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Mark Varrelmann - One of the best experts on this subject based on the ideXlab platform.
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occurrence of resistance breaking strains of beet necrotic yellow vein virus in sugar beet in northwestern europe and identification of a new variant of the viral Pathogenicity Factor p25
Plant Pathology, 2015Co-Authors: Kathrin Bornemann, Mark Varrelmann, B Hanse, M StevensAbstract:Rhizomania, one of the most devastating diseases in sugar beet production, is caused by Beet necrotic yellow vein virus (BNYVV) and transmitted by Polymyxa betae. Previously, disease control was possible by cultivation of sugar beet hybrids carrying a major resistance gene Rz1, which restricts virus accumulation in taproots and suppresses symptom development. Over the last few years, BNYVV strains with four RNA components have arisen, which are able to overcome Rz1-mediated resistance. All strains described so far possess an A67V amino acid exchange within the RNA3-encoded P25 Pathogenicity Factor. In this study, BNYVV was isolated from Rz1 plants, collected in the United Kingdom, the Netherlands and Germany, displaying patches of strong rhizomania symptoms. Sequencing of the coat protein and P25 gene of three isolates showed 100% nucleotide sequence identity and detected AYPR as the P25 tetrad amino acid composition. The ability of this strain to accumulate to higher levels in young plants of Rz1 resistant but not in Rz1 + Rz2 resistant genotypes was initially demonstrated in a greenhouse assay in natural field soil from the Netherlands. This strain was loaded into a virus-free P. betae population and compared to reference strains. The AYPR strain retained its resistance-breaking ability in the Rz1 genotypes and displayed replication at a higher rate compared to the Rz1-resistance-breaking P type. The strain origin is unclear and it remains speculative whether the occurrence at different geographic locations is the result of independent selection or displacement of infested soil.
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effect of sugar beet genotype on the beet necrotic yellow vein virus p25 Pathogenicity Factor and evidence for a fitness penalty in resistance breaking strains
Molecular Plant Pathology, 2013Co-Authors: Kathrin Bornemann, Mark VarrelmannAbstract:Summary Beet necrotic yellow vein virus (BNYVV), vectored by Polymyxa betae, causes rhizomania in sugar beet. For disease control, the cultivation of hybrids carrying Rz1 resistance is crucial, but is compromised by resistance-breaking (RB) strains with specific mutations in the P25 protein at amino acids 67–70 (tetrad). To obtain evidence for P25 variability from soil-borne populations, where the virus persists for decades, populations with wild-type (WT) and RB properties were analysed by P25 deep sequencing. The level of P25 variation in the populations analysed did not correlate with RB properties. Remarkably, one WT population contained P25 with RB mutations at a frequency of 11%. To demonstrate selection by Rz1 and the influence of RB mutations on relative fitness, competition experiments between strains were performed. Following a mixture of strains with four RNAs, a shift in tetrad variants was observed, suggesting that strains did not mix or transreplicate. The plant genotype exerted a clear influence on the frequency of RB tetrads. In Rz1 plants, the RB variants outcompeted the WT variants, and mostly vice versa in susceptible plants, demonstrating a relative fitness penalty of RB mutations. The strong genotype effect supports the hypothesized Rz1 RB strain selection with four RNAs, suggesting that a certain tetrad needs to become dominant in a population to influence its properties. Tetrad selection was not observed when an RB strain, with an additional P26 protein encoded by a fifth RNA, competed with a WT strain, supporting its role as a second BNYVV Pathogenicity Factor and suggesting the reassortment of both types.
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the p25 Pathogenicity Factor of beet necrotic yellow vein virus targets the sugar beet 26s proteasome involved in the induction of a hypersensitive resistance response via interaction with an f box protein
Molecular Plant-microbe Interactions, 2012Co-Authors: Heike Thiel, Kamal Hleibieh, David Gilmer, Mark VarrelmannAbstract:P25, a Beet necrotic yellow vein virus (BNYVV) Pathogenicity Factor, interacts with a sugar beet protein with high homology to Arabidopsis thaliana kelch repeat containing F-box family proteins (FBK) of unknown function in yeast. FBK are members of the Skp1-Cullin-F-box (SCF) complex that mediate protein degradation. Here, we confirm this sugar beet FBK–P25 interaction in vivo and in vitro and provide evidence for in planta interaction and similar subcellular distribution in Nicotiana tabacum leaf cells. P25 even interacts with an FBK from A. thaliana, a BNYVV nonhost. FBK functional classification was possible by demonstrating the interaction with A. thaliana orthologs of Skp1-like (ASK) genes, a member of the SCF E3 ligase. By means of a yeast two-hybrid bridging assay, a direct effect of P25 on SCF-complex formation involving ASK1 protein was demonstrated. FBK transient Agrobacterium tumefaciens–mediated expression in N. benthamiana leaves induced a hypersensitive response. The full-length F-box protei...
