RESUMO
Ebola viruses (EBOVs) assemble into filamentous virions, whose shape and stability are determined by the matrix viral protein 40 (VP40). Virus entry into host cells occurs via membrane fusion in late endosomes; however, the mechanism of how the remarkably long virions undergo uncoating, including virion disassembly and nucleocapsid release into the cytosol, remains unknown. Here, we investigate the structural architecture of EBOVs entering host cells and discover that the VP40 matrix disassembles prior to membrane fusion. We reveal that VP40 disassembly is caused by the weakening of VP40-lipid interactions driven by low endosomal pH that equilibrates passively across the viral envelope without a dedicated ion channel. We further show that viral membrane fusion depends on VP40 matrix integrity, and its disassembly reduces the energy barrier for fusion stalk formation. Thus, pH-driven structural remodeling of the VP40 matrix acts as a molecular switch coupling viral matrix uncoating to membrane fusion during EBOV entry.
Assuntos
Ebolavirus , Doença pelo Vírus Ebola , Humanos , Doença pelo Vírus Ebola/metabolismo , Fusão de Membrana , Proteínas do Core Viral/metabolismo , Endossomos/metabolismo , Proteínas da Matriz ViralRESUMO
Determining the label to target ratio, also known as the degree of labeling (DOL), is crucial for quantitative fluorescence microscopy and a high DOL with minimal unspecific labeling is beneficial for fluorescence microscopy in general. Yet robust, versatile and easy-to-use tools for measuring cell-specific labeling efficiencies are not available. Here we present a DOL determination technique named protein-tag DOL (ProDOL), which enables fast quantification and optimization of protein-tag labeling. With ProDOL various factors affecting labeling efficiency, including substrate type, incubation time and concentration, as well as sample fixation and cell type can be easily assessed. We applied ProDOL to investigate how human immunodeficiency virus-1 pathogenesis factor Nef modulates CD4 T cell activation measuring total and activated copy numbers of the adapter protein SLP-76 in signaling microclusters. ProDOL proved to be a versatile and robust tool for labeling calibration, enabling determination of labeling efficiencies, optimization of strategies and quantification of protein stoichiometry.
RESUMO
The implementation of antiretroviral therapy (ART) has effectively restricted the transmission of Human Immunodeficiency Virus (HIV) and improved overall clinical outcomes. However, a complete cure for HIV remains out of reach, as the virus persists in a stable pool of infected cell reservoir that is resistant to therapy and thus a main barrier towards complete elimination of viral infection. While the mechanisms by which host proteins govern viral gene expression and latency are well-studied, the emerging regulatory functions of non-coding RNAs (ncRNA) in the context of T cell activation, HIV gene expression and viral latency have not yet been thoroughly explored. Here, we report the identification of the Cytoskeleton Regulator (CYTOR) long non-coding RNA (lncRNA) as an activator of HIV gene expression that is upregulated following T cell stimulation. Functional studies show that CYTOR suppresses viral latency by directly binding to the HIV promoter and associating with the cellular positive transcription elongation factor (P-TEFb) to activate viral gene expression. CYTOR also plays a global role in regulating cellular gene expression, including those involved in controlling actin dynamics. Depletion of CYTOR expression reduces cytoplasmic actin polymerization in response to T cell activation. In addition, treating HIV-infected cells with pharmacological inhibitors of actin polymerization reduces HIV gene expression. We conclude that both direct and indirect effects of CYTOR regulate HIV gene expression.
Assuntos
Regulação Viral da Expressão Gênica , Infecções por HIV , HIV-1 , RNA Longo não Codificante , Latência Viral , Humanos , Infecções por HIV/virologia , Infecções por HIV/genética , HIV-1/genética , HIV-1/fisiologia , Células Jurkat , Ativação Linfocitária , Regiões Promotoras Genéticas , RNA Longo não Codificante/genéticaRESUMO
CD4+ T cells are central mediators of adaptive and innate immune responses and constitute a major reservoir for human immunodeficiency virus (HIV) in vivo. Detailed investigations of resting human CD4+ T cells have been precluded by the absence of efficient approaches for genetic manipulation limiting our understanding of HIV replication and restricting efforts to find a cure. Here we report a method for rapid, efficient, activation-neutral gene editing of resting, polyclonal human CD4+ T cells using optimized cell cultivation and nucleofection conditions of Cas9-guide RNA ribonucleoprotein complexes. Up to six genes, including HIV dependency and restriction factors, were knocked out individually or simultaneously and functionally characterized. Moreover, we demonstrate the knock in of double-stranded DNA donor templates into different endogenous loci, enabling the study of the physiological interplay of cellular and viral components at single-cell resolution. Together, this technique allows improved molecular and functional characterizations of HIV biology and general immune functions in resting CD4+ T cells.
