-scale SLG substrate, which would bring about a revolution in Moore's law of integrated circuits, not by decreasing the feature size of line width, but by piling up the atomic-scale-thickness of an SLG circuit board layer by layer. Employing scanning electrochemical microscopy (SECM), we have demonstrated that the electrochemically induced brominating addition reaction can open and regulate the band gap of SLG by forming SLG bromide (SLGBr). The SLG/SLGBr/SLG Schottky junction shows excellent performance in current rectification, and the rectification potential region can be regulated by tuning the degree of bromination of SLG. This work provides a feasible and effective way to regulate the band gap of SLG, which would open new applications for SLG in micro-nano electronics and ultra-large-scale integrated circuits (ULSI).

Plasmon-Mediated Photoelectrochemical Hot-Hole Oxidation Coupling Reactions of Adenine on Nanostructured Silver Electrodes.

Surface-enhanced Raman spectroscopy (SERS) has been widely applied in the identification and characterization of DNA structures with high efficiency. Especially, the SERS signals of the adenine group have exhibited high detection sensitivity in several biomolecular systems. However, there is still no unanimous conclusion regarding the interpretation of some special kinds of SERS signals of adenine and its derivatives on silver colloids and electrodes. This Letter presents a new photochemical azo coupling reaction for adenyl residues, in which the adenine is selectively oxidized to (E)-1,2-di(7H-purin-6-yl) diazene (azopurine) in the presence of silver ions, silver colloids, and electrodes of nanostructures under visible light irradiation. The product, azopurine, is first found to be responsible for the SERS signals. This photoelectrochemical oxidative coupling reaction of adenine and its derivatives is promoted by plasmon-mediated hot holes and is regulated by positive potentials and pH of solutions, which opens up new avenues for studying azo coupling in the photoelectrochemistry of adenine-containing biomolecules on electrode surfaces of plasmonic metal nanostructures.

Task specific monolith for magnetic field-reinforced in-tube solid phase microextraction of mercury species in waters prior to online HPLC quantification.

In this study, a novel sorbent based on task specific monolith doped with Fe3O4 was in situ fabricated in capillary and acted as the extraction medium of magnetic field-reinforced in-tube solid phase microextraction (MFR/IT-SPME) to trap and preconcentrate mercury species which were coordinated with dithizone to form chelates. Various characterization technologies evidenced that the obtained monolithic adsorbent presented porous and super paramagnetic properties, and possessed abundant functional groups. Results evidenced that the implementation of magnetic field during extraction stages enhanced the extraction efficiency of studied Hg chelates from 48.5% to 75.3% to 69.9-94.4%. Under the optimized extraction parameters, the introduced MFR/IT-SPME was online coupled to HPLC/DAD to quantify mercury species at ultra-trace levels in various water samples. Limits of detection varied from 0.0067 µg/L to 0.016 µg/L, and the RSDs for precision were below 7.5%. Additionally, related extraction mechanism was deduced and revealed multiple forces co-contributed to the enrichment. The reliability and accuracy of suggested online approach for speciation analysis of mercury was well proved by confirmatory experiments.

HIV-1 propagation is highly dependent on basal levels of the restriction factor BST2.

BST2 is an interferon-inducible antiviral host protein antagonized by HIV-1 Vpu that entraps nascent HIV-1 virions on the cell surface. Unexpectedly, we find that HIV-1 lacking Nef can revert to full replication competence simply by losing the ability to antagonize BST2. Using gene editing together with cell sorting, we demonstrate that even the propagation of wild-type HIV-1 is strikingly dependent on BST2, including in primary human cells. HIV-1 propagation in BST2−/− populations can be fully rescued by exogenous BST2 irrespective of its capacity to signal and even by an artificial BST2-like protein that shares its virion entrapment activity but lacks sequence homology. Counterintuitively, our results reveal that HIV-1 propagation is critically dependent on basal levels of virion tethering by a key component of innate antiviral immunity.

Primate lentiviral Vpx commandeers DDB1 to counteract a macrophage restriction.

Primate lentiviruses encode four "accessory proteins" including Vif, Vpu, Nef, and Vpr/Vpx. Vif and Vpu counteract the antiviral effects of cellular restrictions to early and late steps in the viral replication cycle. We present evidence that the Vpx proteins of HIV-2/SIV(SM) promote virus infection by antagonizing an antiviral restriction in macrophages. Fusion of macrophages in which Vpx was essential for virus infection, with COS cells in which Vpx was dispensable for virus infection, generated heterokaryons that supported infection by wild-type SIV but not Vpx-deleted SIV. The restriction potently antagonized infection of macrophages by HIV-1, and expression of Vpx in macrophages in trans overcame the restriction to HIV-1 and SIV infection. Vpx was ubiquitylated and both ubiquitylation and the proteasome regulated the activity of Vpx. The ability of Vpx to counteract the restriction to HIV-1 and SIV infection was dependent upon the HIV-1 Vpr interacting protein, damaged DNA binding protein 1 (DDB1), and DDB1 partially substituted for Vpx when fused to Vpr. Our results indicate that macrophage harbor a potent antiviral restriction and that primate lentiviruses have evolved Vpx to counteract this restriction.

Potent Enhancement of HIV-1 Replication by Nef in the Absence of SERINC3 and SERINC5.

It has recently emerged that HIV-1 Nef counteracts the antiviral host proteins SERINC3 and SERINC5. In particular, SERINC5 inhibits the infectivity of progeny virions when incorporated. SERINC3 and SERINC5 are also counteracted by the unrelated murine leukemia virus glycosylated Gag (glycoGag) protein, which possesses a potent Nef-like activity on HIV-1 infectivity. We now report that a minimal glycoGag termed glycoMA can fully substitute for Nef in promoting HIV-1 replication in Jurkat T lymphoid cells, indicating that Nef enhances replication in these cells mainly by counteracting SERINCs. In contrast, the SERINC antagonist glycoMA was unable to substitute for Nef in MOLT-3 T lymphoid cells, in which HIV-1 replication was highly dependent on Nef, and remained so even in the absence of SERINC3 and SERINC5. As in MOLT-3 cells, glycoMA was unable to substitute for Nef in stimulating HIV-1 replication in primary human cells. Although the ability of Nef mutants to promote HIV-1 replication in MOLT-3 cells correlated with the ability to engage endocytic machinery and to downregulate CD4, Nef nevertheless rescued virus replication under conditions where CD4 downregulation did not occur. Taken together, our observations raise the possibility that Nef triggers the endocytosis of a novel antiviral factor that is active against both laboratory-adapted and primary HIV-1 strains.IMPORTANCE The Nef protein of HIV-1 and the unrelated glycoGag protein of a murine leukemia virus similarly prevent the uptake of antiviral host proteins called SERINC3 and SERINC5 into HIV-1 particles, which enhances their infectiousness. We now show that although both SERINC antagonists can in principle similarly enhance HIV-1 replication, glycoGag is unable to substitute for Nef in primary human cells and in a T cell line called MOLT-3. In MOLT-3 cells, Nef remained crucial for HIV-1 replication even in the absence of SERINC3 and SERINC5. The pronounced effect of Nef on HIV-1 spreading in MOLT-3 cells correlated with the ability of Nef to engage cellular endocytic machinery and to downregulate the HIV-1 receptor CD4 but nevertheless persisted in the absence of CD4 downregulation. Collectively, our results provide evidence for a potent novel restriction activity that affects even relatively SERINC-resistant HIV-1 isolates and is counteracted by Nef.