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The P25 Pathogenicity Factor of Beet necrotic yellow vein virus Targets the Sugar Beet 26S Proteasome Involved in the Induction of a Hypersensitive Resistance Response via Interaction with an F-box Protein
Molecular plant-microbe interactions : MPMI, 2012Co-Authors: Heike Thiel, Kamal Hleibieh, David Gilmer, Mark VarrelmannAbstract:P25, a Beet necrotic yellow vein virus (BNYVV) Pathogenicity Factor, interacts with a sugar beet protein with high homology to Arabidopsis thaliana kelch repeat containing F-box family proteins (FBK) of unknown function in yeast. FBK are members of the Skp1-Cullin-F-box (SCF) complex that mediate protein degradation. Here, we confirm this sugar beet FBK-P25 interaction in vivo and in vitro and provide evidence for in planta interaction and similar subcellular distribution in Nicotiana tabacum leaf cells. P25 even interacts with an FBK from A. thaliana, a BNYVV nonhost. FBK functional classification was possible by demonstrating the interaction with A. thaliana orthologs of Skp1-like (ASK) genes, a member of the SCF E3 ligase. By means of a yeast two-hybrid bridging assay, a direct effect of P25 on SCF-complex formation involving ASK1 protein was demonstrated. FBK transient Agrobacterium tumefaciens-mediated expression in N. benthamiana leaves induced a hypersensitive response. The full-length F-box protein consists of one F-box domain followed by two kelch repeats, which alone were unable to interact with P25 in yeast and did not lead to cell-death induction. The results support the idea that P25 is involved in virus Pathogenicity in sugar beet and suggest suppression of resistance response.
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identification of beet necrotic yellow vein virus p25 Pathogenicity Factor interacting sugar beet proteins that represent putative virus targets or components of plant resistance
Molecular Plant-microbe Interactions, 2009Co-Authors: Heike Thiel, Mark VarrelmannAbstract:Beet necrotic yellow vein virus (BNYVV) induces the most important disease threatening sugar beet. The growth of partially resistant hybrids carrying monogenic dominant resistance genes stabilize yield but are unable to entirely prevent virus infection and replication. P25 is responsible for symptom development and previous studies have shown that recently occurring resistance-breaking isolates possess increased P25 variability. To better understand the viral Pathogenicity Factor's interplay with plant proteins and to possibly unravel the molecular basis of sugar beet antivirus resistance, P25 was applied in a yeast two-hybrid screen of a resistant sugar beet cDNA library. This screen identified candidate proteins recognized as orthologues from other plant species which are known to be expressed following pathogen infection and involved in plant defense response. Most of the candidates potentially related to host-pathogen interactions were involved in the ubiquitylation process and plants response to stress, and were part of cell and metabolism components. The interaction of several candidate genes with P25 was confirmed in Nicotiana benthamiana leaf cells by transient agrobacterium-mediated expression applying bimolecular fluorescence complementation assay. The putative functions of several of the candidates identified support previous findings and present first targets for understanding the BNYVV Pathogenicity and antivirus resistance mechanism.
Sidi Chen - One of the best experts on this subject based on the ideXlab platform.
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nonstructural protein 1 of sars cov 2 is a potent Pathogenicity Factor redirecting host protein synthesis machinery toward viral rna
Molecular Cell, 2020Co-Authors: Shuai Yuan, Lei Peng, Jonathan J Park, Swapnil C Devarkar, Matthew B Dong, Qi Shen, Sidi ChenAbstract:The causative virus of the COVID-19 pandemic, SARS-CoV-2, uses its nonstructural protein 1 (Nsp1) to suppress cellular, but not viral, protein synthesis through yet unknown mechanisms. We show here that among all viral proteins, Nsp1 has the largest impact on host viability in the cells of human lung origin. Differential expression analysis of mRNA-seq data revealed that Nsp1 broadly alters the cellular transcriptome. Our cryo-EM structure of the Nsp1-40S ribosome complex shows that Nsp1 inhibits translation by plugging the mRNA entry channel of the 40S. We also determined the structure of the 48S preinitiation complex formed by Nsp1, 40S, and the cricket paralysis virus internal ribosome entry site (IRES) RNA, which shows that it is nonfunctional because of the incorrect position of the mRNA 3' region. Our results elucidate the mechanism of host translation inhibition by SARS-CoV-2 and advance understanding of the impacts from a major Pathogenicity Factor of SARS-CoV-2.
