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(A) The chemical structure of 2c. (B) The infectivity of the NL43 wild-type (WT) and Nef-defective mutant (ΔNef) viruses to the target TZM-bl cells was compared by varying the concentration of p24 Gag protein and is expressed as a percentage of the value for the sample on the far left. Data are shown as the mean±SD of triplicate assays and are representative of two independent experiments with similar results. (C) 2c was added to 293 cells producing NL43-WT or ΔNef viruses at the indicated concentrations for 2 days, and the concentration of p24 Gag protein in the cell supernatants was determined by ELISA (bar graph). Data are shown as the mean±SD of triplicate assays and are representative of two independent experiments with similar results. Alternatively, the producer cells were lysed and analyzed for the expression of Gag and Nef by Western blotting (lower blots). The actin blot was used as a loading control. (D) The infectivity of NL43-WT (upper) or ΔNef viruses (lower) produced by 293 cells in the absence or presence of the indicated concentrations of 2c was determined using TZM-bl cells as the target cells. The WT and ΔNef viruses were inoculated by changing the p24 concentration (2 or 4 ng/ml and 8 or 16 ng/ml for the WT and ΔNef viruses, respectively) so that the two viruses were similarly infective to the target cells. Infectivity is expressed as a percentage of the value for the sample on the far left. Data are shown as the mean±SD of triplicate assays and are representative of three independent experiments with similar results. *p<0.05.
Source publication
Nef is a multifunctional HIV-1 protein that accelerates progression to AIDS, and enhances the infectivity of progeny viruses through a mechanism that is not yet understood. Here, we show that the small molecule compound 2c reduces Nef-mediated viral infectivity enhancement. When added to viral producer cells, 2c did not affect the efficiency of vir...
Similar publications
HIV-1 Nef is essential for AIDS pathogenesis, but this viral protein is not targeted by antiviral strategies. The functions of Nef are largely related to perturbations of intracellular trafficking and signaling pathways through leucine-based and polyproline motifs that are required for interactions with clathrin-associated adaptor protein complexes...
Citations
... As expected, 2C had no effect on HIV-1-induced CD4 downregulation, which does not involve Src-family kinases and relies on the AP-2 endocytic adaptor [31]. In addition to rescue of cell-surface MHC-I, 2C was also shown to block HIV infectivity in a Nef-dependent manner [50]. These proof-of-concept studies were among the first to establish that inhibition of Nef-dependent kinase signaling is a viable approach to the development of Nef antagonists with the potential to reverse MHC-I display and immune escape. ...
While antiretroviral drugs have transformed the lives of HIV-infected individuals, chronic treatment is required to prevent rebound from viral reservoir cells. People living with HIV also are at higher risk for cardiovascular and neurocognitive complications, as well as cancer. Finding a cure for HIV-1 infection is therefore an essential goal of current AIDS research. This review is focused on the discovery of pharmacological inhibitors of the HIV-1 Nef accessory protein. Nef is well known to enhance HIV-1 infectivity and replication, and to promote immune escape of HIV-infected cells by preventing cell surface MHC-I display of HIV-1 antigens. Recent progress shows that Nef inhibitors not only suppress HIV-1 replication, but also restore sufficient MHC-I to the surface of infected cells to trigger a cytotoxic T lymphocyte response. Combining Nef inhibitors with latency reversal agents and therapeutic vaccines may provide a path to clearance of viral reservoirs.
... We used two recombinant HIV-1 viruses, the wild-type NL(AD8) strain (19) and the VSV envelope glycoprotein (VSV-G)-pseudotyped viruses, which are capable of infecting independently of the HIV-1 receptor CD4 (20). 293A cells (Invitrogen) cultured with DMEM with 10% FCS were employed as viral producer cells, as described previously (20). ...