Assuntos
Linfócitos T CD4-Positivos/fisiologia , Sistemas CRISPR-Cas/genética , Edição de Genes/métodos , Infecções por HIV/genética , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/virologia , Proteína 9 Associada à CRISPR/genética , Movimento Celular/genética , Células Cultivadas , DNA , Técnicas de Inativação de Genes , Infecções por HIV/metabolismo , Humanos , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Proteínas de Resistência a Myxovirus/genética , Proteínas de Resistência a Myxovirus/metabolismo , RNA Guia de Cinetoplastídeos , Proteína 1 com Domínio SAM e Domínio HD/genética , Transgenes , Fatores de Poliadenilação e Clivagem de mRNA/genética , Fatores de Poliadenilação e Clivagem de mRNA/metabolismoRESUMO
Immature dendritic cells (iDCs) migrate in microenvironments with distinct cell and extracellular matrix densities in vivo and contribute to HIV-1 dissemination and mounting of antiviral immune responses. Here, we find that, compared to standard 2D suspension cultures, 3D collagen as tissue-like environment alters iDC properties and their response to HIV-1 infection. iDCs adopt an elongated morphology with increased deformability in 3D collagen at unaltered activation, differentiation, cytokine secretion, or responsiveness to LPS. While 3D collagen reduces HIV-1 particle uptake by iDCs, fusion efficiency is increased to elevate productive infection rates due to elevated cell surface exposure of the HIV-1-binding receptor DC-SIGN. In contrast, 3D collagen reduces HIV transfer to CD4 T cells from iDCs. iDC adaptations to 3D collagen include increased pro-inflammatory cytokine production and reduced antiviral gene expression in response to HIV-1 infection. Adhesion to a 2D collagen matrix is sufficient to increase iDC deformability, DC-SIGN exposure, and permissivity to HIV-1 infection. Thus, mechano-physical cues of 2D and 3D tissue-like collagen environments regulate iDC function and shape divergent roles during HIV-1 infection.
Assuntos
Infecções por HIV , HIV-1 , Humanos , Citocinas/metabolismo , Colágeno/metabolismo , Antivirais , Células DendríticasRESUMO
Failures to produce neutralizing antibodies upon HIV-1 infection result in part from B-cell dysfunction due to unspecific B-cell activation. How HIV-1 affects antigen-specific B-cell functions remains elusive. Using an adoptive transfer mouse model and ex vivo HIV infection of human tonsil tissue, we found that expression of the HIV-1 pathogenesis factor NEF in CD4 T cells undermines their helper function and impairs cognate B-cell functions including mounting of efficient specific IgG responses. NEF interfered with T cell help via a specific protein interaction motif that prevents polarized cytokine secretion at the T-cell-B-cell immune synapse. This interference reduced B-cell activation and proliferation and thus disrupted germinal center formation and affinity maturation. These results identify NEF as a key component for HIV-mediated dysfunction of antigen-specific B cells. Therapeutic targeting of the identified molecular surface in NEF will facilitate host control of HIV infection.
Assuntos
Linfócitos B/imunologia , Linfócitos T CD4-Positivos/imunologia , Infecções por HIV/imunologia , Animais , Células HEK293 , HIV-1 , Humanos , Evasão da Resposta Imune/imunologia , Ativação Linfocitária , Masculino , Camundongos , Camundongos Endogâmicos C57BLRESUMO
MOTIVATION: Live-cell microscopy has become an essential tool for analyzing dynamic processes in various biological applications. Thereby, high-throughput and automated tracking analyses allow the simultaneous evaluation of large numbers of objects. However, to critically assess the influence of individual objects on calculated summary statistics, and to detect heterogeneous dynamics or possible artifacts, such as misclassified or -tracked objects, a direct mapping of gained statistical information onto the actual image data would be necessary. RESULTS: We present VisuStatR as a platform independent software package that allows the direct visualization of time-resolved summary statistics of morphological characteristics or motility dynamics onto raw images. The software contains several display modes to compare user-defined summary statistics and the underlying image data in various levels of detail. AVAILABILITY AND IMPLEMENTATION: VisuStatR is a free and open-source R-package, containing a user-friendly graphical-user interface and is available via GitHub at https://github.com/grrchrr/VisuStatR/ under the MIT+ license. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
Assuntos
Microscopia , Software , Artefatos , LicenciamentoRESUMO