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nonstructural protein 1 of sars cov 2 is a potent Pathogenicity Factor redirecting host protein synthesis machinery toward viral rna
bioRxiv, 2020Co-Authors: Shuai Yuan, Lei Peng, Jonathan J Park, Swapnil C Devarkar, Matthew B Dong, Sidi Chen, Ivan B Lomakin, Yong XiongAbstract:Summary The COVID-19 pandemic affects millions of people worldwide with a rising death toll. The causative agent, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), uses its nonstructural protein 1 (Nsp1) to redirect host translation machinery to the viral RNA by binding to the ribosome and suppressing cellular, but not viral, protein synthesis through yet unknown mechanisms. We show here that among all viral proteins, Nsp1 has the largest impact on host viability in the cells of human lung origin. Differential expression analysis of mRNA-seq data revealed that Nsp1 broadly alters the transcriptome in human cells. The changes include repression of major gene clusters in ribosomal RNA processing, translation, mitochondria function, cell cycle and antigen presentation; and induction of Factors in transcriptional regulation. We further gained a mechanistic understanding of the Nsp1 function by determining the cryo-EM structure of the Nsp1-40S ribosomal subunit complex, which shows that Nsp1 inhibits translation by plugging the mRNA entry channel of the 40S. We also determined the cryo-EM structure of the 48S preinitiation complex (PIC) formed by Nsp1, 40S, and the cricket paralysis virus (CrPV) internal ribosome entry site (IRES) RNA, which shows that this 48S PIC is nonfunctional due to the incorrect position of the 3’ region of the mRNA. Results presented here elucidate the mechanism of host translation inhibition by SARS-CoV-2, provide insight into viral protein synthesis, and furnish a comprehensive understanding of the impacts from one of the most potent Pathogenicity Factors of SARS-CoV-2. Highlights ORF screen identified Nsp1 as a major cellular Pathogenicity Factor of SARS-CoV-2 Nsp1 broadly alters the gene expression programs in human cells Nsp1 inhibits translation by blocking mRNA entry channel Nsp1 prevents physiological conformation of the 48S PIC
Oliver T. Fackler - One of the best experts on this subject based on the ideXlab platform.
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molecular design functional characterization and structural basis of a protein inhibitor against the hiv 1 Pathogenicity Factor nef
PLOS ONE, 2011Co-Authors: Sebastian Breuer, Oliver T. Fackler, Simone I Schievink, Antje Schulte, Wulf Blankenfeldt, Matthias GeyerAbstract:Increased spread of HIV-1 and rapid emergence of drug resistance warrants development of novel antiviral strategies. Nef, a critical viral Pathogenicity Factor that interacts with host cell Factors but lacks enzymatic activity, is not targeted by current antiviral measures. Here we inhibit Nef function by simultaneously blocking several highly conserved protein interaction surfaces. This strategy, referred to as “wrapping Nef”, is based on structure-function analyses that led to the identification of four target sites: (i) SH3 domain interaction, (ii) interference with protein transport processes, (iii) CD4 binding and (iv) targeting to lipid membranes. Screening combinations of Nef-interacting domains, we developed a series of small Nef interacting proteins (NIs) composed of an SH3 domain optimized for binding to Nef, fused to a sequence motif of the CD4 cytoplasmic tail and combined with a prenylation signal for membrane association. NIs bind to Nef in the low nM affinity range, associate with Nef in human cells and specifically interfere with key biological activities of Nef. Structure determination of the Nef-inhibitor complex reveals the molecular basis for binding specificity. These results establish Nef-NI interfaces as promising leads for the development of potent Nef inhibitors.
