Drebrin restricts rotavirus entry by inhibiting dynamin-mediated endocytosis

Despite the wide administration of several effective vaccines, rotavirus (RV) remains the single most important etiological agent of severe diarrhea in infants and young children worldwide, with an annual mortality of over 200,000 people. RV attachment and internalization into target cells is mediated by its outer capsid protein VP4. To better understand the molecular details of RV entry, we performed tandem affinity purification coupled with high-resolution mass spectrometry to map the host proteins that interact with VP4. We identified an actin-binding protein, drebrin (DBN1), that coprecipitates and colocalizes with VP4 during RV infection. Importantly, blocking DBN1 function by siRNA silencing, CRISPR knockout (KO), or chemical inhibition significantly increased host cell susceptibility to RV infection.Dbn1KO mice exhibited higher incidence of diarrhea and more viral antigen shedding in their stool samples compared with the wild-type littermates. In addition, we found that uptake of other dynamin-dependent cargos, including transferrin, cholera toxin, and multiple viruses, was also enhanced in DBN1-deficient cells. Inhibition of cortactin or dynamin-2 abrogated the increased virus entry observed in DBN1-deficient cells, suggesting that DBN1 suppresses dynamin-mediated endocytosis via interaction with cortactin. Our study unveiled an unexpected role of DBN1 in restricting the entry of RV and other viruses into host cells and more broadly to function as a crucial negative regulator of diverse dynamin-dependent endocytic pathways.

Download Full-text

GSK-3α Inhibition in Drug-Resistant CML Cells Promotes Susceptibility to NK Cell-Mediated Lysis in an NKG2D- and NKp30-Dependent Manner

Cancers ◽

10.3390/cancers13081802 ◽

2021 ◽

Vol 13 (8) ◽

pp. 1802

Author(s):

Nayoung Kim ◽

Mi Yeon Kim ◽

Woo Seon Choi ◽

Eunbi Yi ◽

Hyo Jung Lee ◽

...

Keyword(s):

Nk Cells ◽

Nk Cell ◽

Negative Regulator ◽

Target Cells ◽

Dependent Manner ◽

Cell Functions ◽

Cell Based Therapy ◽

Activating Receptors ◽

Gsk 3Β

Natural killer (NK) cells are innate cytotoxic lymphocytes that provide early protection against cancer. NK cell cytotoxicity against cancer cells is triggered by multiple activating receptors that recognize specific ligands expressed on target cells. We previously demonstrated that glycogen synthase kinase (GSK)-3β, but not GSK-3α, is a negative regulator of NK cell functions via diverse activating receptors, including NKG2D and NKp30. However, the role of GSK-3 isoforms in the regulation of specific ligands on target cells is poorly understood, which remains a challenge limiting GSK-3 targeting for NK cell-based therapy. Here, we demonstrate that GSK-3α rather than GSK-3β is the primary isoform restraining the expression of NKG2D ligands, particularly ULBP2/5/6, on tumor cells, thereby regulating their susceptibility to NK cells. GSK-3α also regulated the expression of the NKp30 ligand B7-H6, but not the DNAM-1 ligands PVR or nectin-2. This regulation occurred independently of BCR-ABL1 mutation that confers tyrosine kinase inhibitor (TKI) resistance. Mechanistically, an increase in PI3K/Akt signaling in concert with c-Myc was required for ligand upregulation in response to GSK-3α inhibition. Importantly, GSK-3α inhibition improved cancer surveillance by human NK cells in vivo. Collectively, our results highlight the distinct role of GSK-3 isoforms in the regulation of NK cell reactivity against target cells and suggest that GSK-3α modulation could be used to enhance tumor cell susceptibility to NK cells in an NKG2D- and NKp30-dependent manner.

Download Full-text

Fluorescence-Reported Allelic Exchange Mutagenesis-Mediated Gene Deletion Indicates a Requirement for Chlamydia trachomatis Tarp during In Vivo Infectivity and Reveals a Specific Role for the C Terminus during Cellular Invasion

Infection and Immunity ◽

10.1128/iai.00841-19 ◽

2020 ◽

Vol 88 (5) ◽

Author(s):

Susmita Ghosh ◽

Elizabeth A. Ruelke ◽

Joshua C. Ferrell ◽

Maria D. Bodero ◽

Kenneth A. Fields ◽

...