... We used two recombinant HIV-1 viruses, the wild-type NL(AD8) strain (19) and the VSV envelope glycoprotein (VSV-G)-pseudotyped viruses, which are capable of infecting independently of the HIV-1 receptor CD4 (20). 293A cells (Invitrogen) cultured with DMEM with 10% FCS were employed as viral producer cells, as described previously (20). Briefly, the cells were transfected with HIV-1 proviral plasmids using the Lipofectamine 2000 reagent (Invitrogen). ...
... The proviral plasmid for the CCR5-tropic NL(AD8) strain was obtained through the National Institutes of Health (NIH) AIDS Reagent Program (Division of AIDS, National Institute of Allergy and Infectious Diseases, NIH). To prepare the VSV-Gpseudotyped viruses, the 293A cells were cotransfected with an NL43 strain-based envelope (Env)-defective mutant proviral plasmid (pNL-Kp) and VSV-G expression plasmid (20). ...
The proinflammatory cytokine IL-32 is elevated in the plasma and tissues of HIV-1-infected individuals. However, its significance in HIV-1 infection remains unclear because IL-32 inhibits and stimulates viral production in monocyte-derived macrophages (MDMs) and CD4+ T cells, respectively. In this study, we initially found that the inhibitory effect on human MDMs depends on SAMHD1, a dNTP triphosphohydrolase that inhibits viral reverse transcription. IL-32 increased the unphosphorylated active form of SAMHD1, which was consistent with the reduced expression of the upstream cyclin-dependent kinases. Indeed, IL-32 lost its anti-HIV-1 activity in MDMs when SAMHD1 was depleted. These results explain why IL-32 inhibits HIV-1 in MDMs but not CD4+ T cells, because SAMHD1 restricts HIV-1 in noncycling MDMs but not in cycling CD4+ T cells. Another unique feature of IL-32 is the induction of the immunosuppressive molecule IDO1, which is beneficial for HIV-1 infection. In this study, we found that IL-32 also upregulates other immunosuppressive molecules, including PD-L1, in MDMs. Moreover, IL-32 promoted the motility of MDMs, which potentially facilitates intercellular HIV-1 transmission. Our findings indicate that IL-32 has both the direct inhibitory effect on HIV-1 production in MDMs and the indirect stimulatory effects through phenotypic modulation of MDMs, and they suggest that the stimulatory effects may outweigh the inhibitory effect because the window for IL-32 to inhibit HIV-1 is relatively confined to SAMHD1-mediated reverse transcription suppression in the viral life cycle.
... The hK v 1.5 channel modelling, desflurane or sevoflurane docking, and three-dimensional representation were conducted using the Molecu- Goto, Kataoka, Muta, & Hirayama, 2008). We adopted the AMBER10:EHT force field to set partial charge and force-field parameters, and used the generalized born/volume integral (GB/VI) implicit solvent model (Labute, 2008) Furthermore, we calculated the docking pose of desflurane or sevoflurane by predicting its active-site within the inner cavity of the channel pore using a Site Finder (Chutiwitoonchai et al., 2011;Hiasa et al., 2013). The amino acids with side chain atoms located within 4.5 Å of the docked anaesthetics were regarded as having a potential contact. ...
Background and Purpose
Volatile anaesthetics have been shown to differentially modulate mammalian Shaker‐related voltage‐gated potassium (Kv1.x) channels. This study was designed to investigate molecular and cellular mechanisms underlying the modulatory effects of desflurane or sevoflurane on human Kv1.5 (hKv1.5) channels.
Experimental Approach
Thirteen single‐point mutations were constructed within pore domain of hKv1.5 channel using site‐directed mutagenesis. The effects of desflurane or sevoflurane on heterologously expressed wild‐type and mutant hKv1.5 channels were examined by whole‐cell patch‐clamp technique. A computer simulation was conducted to predict the docking pose of desflurane or sevoflurane within hKv1.5 channel.