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the hiv 1 Pathogenicity Factor nef interferes with maturation of stimulatory t lymphocyte contacts by modulation of n wasp activity
Journal of Biological Chemistry, 2006Co-Authors: Claudia Haller, Nico Michel, Oliver T Keppler, Susanne Rauch, Sebastian Hannemann, Maik J Lehmann, Oliver T. FacklerAbstract:The Nef protein is a key determinant of human immunodeficiency virus (HIV) Pathogenicity that, among other activities, sensitizes T-lymphocytes for optimal virus production. The initial events by which Nef modulates the T-cell receptor (TCR) cascade are poorly understood. TCR engagement triggers actin rearrangements that control receptor clustering for signal initiation and dynamic organization of signaling protein complexes to form an immunological synapse. Here we report that Nef potently interferes with cell spreading and formation of actin-rich circumferential rings in T-lymphocytes upon surface-supported TCR stimulation. These effects were conserved among Nef proteins from different lentiviruses and occurred in HIV-1-infected primary human T-lymphocytes. This novel Nef activity critically depended on its Src homology 3 domain binding motif and required efficient association with Pak2 activity. Notably, whereas overall signaling microcluster formation immediately following TCR engagement occurred normally in Nef-expressing cells, the viral protein inhibited the concomitant activation of the actin organizer N-Wasp. During the subsequent maturation phase of the stimulatory contact, Nef interfered with the translocation of N-Wasp to the cell periphery, the overall induction of tyrosine phosphorylation, and the selective recruitment of phosphorylated LAT to stimulatory contacts. Consistent with such a critical role of N-Wasp in this process, Nef also blocked morphological changes induced by the known N-Wasp regulators Rac1 and Cdc42. Together, our results demonstrate that Nef alters both the amount and composition of signaling microclusters. We propose modulation of actin dynamics as an important mechanism for Nef-induced alterations of TCR signaling.
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the nef protein of human immunodeficiency virus is a broad spectrum modulator of chemokine receptor cell surface levels that acts independently of classical motifs for receptor endocytosis and galphai signaling
Molecular Biology of the Cell, 2006Co-Authors: Nico Michel, Oliver T. Fackler, Kerstin Ganter, Stephanie Venzke, Julia Bitzegeio, Oliver T KepplerAbstract:Chemokine receptors (CKRs) are important physiological mediators of immune defense, inflammatory responses, and angiogenesis, and they have also been implicated in a number of viral disease processes. Here, we report that the Nef protein of human immunodeficiency virus (HIV) reduces cell surface levels of eight different members of the CC- and CXC-family of CKRs by up to 92%. This broad-range activity required specific elements in HIV(SF2) Nef, including the proline-rich motif P73P76P79P82 as well as the acidic cluster motif E66E67E68E69, and Nef expression induced a marked perinuclear accumulation of CKRs. Surprisingly, receptor mutagenesis demonstrated that the cytoplasmic tail of CCR5 and CXCR4, which is critical for basal and ligand-mediated endocytosis, was completely dispensable for this Nef activity. In contrast, triple-mutation of the highly conserved DRY motif in the second intracellular CKR loop abolished the Nef-mediated down-regulation of CXCR4 independently of this motif's role in CKR binding to heterotrimeric G proteins and signaling via the Galphai subunit. Thus, we identify the lentiviral Pathogenicity Factor Nef as a unique and broad-range modulator of CKR cell surface levels. Nef uses a mechanism that is distinct from well-established pathways orchestrating CKR metabolism and offers an interesting tool to study the multifaceted biology of CKRs.
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the nef protein of human immunodeficiency virus establishes superinfection immunity by a dual strategy to downregulate cell surface ccr5 and cd4
Current Biology, 2005Co-Authors: Nico Michel, Oliver T. Fackler, Stephanie Venzke, Ina Allespach, Oliver T KepplerAbstract:Summary Background : Viruses frequently render cells refractory to subsequent infection with the same virus. This state of superinfection immunity counteracts potentially detrimental consequences for the infected cell and facilitates high-level replication and viral spread in the host. Results: Here, we show that human immunodeficiency virus (HIV) employs its early gene product Nef to efficiently interfere with superinfection at the viral-entry step. In this context, we identify the downregulation of cell-surface CCR5, the major HIV coreceptor, as a novel and highly conserved activity of Nef. Nef targets the CCR5 coreceptor and the HIV binding receptor CD4 via distinct cellular machineries to enhance the endocytosis rate of both HIV receptor components and to accelerate their degradation. Functionally, these genetically separable actions by Nef synergized to efficiently protect cells from HIV superinfection at the level of fusion of the viral envelope with the plasma membrane. Conclusions: HIV has evolved two independent activities for Nef to downregulate the receptor complex and to facilitate its efficient replication and spread. This evasion strategy likely represents a mechanism by which the Pathogenicity Factor Nef elevates viral replication in vivo and thus promotes AIDS pathogenesis.