HIV-1 has to overcome physical barriers posed by host cell restriction factors (RFs) for efficient replication. Some RFs, including Trim5α and tetherin, trigger antiviral signaling in addition to directly impairing HIV replication. SERINC5 (S5) is an RF that is incorporated into HIV-1 particles to potently impair their infectivity and is efficiently antagonized by the viral pathogenesis factor Nef. Since effects of S5 on HIV-1 infectivity were mostly studied in reporter cell lines, we analyzed the effects of S5 during infection of primary HIV-1 target cells. In activated CD4+ T lymphocytes, virion incorporation of S5 only moderately impaired virion infectivity and was not associated with altered innate immune recognition. In contrast, in monocyte-derived macrophages, S5 virion incorporation potentiated the production of proinflammatory cytokines with very potent but donor-dependent effects on virion infectivity. Nef counteracted effects of S5 on both cytokine production and virion infectivity. Similar S5-induced cytokine production was observed in immature monocyte-derived dendritic cells. Notably, S5-mediated enhancement of cytokine production was not linked to the efficacy of productive infection and could be overcome by using vesicular stomatitis virus glycoprotein (VSV-G) but not infectivity restriction-insensitive HIV-1 Env for cell entry. Moreover, inhibiting entry of S5-negative HIV-1 ΔNef particles increased proinflammatory cytokine production comparably to virion incorporation of S5. Together, these results describe the sensitization of noninfectious HIV-1 particles to proinflammatory cytokine production by myeloid target cells as an additional and Nef-sensitive activity of S5. Moreover, the study reveals important cell-type and donor-dependent differences in the sensitivity of HIV target cells for antiviral effects of S5.IMPORTANCE SERINC5 (S5) is a host cell restriction factor (RF) that impairs the infectivity of HIV-1 particles in target cell lines. To assess the potential physiological relevance of this restriction, we assessed the effects of S5 on HIV-1 infection of relevant primary human target cells. We found that effects of S5 on infection of CD4+ T lymphocytes were negligible. In myeloid target cells, however, virion incorporation of S5 potently suppressed infectivity and promoted innate immune recognition of HIV-1 particles characterized by proinflammatory cytokine production. Both effects were not observed in cells of all donors analyzed, were exerted independently of one another, and were counteracted by the HIV-1 pathogenesis factor Nef. These results identify the sensitization of HIV-1 particles for innate immune recognition by myeloid target cells as a novel activity of S5 and emphasize the need to study RF function in the context of primary target cells and taking donor variabilities into account.
Assuntos
Citocinas/metabolismo , Infecções por HIV/virologia , HIV-1/fisiologia , Interações entre Hospedeiro e Microrganismos , Proteínas de Membrana/metabolismo , Produtos do Gene nef do Vírus da Imunodeficiência Humana/metabolismo , Linfócitos T CD4-Positivos/imunologia , Células Dendríticas/imunologia , Células HEK293 , Humanos , Leucócitos Mononucleares/imunologia , Macrófagos/imunologia , Células Mieloides/imunologia , Vírion/metabolismoRESUMO
HIV-1 Nef promotes virus spread and disease progression by altering host cell transport and signaling processes through interaction with multiple host cell proteins. The N-terminal region in HIV-1 Nef encompassing residues 12 to 39 has been implicated in many Nef activities, including disruption of CD4 T lymphocyte polarization and homing to lymph nodes, antagonism of SERINC5 restriction to virion infectivity, downregulation of cell surface CD4 and major histocompatibility complex class I (MHC-I), release of Nef-containing extracellular vesicles, and phosphorylation of Nef by recruitment of the Nef-associated kinase complex (NAKC). How this region mediates these pleiotropic functions is unclear. Characterization of a panel of alanine mutants spanning the N-terminal region to identify specific functional determinants revealed this region to be dispensable for effects of Nef from HIV-1 strain SF2 (HIV-1SF2Nef) on T cell actin organization and chemotaxis, retargeting of the host cell kinase Lck to the trans-Golgi network, and incorporation of Nef into extracellular vesicles. MHC-I downmodulation was specific to residue M20, and inhibition of T cell polarization by Nef required the integrity of the entire region. In contrast, downmodulation of cell surface CD4 and SERINC5 antagonism were mediated by a specific motif encompassing residues 32 to 39 that was also essential for efficient HIV replication in primary CD4 T lymphocytes. Finally, Nef phosphorylation via association with the NAKC was mediated by two EP repeats within residues 24 to 29 but was dispensable for other functions. These results identify the N-terminal region as a multifunctional interaction module for at least three different host cell ligands that mediate independent functions of HIV-1SF2Nef to facilitate immune evasion and virus spread.IMPORTANCE HIV-1 Nef critically determines virus spread and disease progression in infected individuals by acting as a protein interaction adaptor via incompletely defined mechanisms and ligands. Residues 12 to 39 near the N terminus of Nef have been described as an interaction platform for the Nef-associated kinase complex (NAKC) and were recently identified as essential determinants for a broad range of Nef activities. Here, we report a systematic mapping of this amino acid stretch that revealed the presence of three independent interaction motifs with specific ligands and activities. While downmodulation of cell surface MHC-I depends on M20, two EP repeats are the minimal binding site for the NAKC, and residues 32 to 39 mediate antagonism of the host cell restriction factor SERINC5 as well as downmodulation of cell surface CD4. These results reveal that the N-terminal region of HIV-1SF2Nef is a versatile and multifunctional protein interaction module that exerts essential functions of the pathogenicity factor via independent mechanisms.