Key Results
Both desflurane and sevoflurane increased hKv1.5 current at mild depolarizations but decreased it at strong depolarizations, indicating that these anaesthetics produce both stimulatory and inhibitory actions on hKv1.5 channels. The inhibitory effect of desflurane or sevoflurane on hKv1.5 channels arose primarily from its open‐channel blocking action. The inhibitory action of desflurane or sevoflurane on hKv1.5 channels was significantly attenuated in T480A, V505A, and I508A mutant channels, compared with wild‐type channel. Computational docking simulation predicted that desflurane or sevoflurane resides within the inner cavity of channel pore and has contact with Thr479, Thr480, Val505, and Ile508.
Conclusion and Implications
Desflurane and sevoflurane exert an open‐channel blocking action on hKv1.5 channels by functionally interacting with specific amino acids located within the channel pore. This study thus identifies a novel molecular basis mediating inhibitory modulation of hKv1.5 channels by desflurane and sevoflurane.
... Killing: conversely, the inhibition of Nef's activity using molecular adjuvants, such as 2c-like compounds (red hexagon) that block the interaction between Nef and SFKs (−Nef ) will restore cell surface levels of MHC-I which in turn will promote HIV antigen presentation (purple dot) and enhance susceptibility to HIV specific CD8 + -CTL viral replication is also dependent on Nef-SFK interactions. Cells expressing Nef AxxA 75 were deficient in Nef-mediated enhancement of viral replication by fivefold [19]. In addition, we described how disrupting the Nef-SFK interaction with the small molecule 2c blocked Nef-mediated MHC-I downregulation in human primary CD4 + T-cells [13]. ...
... In addition, we described how disrupting the Nef-SFK interaction with the small molecule 2c blocked Nef-mediated MHC-I downregulation in human primary CD4 + T-cells [13]. Subsequently, 2c was also reported to inhibit Nef PxxP 75 -dependent HIV-1 infectivity and replication in macrophages [19]. Similar results were obtained in human monocytes using diphenylfuropyriminde compounds, which block Nef PxxP 75 -dependent HIV replication by directly inhibiting SFK activity [20]. ...
The success of many current vaccines relies on a formulation that incorporates an immune activating adjuvant. This will hold true for the design of a successful therapeutic HIV vaccine targeted at controlling reactivated virus following cessation of combined antiretroviral therapy (cART). The HIV accessory protein Nef functions by interfering with HIV antigen presentation through the major histocompatibility complex I (MHC-I) pathway thereby suppressing CD8⁺ cytotoxic T cell (CTL)-mediated killing of HIV infected cells. Thus, this important impediment to HIV vaccine success must be circumvented. This review covers our current knowledge of Nef inhibitors that may serve as immune adjuvants that will specifically restore and enhance CTL-mediated killing of reactivated HIV infected cells as part of an overall vaccine strategy to affect a cure for HIV infection.
... rHIV-1 was prepared as described previously (30). HEK293 cells cultured with DMEM-10% FCS were used as viral producer cells. ...
... Thus, we focused on the initial phase of infection in the following experiments. The WT virus replicated more efficiently in MDMs than DNef virus (Fig. 1B, 1C) as reported previously (30). However, even when the replication of these two viruses was adjusted to equivalent levels (25 and 100 ng/ml input Gag for WT and DNef viruses, respectively; see Fig. 1B), the percentage of TNT-positive MDMs in WT virus-infected cultures was higher than that in DNef virus-infected cultures (Fig. 1D). ...