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Rodent cells support key functions of the human immunodeficiency virus type 1 Pathogenicity Factor Nef.
Journal of Virology, 2005Co-Authors: Oliver T Keppler, Ina Allespach, Lismarie Schüller, David Fenard, Warner C. Greene, Oliver T. FacklerAbstract:After infection with human immunodeficiency virus (HIV), progression toward immunodeficiency is governed by a complex interplay of viral and host determinants. The viral accessory protein Nef is a key Factor for the development of AIDS. Strains of HIV and simian immunodeficiency virus that lack functional nef genes either do not induce AIDS or do so only after a significant delay. The validity of a transgenic-small-animal model for de novo infection by HIV will depend on its ability to recapitulate the actions of critical Factors of viral Pathogenicity, such as Nef. We assessed the ability of rat, mouse, and hamster cells to support key effector functions of Nef. In cell lines from rodents, the subcellular distribution of wild-type HIV type 1 strain SF2 Nef and mutants was comparable to that in human cells. Nef downregulated human CD4 from the cell surface, was associated with p21-activated kinase activity, and enhanced the infectivity of HIV-1 virions. Importantly, these Nef-induced effects, as well as the downregulation of rat CD4 and major histocompatibility complex class I molecules, could also be demonstrated in primary T lymphocytes and macrophages from human CD4-transgenic rats. Thus, HIV-1 Nef exerts key functions in rodent cells. In line with our ongoing efforts to establish a transgenic-rat model of HIV disease, these results indicate that important aspects of viral pathogenesis could be addressed in a transgenic-rodent model permissive for de novo infection and that such a model would be valuable for evaluating the function of Nef in vivo.
Heike Thiel - One of the best experts on this subject based on the ideXlab platform.
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the p25 Pathogenicity Factor of beet necrotic yellow vein virus targets the sugar beet 26s proteasome involved in the induction of a hypersensitive resistance response via interaction with an f box protein
Molecular Plant-microbe Interactions, 2012Co-Authors: Heike Thiel, Kamal Hleibieh, David Gilmer, Mark VarrelmannAbstract:P25, a Beet necrotic yellow vein virus (BNYVV) Pathogenicity Factor, interacts with a sugar beet protein with high homology to Arabidopsis thaliana kelch repeat containing F-box family proteins (FBK) of unknown function in yeast. FBK are members of the Skp1-Cullin-F-box (SCF) complex that mediate protein degradation. Here, we confirm this sugar beet FBK–P25 interaction in vivo and in vitro and provide evidence for in planta interaction and similar subcellular distribution in Nicotiana tabacum leaf cells. P25 even interacts with an FBK from A. thaliana, a BNYVV nonhost. FBK functional classification was possible by demonstrating the interaction with A. thaliana orthologs of Skp1-like (ASK) genes, a member of the SCF E3 ligase. By means of a yeast two-hybrid bridging assay, a direct effect of P25 on SCF-complex formation involving ASK1 protein was demonstrated. FBK transient Agrobacterium tumefaciens–mediated expression in N. benthamiana leaves induced a hypersensitive response. The full-length F-box protei...
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The P25 Pathogenicity Factor of Beet necrotic yellow vein virus Targets the Sugar Beet 26S Proteasome Involved in the Induction of a Hypersensitive Resistance Response via Interaction with an F-box Protein
Molecular plant-microbe interactions : MPMI, 2012Co-Authors: Heike Thiel, Kamal Hleibieh, David Gilmer, Mark VarrelmannAbstract:P25, a Beet necrotic yellow vein virus (BNYVV) Pathogenicity Factor, interacts with a sugar beet protein with high homology to Arabidopsis thaliana kelch repeat containing F-box family proteins (FBK) of unknown function in yeast. FBK are members of the Skp1-Cullin-F-box (SCF) complex that mediate protein degradation. Here, we confirm this sugar beet FBK-P25 interaction in vivo and in vitro and provide evidence for in planta interaction and similar subcellular distribution in Nicotiana tabacum leaf cells. P25 even interacts with an FBK from A. thaliana, a BNYVV nonhost. FBK functional classification was possible by demonstrating the interaction with A. thaliana orthologs of Skp1-like (ASK) genes, a member of the SCF E3 ligase. By means of a yeast two-hybrid bridging assay, a direct effect of P25 on SCF-complex formation involving ASK1 protein was demonstrated. FBK transient Agrobacterium tumefaciens-mediated expression in N. benthamiana leaves induced a hypersensitive response. The full-length F-box protein consists of one F-box domain followed by two kelch repeats, which alone were unable to interact with P25 in yeast and did not lead to cell-death induction. The results support the idea that P25 is involved in virus Pathogenicity in sugar beet and suggest suppression of resistance response.