Keyword(s):

Chlamydia Trachomatis ◽

Host Cell ◽

Host Cells ◽

Actin Binding ◽

Wild Type ◽

Content Type ◽

Allelic Exchange ◽

Binding Domains

ABSTRACT The translocated actin recruiting phosphoprotein (Tarp) is a multidomain type III secreted effector used by Chlamydia trachomatis. In aggregate, existing data suggest a role of this effector in initiating new infections. As new genetic tools began to emerge to study chlamydial genes in vivo, we speculated as to what degree Tarp function contributes to Chlamydia’s ability to parasitize mammalian host cells. To address this question, we generated a complete tarP deletion mutant using the fluorescence-reported allelic exchange mutagenesis (FRAEM) technique and complemented the mutant in trans with wild-type tarP or mutant tarP alleles engineered to harbor in-frame domain deletions. We provide evidence for the significant role of Tarp in C. trachomatis invasion of host cells. Complementation studies indicate that the C-terminal filamentous actin (F-actin)-binding domains are responsible for Tarp-mediated invasion efficiency. Wild-type C. trachomatis entry into HeLa cells resulted in host cell shape changes, whereas the tarP mutant did not. Finally, using a novel cis complementation approach, C. trachomatis lacking tarP demonstrated significant attenuation in a murine genital tract infection model. Together, these data provide definitive genetic evidence for the critical role of the Tarp F-actin-binding domains in host cell invasion and for the Tarp effector as a bona fide C. trachomatis virulence factor.

Download Full-text

Binding of Brucella protein, Bp26, to select extracellular matrix molecules

BMC Molecular and Cell Biology ◽

10.1186/s12860-019-0239-7 ◽

2019 ◽

Vol 20 (1) ◽

Cited By ~ 4

Author(s):

Yasmin ElTahir ◽

Amna Al-Araimi ◽

Remya R. Nair ◽

Kaija J. Autio ◽

Hongmin Tu ◽

...

Keyword(s):

Extracellular Matrix ◽

Type I Collagen ◽

Host Cells ◽

Mouse Serum ◽

Enzyme Linked Immunosorbent Assay ◽

Target Cells ◽

Type I ◽

Dependent Manner ◽

Biolayer Interferometry

Abstract Background Brucella is a facultative intracellular pathogen responsible for zoonotic disease brucellosis. Little is known about the molecular basis of Brucella adherence to host cells. In the present study, the possible role of Bp26 protein as an adhesin was explored. The ability of Brucella protein Bp26 to bind to extracellular matrix (ECM) proteins was determined by enzyme-linked immunosorbent assay (ELISA) and biolayer interferometry (BLI). Results ELISA experiments showed that Bp26 bound in a dose-dependent manner to both immobilized type I collagen and vitronectin. Bp26 bound weakly to soluble fibronectin but did not bind to immobilized fibronectin. No binding to laminin was detected. Biolayer interferometry showed high binding affinity of Bp26 to immobilized type I collagen and no binding to fibronectin or laminin. Mapping of Bp26 antigenic epitopes by biotinylated overlapping peptides spanning the entire sequence of Bp26 using anti Bp26 mouse serum led to the identification of five linear epitopes. Collagen and vitronectin bound to peptides from several regions of Bp26, with many of the binding sites for the ligands overlapping. The strongest binding for anti-Bp26 mouse serum, collagen and vitronectin was to the peptides at the C-terminus of Bp26. Fibronectin did not bind to any of the peptides, although it bound to the whole Bp26 protein. Conclusions Our results highlight the possible role of Bp26 protein in the adhesion process of Brucella to host cells through ECM components. This study revealed that Bp26 binds to both immobilized and soluble type I collagen and vitronectin. It also binds to soluble but not immobilized fibronectin. However, Bp26 does not bind to laminin. These are novel findings that offer insight into understanding the interplay between Brucella and host target cells, which may aid in future identification of a new target for diagnosis and/or vaccine development and prevention of brucellosis.