Assuntos
HIV-1/genética , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/genética , Domínios Proteicos , Vírion/genética , Produtos do Gene nef do Vírus da Imunodeficiência Humana/genética , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Sítios de Ligação , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/virologia , Células COS , Chlorocebus aethiops , Expressão Gênica , Células HEK293 , HIV-1/metabolismo , Humanos , Evasão da Resposta Imune , Células Jurkat , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/metabolismo , Mutação , Cultura Primária de Células , Ligação Proteica , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Vírion/metabolismo , Produtos do Gene nef do Vírus da Imunodeficiência Humana/química , Produtos do Gene nef do Vírus da Imunodeficiência Humana/metabolismoRESUMO
SERINC5 is a host restriction factor that impairs infectivity of HIV-1 and other primate lentiviruses and is counteracted by the viral accessory protein Nef. However, the importance of SERINC5 antagonism for viral replication and cytopathicity remained unclear. Here, we show that the Nef protein of the highly divergent SIVcol lineage infecting mantled guerezas (Colobus guereza) is a potent antagonist of SERINC5, although it lacks the CD4, CD3 and CD28 down-modulation activities exerted by other primate lentiviral Nefs. In addition, SIVcol Nefs decrease CXCR4 cell surface expression, suppress TCR-induced actin remodeling, and counteract Colobus but not human tetherin. Unlike HIV-1 Nef proteins, SIVcol Nef induces efficient proteasomal degradation of SERINC5 and counteracts orthologs from highly divergent vertebrate species, such as Xenopus frogs and zebrafish. A single Y86F mutation disrupts SERINC5 and tetherin antagonism but not CXCR4 down-modulation by SIVcol Nef, while mutation of a C-proximal di-leucine motif has the opposite effect. Unexpectedly, the Y86F change in SIVcol Nef had little if any effect on viral replication and CD4+ T cell depletion in preactivated human CD4+ T cells and in ex vivo infected lymphoid tissue. However, SIVcol Nef increased virion infectivity up to 10-fold and moderately increased viral replication in resting peripheral blood mononuclear cells (PBMCs) that were first infected with HIV-1 and activated three or six days later. In conclusion, SIVcol Nef lacks several activities that are conserved in other primate lentiviruses and utilizes a distinct proteasome-dependent mechanism to counteract SERINC5. Our finding that evolutionarily distinct SIVcol Nefs show potent anti-SERINC5 activity supports a relevant role of SERINC5 antagonism for viral fitness in vivo. Our results further suggest this Nef function is particularly important for virion infectivity under conditions of limited CD4+ T cell activation.
Assuntos
Linfócitos T CD4-Positivos/virologia , Produtos do Gene nef/fisiologia , HIV-1/fisiologia , Tecido Linfoide/virologia , Proteínas de Membrana/metabolismo , Replicação Viral/genética , Animais , Linfócitos T CD4-Positivos/metabolismo , Células Cultivadas , Colobus/virologia , Células HEK293 , Humanos , Células Jurkat , Proteínas de Membrana/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteólise , Vírus da Imunodeficiência Símia/genéticaRESUMO
Most biological systems are difficult to analyse due to a multitude of interacting components and the concomitant lack of information about the essential dynamics. Finding appropriate models that provide a systematic description of such biological systems and that help to identify their relevant factors and processes can be challenging given the sheer number of possibilities. Model selection algorithms that evaluate the performance of a multitude of different models against experimental data provide a useful tool to identify appropriate model structures. However, many algorithms addressing the analysis of complex dynamical systems, as they are often used in biology, compare a preselected number of models or rely on exhaustive searches of the total model space which might be unfeasible dependent on the number of possibilities. Therefore, we developed an algorithm that is able to perform model selection on complex systems and searches large model spaces in a dynamical way. Our algorithm includes local and newly developed non-local search methods that can prevent the algorithm from ending up in local minima of the model space by accounting for structurally similar processes. We tested and validated the algorithm based on simulated data and showed its flexibility for handling different model structures. We also used the algorithm to analyse experimental data on the cell proliferation dynamics of CD4+ and CD8+ T cells that were cultured under different conditions. Our analyses indicated dynamical changes within the proliferation potential of cells that was reduced within tissue-like 3D ex vivo cultures compared to suspension. Due to the flexibility in handling various model structures, the algorithm is applicable to a large variety of different biological problems and represents a useful tool for the data-oriented evaluation of complex model spaces.