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identification of beet necrotic yellow vein virus p25 Pathogenicity Factor interacting sugar beet proteins that represent putative virus targets or components of plant resistance
Molecular Plant-microbe Interactions, 2009Co-Authors: Heike Thiel, Mark VarrelmannAbstract:Beet necrotic yellow vein virus (BNYVV) induces the most important disease threatening sugar beet. The growth of partially resistant hybrids carrying monogenic dominant resistance genes stabilize yield but are unable to entirely prevent virus infection and replication. P25 is responsible for symptom development and previous studies have shown that recently occurring resistance-breaking isolates possess increased P25 variability. To better understand the viral Pathogenicity Factor's interplay with plant proteins and to possibly unravel the molecular basis of sugar beet antivirus resistance, P25 was applied in a yeast two-hybrid screen of a resistant sugar beet cDNA library. This screen identified candidate proteins recognized as orthologues from other plant species which are known to be expressed following pathogen infection and involved in plant defense response. Most of the candidates potentially related to host-pathogen interactions were involved in the ubiquitylation process and plants response to stress, and were part of cell and metabolism components. The interaction of several candidate genes with P25 was confirmed in Nicotiana benthamiana leaf cells by transient agrobacterium-mediated expression applying bimolecular fluorescence complementation assay. The putative functions of several of the candidates identified support previous findings and present first targets for understanding the BNYVV Pathogenicity and antivirus resistance mechanism.
Oliver T Keppler - One of the best experts on this subject based on the ideXlab platform.
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the hiv 1 Pathogenicity Factor nef interferes with maturation of stimulatory t lymphocyte contacts by modulation of n wasp activity
Journal of Biological Chemistry, 2006Co-Authors: Claudia Haller, Nico Michel, Oliver T Keppler, Susanne Rauch, Sebastian Hannemann, Maik J Lehmann, Oliver T. FacklerAbstract:The Nef protein is a key determinant of human immunodeficiency virus (HIV) Pathogenicity that, among other activities, sensitizes T-lymphocytes for optimal virus production. The initial events by which Nef modulates the T-cell receptor (TCR) cascade are poorly understood. TCR engagement triggers actin rearrangements that control receptor clustering for signal initiation and dynamic organization of signaling protein complexes to form an immunological synapse. Here we report that Nef potently interferes with cell spreading and formation of actin-rich circumferential rings in T-lymphocytes upon surface-supported TCR stimulation. These effects were conserved among Nef proteins from different lentiviruses and occurred in HIV-1-infected primary human T-lymphocytes. This novel Nef activity critically depended on its Src homology 3 domain binding motif and required efficient association with Pak2 activity. Notably, whereas overall signaling microcluster formation immediately following TCR engagement occurred normally in Nef-expressing cells, the viral protein inhibited the concomitant activation of the actin organizer N-Wasp. During the subsequent maturation phase of the stimulatory contact, Nef interfered with the translocation of N-Wasp to the cell periphery, the overall induction of tyrosine phosphorylation, and the selective recruitment of phosphorylated LAT to stimulatory contacts. Consistent with such a critical role of N-Wasp in this process, Nef also blocked morphological changes induced by the known N-Wasp regulators Rac1 and Cdc42. Together, our results demonstrate that Nef alters both the amount and composition of signaling microclusters. We propose modulation of actin dynamics as an important mechanism for Nef-induced alterations of TCR signaling.