Download Full-text

Actin depolymerizing factor controls actin turnover and gliding motility in Toxoplasma gondii

Molecular Biology of the Cell ◽

10.1091/mbc.e10-12-0939 ◽

2011 ◽

Vol 22 (8) ◽

pp. 1290-1299 ◽

Cited By ~ 42

Author(s):

Simren Mehta ◽

L. David Sibley

Keyword(s):

Toxoplasma Gondii ◽

Actin Filaments ◽

Gliding Motility ◽

Conditional Knockout ◽

Host Cells ◽

Actin Binding ◽

Actin Depolymerizing Factor

Apicomplexan parasites rely on actin-based gliding motility to move across the substratum, cross biological barriers, and invade their host cells. Gliding motility depends on polymerization of parasite actin filaments, yet ∼98% of actin is nonfilamentous in resting parasites. Previous studies suggest that the lack of actin filaments in the parasite is due to inherent instability, leaving uncertain the role of actin-binding proteins in controlling dynamics. We have previously shown that the single allele of Toxoplasma gondii actin depolymerizing factor (TgADF) has strong actin monomer–sequestering and weak filament-severing activities in vitro. Here we used a conditional knockout strategy to investigate the role of TgADF in vivo. Suppression of TgADF led to accumulation of actin-rich filaments that were detected by immunofluorescence and electron microscopy. Parasites deficient in TgADF showed reduced speed of motility, increased aberrant patterns of motion, and inhibition of sustained helical gliding. Lack of TgADF also led to severe defects in entry and egress from host cells, thus blocking infection in vitro. These studies establish that the absence of stable actin structures in the parasite are not simply the result of intrinsic instability, but that TgADF is required for the rapid turnover of parasite actin filaments, gliding motility, and cell invasion.

Download Full-text

PP2A Facilitates Porcine Reproductive and Respiratory Syndrome Virus Replication by Deactivating irf3 and Limiting Type I Interferon Production

Viruses ◽

10.3390/v11100948 ◽

2019 ◽

Vol 11 (10) ◽

pp. 948 ◽

Cited By ~ 1

Author(s):

Jiayu Xu ◽

Lu Zhang ◽

Yunfei Xu ◽

He Zhang ◽

Junxin Gao ◽

...

Keyword(s):

Virus Infection ◽

Therapeutic Target ◽

Type I Interferon ◽

Negative Regulator ◽

Host Cells ◽

Respiratory Syndrome Virus ◽

Target Cells ◽

Type I ◽

Dependent Manner ◽

Pp2a Activity

Protein phosphatase 2A (PP2A), a major serine/threonine phosphatase in mammalian cells, is known to regulate the kinase-driven intracellular signaling pathways. Emerging evidences have shown that the PP2A phosphatase functions as a bona-fide therapeutic target for anticancer therapy, but it is unclear whether PP2A affects a porcine reproductive and respiratory syndrome virus infection. In the present study, we demonstrated for the first time that inhibition of PP2A activity by either inhibitor or small interfering RNA duplexes in target cells significantly reduced their susceptibility to porcine reproductive and respiratory syndrome virus (PRRSV) infection. Further analysis revealed that inhibition of PP2A function resulted in augmented production of type I interferon (IFN). The mechanism is that inhibition of PP2A activity enhances the levels of phosphorylated interferon regulatory factor 3, which activates the transcription of IFN-stimulated genes. Moreover, inhibition of PP2A activity mainly blocked PRRSV replication in the early stage of viral life cycle, after virus entry but before virus release. Using type I IFN receptor 2 specific siRNA in combination with PP2A inhibitor, we confirmed that the effect of PP2A on viral replication within target cells was an interferon-dependent manner. Taken together, these findings demonstrate that PP2A serves as a negative regulator of host cells antiviral responses and provides a novel therapeutic target for virus infection.

Download Full-text

GDF11 expressed in the adult brain negatively regulates hippocampal neurogenesis

10.21203/rs.3.rs-148652/v1 ◽

2021 ◽

Author(s):

Brittany A Mayweather ◽

Sean M Buchanan ◽

Lee L Rubin

Keyword(s):