Assuntos
Algoritmos , Modelos Biológicos , Biologia de Sistemas/estatística & dados numéricos , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD8-Positivos/citologia , Técnicas de Cultura de Células/métodos , Proliferação de Células , Biologia Computacional , Simulação por Computador , HumanosRESUMO
HIV-1 Nef is a multifunctional protein that optimizes virus spread and promotes immune evasion of infected cells to accelerate disease progression in AIDS patients. As one of its activities, Nef reduces the motility of infected CD4+ T lymphocytes in confined space. In vivo, Nef restricts T lymphocyte homing to lymph nodes as it reduces the ability for extravasation at the diapedesis step. Effects of Nef on T lymphocyte motility are typically mediated by its ability to reduce actin remodeling. However, interference with diapedesis does not depend on residues in Nef required for inhibition of host cell actin dynamics. In search for an alternative mechanism by which Nef could alter T lymphocyte extravasation, we noted that the viral protein interferes with the polarization of primary human CD4+ T lymphocytes upon infection with HIV-1. Expression of Nef alone is sufficient to disrupt T cell polarization, and this effect is conserved among lentiviral Nef proteins. Nef acts by arresting the oscillation of CD4+ T cells between polarized and nonpolarized morphologies. Mapping studies identified the binding site for the Nef-associated kinase complex (NAKC) as critical determinant of this Nef activity and a NAKC-binding-deficient Nef variant fails to impair CD4+ T lymphocyte extravasation and homing to lymph nodes. These results thus imply the disruption of T lymphocyte polarity via its NAKC binding site as a novel mechanism by which lentiviral Nef proteins alter T lymphocyte migration in vivo.
Assuntos
Linfócitos T CD4-Positivos/virologia , Polaridade Celular/imunologia , Quimiotaxia de Leucócito/imunologia , Migração Transendotelial e Transepitelial/imunologia , Produtos do Gene nef do Vírus da Imunodeficiência Humana/metabolismo , Animais , Sítios de Ligação , Linfócitos T CD4-Positivos/imunologia , Humanos , Linfonodos/imunologia , Ativação Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Early after entry into monocytes, macrophages, dendritic cells, and resting CD4 T cells, HIV encounters a block, limiting reverse transcription (RT) of the incoming viral RNA genome. In this context, dNTP triphosphohydrolase SAM domain and HD domain-containing protein 1 (SAMHD1) has been identified as a restriction factor, lowering the concentration of dNTP substrates to limit RT. The accessory lentiviral protein X (Vpx) proteins from the major simian immunodeficiency virus of rhesus macaque, sooty mangabey, and HIV-2 (SIVsmm/SIVmac/HIV-2) lineage packaged into virions target SAMHD1 for proteasomal degradation, increase intracellular dNTP pools, and facilitate HIV cDNA synthesis. We find that virion-packaged Vpx proteins from a second SIV lineage, SIV of red-capped mangabeys or mandrills (SIVrcm/mnd-2), increased HIV infection in resting CD4 T cells, but not in macrophages, and, unexpectedly, acted in the absence of SAMHD1 degradation, dNTP pool elevation, or changes in SAMHD1 phosphorylation. Vpx rcm/mnd-2 virion incorporation resulted in a dramatic increase of HIV-1 RT intermediates and viral cDNA in infected resting CD4 T cells. These analyses also revealed a barrier limiting HIV-1 infection of resting CD4 T cells at the level of nuclear import. Single amino acid changes in the SAMHD1-degrading Vpx mac239 allowed it to enhance early postentry steps in a Vpx rcm/mnd-2-like fashion. Moreover, Vpx enhanced HIV-1 infection of SAMHD1-deficient resting CD4 T cells of a patient with Aicardi-Goutières syndrome. These results indicate that Vpx, in addition to SAMHD1, overcomes a previously unappreciated restriction for lentiviruses at the level of RT that acts independently of dNTP concentrations and is specific to resting CD4 T cells.