Tunneling nanotubes (TNTs), the long membrane extensions connecting distant cells, have emerged as a novel form of cell-to-cell communication. However, it is not fully understood how and to what extent TNTs contribute to intercellular spread of pathogens including HIV-1. In this study, we show that HIV-1 promotes TNT formation per se via its protein Nef and a cellular protein M-Sec, which appears to mediate approximately half of viral spread among monocyte-derived macrophages (MDMs). A small compound that inhibits M-Sec-induced TNT formation reduced HIV-1 production by almost half in MDMs. Such inhibition was not observed with Nef-deficient mutant HIV-1 that fails to promote TNT formation and replicates less efficiently than the wild-type HIV-1 in MDMs. The TNT inhibitor-sensitive/Nef-promoting viral production was also observed in a T cell line ectopically expressing M-Sec, but not in another M-Sec(-) T cell line. Our results suggest the importance of TNTs in HIV-1 spread among MDMs and might answer the long-standing question how Nef promotes HIV-1 production in a cell type-specific manner.
... Primary viruses were expanded as described previously (42). Recombinant viruses such as JRFL, AD8, and NL(AD8) were prepared using HEK293 cells as viral producer cells (43). HIV-1 replication assay with fibrocytes was performed as described for macrophages (40). ...
... The cell lysates were analyzed for Gag by Western blotting. The viral infectivity was assessed using TZM-bl cells as described previously (43). ...
Fibrocytes (fibroblastic leukocytes) are recently identified as unique hematopoietic cells with features of both macrophages and fibroblasts. Fibrocytes are known to contribute to the remodeling or fibrosis of various injured tissues. However, their role in viral infection is not fully understood. In this study, we show that differentiated fibrocytes are phenotypically distinguishable from macrophages but can be infected with HIV-1. Importantly, fibrocytes exhibited persistently infected cell-like phenotypes, the degree of which was more apparent than macrophages. The infected fibrocytes produced replication-competent HIV-1, but expressed HIV-1 mRNA at low levels and strongly resisted HIV-1-induced cell death, which enabled them to support an extremely long-term HIV-1 production at low but steady levels. More importantly, our results suggested that fibrocytes were susceptible to HIV-1 regardless of their differentiation state, in contrast to the fact that monocytes become susceptible to HIV-1 after the differentiation into macrophages. Our findings indicate that fibrocytes are the previously unreported HIV-1 host cells, and they suggest the importance of considering fibrocytes as one of the long-lived persistently infected cells for curing HIV-1.
... HIV-1 viruses were prepared as described previously (29,30). HEK293 cells (Invitrogen) were maintained in DMEM-10% FCS and used as viral producer cells. ...
... Then the supernatants containing the viruses were clarified by centrifugation, analyzed for their p24 HIV-1 Gag protein concentrations by ELISA (RET-ROtek p24 Ag ELISA kit; ZeptoMetrix, Buffalo, NY), and kept at 270˚C before use. The HIV-1 infection was performed essentially as described previously (29,30). Macrophages cultured on 24-well tissue culture plates were incubated with 250 ml of the supernatants of 293 cells containing HIV-1 viruses (the number of viruses was normalized by adjusting the p24 Gag protein concentrations to 100 ng/ml) for 2 h at 37˚C. ...
... Then the cells were washed twice with PBS to remove any unbound viruses. Half of the culture medium was replaced with complete medium every 3 d (29). In a selected experiment (see Fig. 4C), the infected macrophages were washed with PBS on day 3, and cultured in the absence or presence of IL-32g or M-CSF. ...
M-CSF promotes the differentiation and survival of macrophages, and preferentially induces anti-inflammatory M2, rather than proinflammatory M1 macrophages. Recently, another cytokine, IL-32, was also shown to promote macrophage differentiation. In this article, we provide the first evidence, to our knowledge, that M-CSF has both additive and inhibitory effects on the macrophage-related activities of IL-32. When added to M-CSF-derived macrophages, M-CSF and IL-32 promoted macrophage survival, which was further enhanced by their combination. However, they had different effects on HIV-1 replication; that is, it was stimulated by M-CSF and inhibited by IL-32. Interestingly, the anti-HIV-1 activity of IL-32 was counteracted by M-CSF. Such inhibitory effect of M-CSF was not observed with IL-32-induced M1-like features including high cytokine/chemokine production and strong expression of the costimulatory molecule CD80. However, IL-32-treated macrophages unexpectedly showed also M2-like features including increased phagocytic activity, and high expression of CD14 and the scavenger receptor CD163, and the expression of CD14 and CD163 was further upregulated by cotreatment with M-CSF. The findings of this study regarding the unique functional interplay between M-CSF and IL-32 increase our understanding of the mechanisms that regulate the survival and M1/M2 ratio of macrophages, as well as HIV-1 replication in macrophages.