Histological Analysis ◽

Hippocampal Neurogenesis ◽

Neural Progenitors ◽

Adult Brain ◽

Adult Hippocampal Neurogenesis ◽

Negative Regulator ◽

Target Cells ◽

Adult Mice ◽

Newborn Neurons

Abstract Growth differentiation factor 11 (GDF11) is a transforming factor-β superfamily member that functions as a negative regulator of neurogenesis during embryonic development. However, when recombinant GDF11 (rGDF11) is administered systemically in aged mice, it promotes neurogenesis, the opposite of its role during development. The goal of the present study was to reconcile this apparent discrepancy by performing the first detailed investigation into the expression of endogenous GDF11 in the adult brain and its effects on neurogenesis. Using quantitative histological analysis, we observed that Gdf11 is highly expressed in adult neurogenic niches and non-neurogenic regions within the hippocampus, choroid plexus, thalamus, habenula, and cerebellum. To investigate the role of endogenous GDF11 during adult hippocampal neurogenesis, we generated a tamoxifen inducible mouse that allowed us to reduce GDF11 levels. Depletion of Gdf11 during adulthood increased proliferation of neural progenitors and decreased the number of newborn neurons in the hippocampus, suggesting that endogenous GDF11 remains a negative regulator of hippocampal neurogenesis in adult mice. These findings further support the idea that circulating systemic GDF11 and endogenously expressed GDF11 in the adult brain have different target cells or mechanisms of action. Our data describe a role for GDF11-dependent signaling in adult neurogenesis that has implications for how GDF11 may be used to treat CNS disease.

Download Full-text

The innate immunity protein C1QBP functions as a negative regulator of circulative transmission of Potato leafroll virus by aphids

10.1101/2020.12.04.412668 ◽

2020 ◽

Author(s):

Stacy L. DeBlasio ◽

Jennifer Wilson ◽

Cecilia Tamborindeguy ◽

Richard S. Johnson ◽

Patricia V. Pinheiro ◽

...

Keyword(s):

Innate Immunity ◽

High Resolution Mass Spectrometry ◽

Affinity Purification ◽

Plant Viruses ◽

Potato Leafroll Virus ◽

Negative Regulator ◽

Virus Protein ◽

Insect Vector ◽

Immunity Protein ◽

Leafroll Virus

ABSTRACTThe vast majority of plant viruses are transmitted by insect vectors with many crucial aspects of the transmission process being mediated by key protein-protein interactions. Yet, very few vector proteins interacting with virus have been identified and functionally characterized. Potato leafroll virus (PLRV) is transmitted most effectively by Myzus persicae, the green peach aphid, in a circulative, non-propagative manner. Using an affinity purification strategy coupled to high-resolution mass spectrometry (AP-MS), we identified 11 proteins from M. persicae displaying high probability of interaction with PLRV and an additional 23 vector proteins with medium confidence interaction scores. Two of these proteins were confirmed to directly interact with the structural proteins of PLRV and other luteovirid species via yeast two-hybrid with an additional vector protein displaying binding specificity. Immunolocalization of one of these direct PLRV-interacting proteins, an orthologue of the human innate immunity protein complement component 1 Q subcomponent-binding protein (C1QBP), shows that MpC1QBP partially co-localizes with PLRV within cytoplasmic puncta and along the periphery of aphid gut epithelial cells. Chemical inhibition of C1QBP in the aphid leads to increased PLRV acquisition and subsequently increased titer in inoculated plants, supporting the role of C1QBP as a negative regulator of PLRV accumulation in M. persicae. We hypothesize that the innate immune function of C1QBP is conserved in aphids and represents the first instance of aphids mounting an immune response to a non-propagative plant virus. This study presents the first use of AP-MS for the in vivo isolation of functionally relevant insect vector-virus protein complexes.

Download Full-text

Unexpected Role of Rotavirus G3P[8] Infection in Causing Severe Diarrhea in a Major Tertiary Referral Hospital in the Prevaccine Era

Archives of Clinical Infectious Diseases ◽

10.5812/archcid.105501 ◽

2021 ◽

Vol 16 (1) ◽

Author(s):

Wassim Chehadeh

Keyword(s):