Assuntos
Infecções por HIV/genética , Transcrição Reversa/genética , Proteína 1 com Domínio SAM e Domínio HD/genética , Proteínas Virais Reguladoras e Acessórias/genética , Animais , Linfócitos T CD4-Positivos/virologia , Genoma Viral/genética , Infecções por HIV/virologia , HIV-1/genética , HIV-1/patogenicidade , HIV-2/genética , HIV-2/patogenicidade , Interações Hospedeiro-Patógeno/genética , Humanos , Macaca mulatta/genética , Macaca mulatta/virologia , Monócitos/virologia , Proteólise , RNA Viral/genética , Vírion/genética , Vírion/patogenicidade , Replicação Viral/genéticaRESUMO
The HIV type 1 pathogenicity factor Nef enhances viral replication by modulating multiple host cell pathways, including tuning the activation state of infected CD4 T lymphocytes to optimize virus spread. For this, Nef inhibits anterograde transport of the Src family kinase (SFK) Lck toward the plasma membrane (PM). This leads to retargeting of the kinase to the trans-Golgi network, whereas the intracellular transport of a related SFK, Fyn, is unaffected by Nef. The 18-amino acid Src homology 4 (SH4) domain membrane anchor of Lck is necessary and sufficient for Nef-mediated retargeting, but other details of this process are not known. The goal of this study was therefore to identify characteristics of SH4 domains responsive to Nef and the transport machinery used. Screening a panel of SFK SH4 domains revealed two groups that were sensitive or insensitive for trans-Golgi network retargeting by Nef as well as the importance of the amino acid at position 8 for determining Nef sensitivity. Anterograde transport of Nef-sensitive domains was characterized by slower delivery to the PM and initial targeting to Golgi membranes, where transport was arrested in the presence of Nef. For Nef-sensitive SH4 domains, ectopic expression of the lipoprotein binding chaperone Unc119a or the GTPase Arl3 or reduction of their endogenous expression phenocopied the effect of Nef. Together, these results suggest that, analogous to K-Ras, Nef-sensitive SH4 domains are transported to the PM by a cycle of solubilization and membrane insertion and that intrinsic properties define SH4 domains as cargo of this Nef-sensitive lipoprotein binding chaperone-GTPase transport cycle.
Assuntos
Membrana Celular/metabolismo , HIV-1/metabolismo , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/metabolismo , Linfócitos T/virologia , Produtos do Gene nef do Vírus da Imunodeficiência Humana/metabolismo , Rede trans-Golgi/metabolismo , Fatores de Ribosilação do ADP/genética , Fatores de Ribosilação do ADP/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Membrana Celular/virologia , Regulação da Expressão Gênica , HIV-1/genética , Interações Hospedeiro-Patógeno , Humanos , Células Jurkat , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/química , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/genética , Cultura Primária de Células , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Transporte Proteico , Proteínas Proto-Oncogênicas c-fyn/genética , Proteínas Proto-Oncogênicas c-fyn/metabolismo , Transdução de Sinais , Linfócitos T/metabolismo , Produtos do Gene nef do Vírus da Imunodeficiência Humana/genética , Rede trans-Golgi/virologiaRESUMO
A first step towards the development of a human immunodeficiency virus (HIV) animal model has been the identification and surmounting of species-specific barriers encountered by HIV along its replication cycle in cells from small animals. Serine incorporator proteins 3 (SERINC3) and 5 (SERINC5) were recently identified as restriction factors that reduce HIV-1 infectivity. Here, we compared the antiviral activity of SERINC3 and SERINC5 among mice, rats and rabbits, and their susceptibility to viral counteraction to their human counterparts. In the absence of viral antagonists, rodent and lagomorph SERINC3 and SERINC5 displayed anti-HIV activity in a similar range to human controls. Vesicular stomatitis virus G protein (VSV-G) pseudotyped virions were considerably less sensitive to restriction by all SERINC3/5 orthologs. Interestingly, HIV-1 Nef, murine leukemia virus (MLV) GlycoGag and equine infectious anemia virus (EIAV) S2 counteracted the antiviral activity of all SERINC3/5 orthologs with similar efficiency. Our results demonstrate that the antiviral activity of SERINC3/5 proteins is conserved in rodents and rabbits, and can be overcome by all three previously reported viral antagonists.
Assuntos
HIV-1/crescimento & desenvolvimento , HIV-1/imunologia , Interações Hospedeiro-Patógeno , Fatores Imunológicos/metabolismo , Produtos do Gene nef do Vírus da Imunodeficiência Humana/metabolismo , Animais , Vetores Genéticos , Camundongos , Coelhos , Ratos , Vesiculovirus/genética , Vesiculovirus/crescimento & desenvolvimentoRESUMO
Motile cells and pathogens migrate in complex environments and yet are mostly studied on simple 2D substrates. In order to mimic the diverse environments of motile cells, a set of assays including substrates of defined elasticity, microfluidics, micropatterns, organotypic cultures, and 3D gels have been developed. We briefly introduce these and then focus on the use of micropatterned pillar arrays, which help to bridge the gap between 2D and 3D. These structures are made from polydimethylsiloxane, a moldable plastic, and their use has revealed new insights into mechanoperception in Caenorhabditis elegans, gliding motility of Plasmodium, swimming of trypanosomes, and nuclear stability in cancer cells. These studies contributed to our understanding of how the environment influences the respective cell and inform on how the cells adapt to their natural surroundings on a cellular and molecular level.