... In einer weiteren Studie wurde in Replikationsversuchen mit 2c ein inhibitorischer Effekt auf die Nef-vermittelte HIV-1 Infektiosität beobachtet, ohne das es zu toxischen oder zytopathischen Effekten kam. Zudem bestätigten kompetitive pull-down-Versuche, dass 2c mit der Hck Bindung an Nef kompetiert (Chutiwitoonchai et al., 2011). ...
... HIV Nef is relatively small, polymorphic protein (27)(28)(29)(30) that is packaged in the virion and is also expressed at high levels early in the viral life cycle. Nef is myristoylated on its Nterminus, which helps to attach it to cellular membranes essential for function [10]. ...
... As expected, HIV-1-induced CD4 downregulation was unaffected [21]. In addition to its effects on MHC-I downregulation, 2c was also observed to interfere with HIV infectivity in a Nef-dependent manner [27]. This observation supports a role for the Nef-assembled multi-kinase complex in both immune evasion and viral infectivity. ...
Although antiretroviral therapy has revolutionized the clinical management of AIDS, life-long treatment is required because these drugs do not eradicate HIV-infected cells. Chronic antiretroviral therapy may not protect AIDS patients from cognitive impairment, raising important quality of life issues. Because of the rise of HIV strains resistant to current drugs and uncertain vaccine prospects, an urgent need exists for the discovery and development of new therapeutic approaches. This review is focused on one such approach, which involves targeting HIV-1 Nef, a viral accessory protein essential for AIDS pathogenesis.
... Separation of these and other functional motifs in the 3D structure of Nef creates a challenge for drug discovery, 10 as small molecules that selectively target individual interfaces may inhibit only a subset of Nef functions. 11,12 Previous biochemical and structural studies have demonstrated that Nef forms homodimers and possibly higherorder oligomers in vitro. 13,14 For example, Nef/SH3 complexes crystallize as dimers, with the Nef/Nef dimerization interface formed by the nearly orthogonal packing of the αB helices of the folded Nef core. ...
Nef is a human immunodeficiency virus 1 (HIV-1) accessory factor essential for viral pathogenesis and AIDS progression. Many Nef functions require dimerization, and small molecules that block Nef dimerization may represent antiretroviral drug leads. Here we describe a cell-based assay for Nef dimerization inhibitors based on bimolecular fluorescence complementation (BiFC). Nef was fused to nonfluorescent, complementary fragments of yellow fluorescent protein (YFP) and coexpressed in the same cell population. Dimerization of Nef resulted in juxtaposition of the YFP fragments and reconstitution of the fluorophore. For automation, the Nef-YFP fusion proteins plus a monomeric red fluorescent protein (mRFP) reporter were expressed from a single vector, separated by picornavirus "2A" linker peptides to permit equivalent translation of all three proteins. Validation studies revealed a critical role for gating on the mRFP-positive subpopulation of transfected cells, as well as use of the mRFP signal to normalize the Nef-BiFC signal. Nef-BiFC/mRFP ratios resulting from cells expressing wild-type versus dimerization-defective Nef were very clearly separated, with Z factors consistently in the 0.6 to 0.7 range. A fully automated pilot screen of the National Cancer Institute Diversity Set III identified several hit compounds that reproducibly blocked Nef dimerization in the low micromolar range. This BiFC-based assay has the potential to identify cell-active small molecules that directly interfere with Nef dimerization and function.