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the nef protein of human immunodeficiency virus is a broad spectrum modulator of chemokine receptor cell surface levels that acts independently of classical motifs for receptor endocytosis and galphai signaling
Molecular Biology of the Cell, 2006Co-Authors: Nico Michel, Oliver T. Fackler, Kerstin Ganter, Stephanie Venzke, Julia Bitzegeio, Oliver T KepplerAbstract:Chemokine receptors (CKRs) are important physiological mediators of immune defense, inflammatory responses, and angiogenesis, and they have also been implicated in a number of viral disease processes. Here, we report that the Nef protein of human immunodeficiency virus (HIV) reduces cell surface levels of eight different members of the CC- and CXC-family of CKRs by up to 92%. This broad-range activity required specific elements in HIV(SF2) Nef, including the proline-rich motif P73P76P79P82 as well as the acidic cluster motif E66E67E68E69, and Nef expression induced a marked perinuclear accumulation of CKRs. Surprisingly, receptor mutagenesis demonstrated that the cytoplasmic tail of CCR5 and CXCR4, which is critical for basal and ligand-mediated endocytosis, was completely dispensable for this Nef activity. In contrast, triple-mutation of the highly conserved DRY motif in the second intracellular CKR loop abolished the Nef-mediated down-regulation of CXCR4 independently of this motif's role in CKR binding to heterotrimeric G proteins and signaling via the Galphai subunit. Thus, we identify the lentiviral Pathogenicity Factor Nef as a unique and broad-range modulator of CKR cell surface levels. Nef uses a mechanism that is distinct from well-established pathways orchestrating CKR metabolism and offers an interesting tool to study the multifaceted biology of CKRs.
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the nef protein of human immunodeficiency virus establishes superinfection immunity by a dual strategy to downregulate cell surface ccr5 and cd4
Current Biology, 2005Co-Authors: Nico Michel, Oliver T. Fackler, Stephanie Venzke, Ina Allespach, Oliver T KepplerAbstract:Summary Background : Viruses frequently render cells refractory to subsequent infection with the same virus. This state of superinfection immunity counteracts potentially detrimental consequences for the infected cell and facilitates high-level replication and viral spread in the host. Results: Here, we show that human immunodeficiency virus (HIV) employs its early gene product Nef to efficiently interfere with superinfection at the viral-entry step. In this context, we identify the downregulation of cell-surface CCR5, the major HIV coreceptor, as a novel and highly conserved activity of Nef. Nef targets the CCR5 coreceptor and the HIV binding receptor CD4 via distinct cellular machineries to enhance the endocytosis rate of both HIV receptor components and to accelerate their degradation. Functionally, these genetically separable actions by Nef synergized to efficiently protect cells from HIV superinfection at the level of fusion of the viral envelope with the plasma membrane. Conclusions: HIV has evolved two independent activities for Nef to downregulate the receptor complex and to facilitate its efficient replication and spread. This evasion strategy likely represents a mechanism by which the Pathogenicity Factor Nef elevates viral replication in vivo and thus promotes AIDS pathogenesis.
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Rodent cells support key functions of the human immunodeficiency virus type 1 Pathogenicity Factor Nef.
Journal of Virology, 2005Co-Authors: Oliver T Keppler, Ina Allespach, Lismarie Schüller, David Fenard, Warner C. Greene, Oliver T. FacklerAbstract:After infection with human immunodeficiency virus (HIV), progression toward immunodeficiency is governed by a complex interplay of viral and host determinants. The viral accessory protein Nef is a key Factor for the development of AIDS. Strains of HIV and simian immunodeficiency virus that lack functional nef genes either do not induce AIDS or do so only after a significant delay. The validity of a transgenic-small-animal model for de novo infection by HIV will depend on its ability to recapitulate the actions of critical Factors of viral Pathogenicity, such as Nef. We assessed the ability of rat, mouse, and hamster cells to support key effector functions of Nef. In cell lines from rodents, the subcellular distribution of wild-type HIV type 1 strain SF2 Nef and mutants was comparable to that in human cells. Nef downregulated human CD4 from the cell surface, was associated with p21-activated kinase activity, and enhanced the infectivity of HIV-1 virions. Importantly, these Nef-induced effects, as well as the downregulation of rat CD4 and major histocompatibility complex class I molecules, could also be demonstrated in primary T lymphocytes and macrophages from human CD4-transgenic rats. Thus, HIV-1 Nef exerts key functions in rodent cells. In line with our ongoing efforts to establish a transgenic-rat model of HIV disease, these results indicate that important aspects of viral pathogenesis could be addressed in a transgenic-rodent model permissive for de novo infection and that such a model would be valuable for evaluating the function of Nef in vivo.