Assuntos
Movimento Celular/fisiologia , Animais , Bioensaio/métodos , Caenorhabditis elegans/patogenicidade , Dimetilpolisiloxanos , Humanos , Plasmodium/patogenicidadeRESUMO
The host-cell restriction factor SERINC5 potently suppresses the infectivity of HIV, type 1 (HIV-1) particles, and is counteracted by the viral pathogenesis factor Nef. However, the molecular mechanism by which SERINC5 restricts HIV-1 particle infectivity is still unclear. Because SERINC proteins have been suggested to facilitate the incorporation of serine during the biosynthesis of membrane lipids and because lipid composition of HIV particles is a major determinant of the infectious potential of the particles, we tested whether SERINC5-mediated restriction of HIV particle infectivity involves alterations of membrane lipid composition. We produced and purified HIV-1 particles from SERINC5293T cells with very low endogenous SERINC5 levels under conditions in which ectopically expressed SERINC5 restricts HIV-1 infectivity and is antagonized by Nef and analyzed both virions and producer cells with quantitative lipid MS. SERINC5 restriction and Nef antagonism were not associated with significant alterations in steady-state lipid composition of producer cells and HIV particles. Sphingosine metabolism kinetics were also unaltered by SERINC5 expression. Moreover, the levels of phosphatidylserine on the surface of HIV-1 particles, which may trigger uptake into non-productive internalization pathways in target cells, did not change upon expression of SERINC5 or Nef. Finally, saturating the phosphatidylserine-binding sites on HIV target cells did not affect SERINC5 restriction or Nef antagonism. These results demonstrate that the restriction of HIV-1 particle infectivity by SERINC5 does not depend on alterations in lipid composition and organization of HIV-1 particles and suggest that channeling serine into lipid biosynthesis may not be a cardinal cellular function of SERINC5.
Assuntos
HIV-1/patogenicidade , Metabolismo dos Lipídeos , Proteínas de Membrana/metabolismo , Vírion/patogenicidade , Produtos do Gene nef do Vírus da Imunodeficiência Humana/metabolismo , Antígenos de Superfície/genética , Antígenos de Superfície/metabolismo , Ligação Competitiva , Linhagem Celular Tumoral , Deleção de Genes , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , HIV-1/química , HIV-1/fisiologia , Humanos , Cinética , Lipossomos , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/genética , Proteínas do Leite/genética , Proteínas do Leite/metabolismo , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Fosfatidilserinas/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Esfingosina/metabolismo , Propriedades de Superfície , Vírion/química , Vírion/fisiologia , Montagem de Vírus , Produtos do Gene nef do Vírus da Imunodeficiência Humana/genéticaRESUMO
AIDS is the result of a constant struggle between the lentivirus HIV and the immune system. Infection with HIV interferes directly with the function of CD4(+) T cells and manipulates the host immune response to the virus. Recent studies indicate that the viral protein Nef, a central player in HIV pathogenesis, impairs the ability of infected lymphocytes to form immunological synapses with antigen-presenting cells and affects T-cell-receptor-mediated stimulation. An integrative picture of the abnormal behaviour of HIV-infected lymphocytes is therefore emerging. We propose that modulating lymphocyte signalling, apoptosis and intracellular trafficking ensures efficient spread of the virus in the hostile environment of the immune system.
Assuntos
Síndrome da Imunodeficiência Adquirida/imunologia , Células Apresentadoras de Antígenos/imunologia , Linfócitos T CD4-Positivos/imunologia , Comunicação Celular/imunologia , Produtos do Gene nef/imunologia , HIV-1/imunologia , Animais , Humanos , Transporte Proteico/imunologia , Receptor Cross-Talk/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Produtos do Gene nef do Vírus da Imunodeficiência HumanaRESUMO
SERINC3 (serine incorporator 3) and SERINC5 are recently identified host cell inhibitors of HIV-1 particle infectivity that are counteracted by the viral pathogenesis factor Nef. Here we confirm that HIV-1 Nef, but not HIV-1 Vpu, antagonizes the particle infectivity restriction of SERINC5. SERINC5 antagonism occurred in parallel with other Nef activities, including cell surface receptor downregulation, trans-Golgi network targeting of Lck, and inhibition of host cell actin dynamics. Interaction motifs with host cell endocytic machinery and the Nef-associated kinase complex, as well as CD4 cytoplasmic tail/HIV-1 protease, were identified as essential Nef determinants for SERINC5 antagonism. Characterization of antagonism-deficient Nef mutants revealed that counteraction of SERINC5 occurs in the absence of retargeting of the restriction factor to intracellular compartments and reduction of SERINC5 cell surface density is insufficient for antagonism. Consistent with virion incorporation of SERINC5 being a prerequisite for its antiviral activity, the infectivity of HIV-1 particles produced in the absence of a SERINC5 antagonist decreased with increasing amounts of virion SERINC5. At low levels of SERINC5 expression, enhancement of virion infectivity by Nef was associated with reduced virion incorporation of SERINC5 and antagonism-defective Nef mutants failed to exclude SERINC5 from virions. However, at elevated levels of SERINC5 expression, Nef maintained infectious HIV particles, despite significant virion incorporation of the restriction factor. These results suggest that in addition to virion exclusion, Nef employs a cryptic mechanism to antagonize virion-associated SERINC5. The involvement of common determinants suggests that the antagonism of Nef to SERINC5 and the downregulation of cell surface CD4 by Nef involve related molecular mechanisms. IMPORTANCE: HIV-1 Nef critically determines virus spread and disease progression in infected individuals by incompletely defined mechanisms. SERINC3 and SERINC5 were recently identified as potent inhibitors of HIV particle infectivity whose antiviral activity is antagonized by HIV-1 Nef. To address the mechanism of SERINC5 antagonism, we identified four molecular determinants of Nef antagonism that are all linked to the mechanism by which Nef downregulates cell surface CD4. Functional characterization of these mutants revealed that endosomal targeting and cell surface downregulation of SERINC5 are dispensable and insufficient for antagonism, respectively. In contrast, virion exclusion and antagonism of SERINC5 were correlated; however, Nef was also able to enhance the infectivity of virions that incorporated robust levels of SERINC5. These results suggest that the antagonism of HIV-1 Nef to SERINC5 restriction of virion infectivity is mediated by a dual mechanism that is related to CD4 downregulation.
Assuntos
HIV-1/fisiologia , HIV-1/patogenicidade , Proteínas de Membrana/fisiologia , Produtos do Gene nef do Vírus da Imunodeficiência Humana/fisiologia , Antígenos CD4/metabolismo , Regulação para Baixo , Genes Virais , Infecções por HIV/imunologia , Infecções por HIV/virologia , HIV-1/genética , Interações Hospedeiro-Patógeno/genética , Interações Hospedeiro-Patógeno/imunologia , Interações Hospedeiro-Patógeno/fisiologia , Humanos , Proteínas de Membrana/genética , Mutação , Vírion/genética , Vírion/fisiologia , Virulência/genética , Virulência/fisiologia , Produtos do Gene nef do Vírus da Imunodeficiência Humana/genéticaRESUMO
UNLABELLED: Production of proinflammatory cytokines indicative of potent recognition by the host innate immune system has long been recognized as a hallmark of the acute phase of HIV-1 infection. The first components of the machinery by which primary HIV target cells sense infection have recently been described; however, the mechanistic dissection of innate immune recognition and viral evasion would be facilitated by an easily accessible cell line model. Here we describe that reconstituted expression of the innate signaling adaptor STING enhanced the ability of the well-established HIV reporter cell line Tzm-bl to sense HIV infection and to convert this information into nuclear translocation of IRF3 as well as expression of cytokine mRNA. STING-dependent immune sensing of HIV-1 required virus entry and reverse transcription but not genome integration. Particularly efficient recognition was observed for an HIV-1 variant lacking expression of the accessory protein Vpr, suggesting a role of the viral protein in circumventing STING-mediated immune signaling. Vpr as well as STING significantly impacted the magnitude and breadth of the cytokine mRNA expression profile induced upon HIV-1 infection. However, cytoplasmic DNA sensing did not result in detectable cytokine secretion in this cell system, and innate immune recognition did not affect infection rates. Despite these deficits in eliciting antiviral effector functions, these results establish Tzm-bl STING and Tzm-bl STING IRF3.GFP cells as useful tools for studies aimed at dissecting mechanisms and regulation of early innate immune recognition of HIV infection. IMPORTANCE: Cell-autonomous immune recognition of HIV infection was recently established as an important aspect by which the host immune system attempts to fend off HIV-1 infection. Mechanistic studies on host cell recognition and viral evasion are hampered by the resistance of many primary HIV target cells to detailed experimental manipulation. We describe here that expression of the signaling adaptor STING renders the well-established HIV reporter cell line Tzm-bl competent for innate recognition of HIV infection. Key characteristics reflected in this cell model include nuclear translocation of IRF3, expression of a broad range of cytokine mRNAs, and an antagonistic activity of the HIV-1 protein Vpr. These results establish Tzm-bl STING and Tzm-bl STING IRF3.GFP cells as a useful tool for studies of innate recognition of HIV infection.