Nucleotide Sequences ◽

Referral Hospital ◽

Tertiary Referral ◽

Tertiary Referral Hospital ◽

Rotavirus A ◽

Severe Diarrhea ◽

National Immunization ◽

Severe Gastroenteritis ◽

Stool Samples

Background: Rotavirus A species is associated with severe gastroenteritis in children. Rotavirus G1P[8] was the most prevalent genotype found in Kuwait in a study conducted between 2005 and 2006. The RotaTeq vaccine was included in the Kuwait national immunization program at the end of 2017. Objectives: Since there is no available data on the rotavirus genotypes circulating before the introduction of the vaccine, we conducted a study to investigate the role of rotaviruses in causing severe diarrhea in children hospitalized in a major tertiary referral hospital in Kuwait during the year 2016. Methods: Viral RNA was isolated from the stool samples of 101 children under five years of age, hospitalized for acute gastroenteritis. Rotavirus VP4 and VP7 dsRNA were detected by RT-PCR, and their partial sequences were analyzed by phylogenic analysis. Results: Rotavirus dsRNA was detected in 24.7% of children with median age of 1 year. The genotype G3P[8] accounted for 47% of cases, followed by G1P[8] (26%), G9P[8] (10.5%), G4P[8] (10.5%), and G9P[4] (5%). Only VP7 nucleotide sequences of rotavirus G3 or G4 type clustered in the same lineage as RotaTeq vaccine, while most VP4 nucleotide sequences of rotavirus P[8] type clustered in a different lineage compared to Rotarix and RotaTeq vaccines. Conclusions: Our findings highlight the role of rotavirus G3P[8] in causing severe diarrhea and invites future investigations to know whether the recent introduction of RotaTeq vaccine in Kuwait selects certain genotypes and subgenomic lineages.

Download Full-text

GDF11 expressed in the adult brain negatively regulates hippocampal neurogenesis

Molecular Brain ◽

10.1186/s13041-021-00845-z ◽

2021 ◽

Vol 14 (1) ◽

Author(s):

Brittany A. Mayweather ◽

Sean M. Buchanan ◽

Lee L. Rubin

Keyword(s):

Histological Analysis ◽

Hippocampal Neurogenesis ◽

Neural Progenitors ◽

Adult Brain ◽

Adult Hippocampal Neurogenesis ◽

Negative Regulator ◽

Target Cells ◽

Adult Mice ◽

Newborn Neurons

AbstractGrowth differentiation factor 11 (GDF11) is a transforming factor-β superfamily member that functions as a negative regulator of neurogenesis during embryonic development. However, when recombinant GDF11 (rGDF11) is administered systemically in aged mice, it promotes neurogenesis, the opposite of its role during development. The goal of the present study was to reconcile this apparent discrepancy by performing the first detailed investigation into the expression of endogenous GDF11 in the adult brain and its effects on neurogenesis. Using quantitative histological analysis, we observed that Gdf11 is most highly expressed in adult neurogenic niches and non-neurogenic regions within the hippocampus, choroid plexus, thalamus, habenula, and cerebellum. To investigate the role of endogenous GDF11 during adult hippocampal neurogenesis, we generated a tamoxifen inducible mouse that allowed us to reduce GDF11 levels. Depletion of Gdf11 during adulthood increased proliferation of neural progenitors and decreased the number of newborn neurons in the hippocampus, suggesting that endogenous GDF11 remains a negative regulator of hippocampal neurogenesis in adult mice. These findings further support the idea that circulating systemic GDF11 and endogenously expressed GDF11 in the adult brain have different target cells or mechanisms of action. Our data describe a role for GDF11-dependent signaling in adult neurogenesis that has implications for how GDF11 may be used to treat CNS disease.

Download Full-text

Cellular and Biochemical Mechanisms of the Retroviral Restriction Factor SAMHD1

ISRN Biochemistry ◽

10.1155/2013/728392 ◽

2013 ◽

Vol 2013 ◽

pp. 1-11 ◽

Cited By ~ 13

Author(s):

Li Wu

Keyword(s):

Host Protein ◽

Nucleic Acid Metabolism ◽

Target Cells ◽

Restriction Factors ◽

Host Proteins ◽

Retroviral Infection ◽

Deoxynucleoside Triphosphate ◽

Biochemical Mechanisms ◽

Hiv 1

Replication of HIV-1 and other retroviruses is dependent on numerous host proteins in the cells. Some of the host proteins, however, function as restriction factors to block retroviral infection of target cells. The host protein SAMHD1 has been identified as the first mammalian deoxynucleoside triphosphate triphosphohydrolase (dNTPase), which blocks the infection of HIV-1 and other retroviruses in non-cycling immune cells. SAMHD1 protein is highly expressed in human myeloid-lineage cells and CD4+ T-lymphocytes, but its retroviral restriction function is only observed in noncycling cells. Recent studies have revealed biochemical mechanisms of SAMHD1-mediated retroviral restriction. In this review, the latest progress on SAMHD1 research is summarized and the mechanisms by which SAMHD1 mediates retroviral restriction are analyzed. Although the physiological function of SAMHD1 is largely unknown, this review provides perspectives about the role of endogenous SAMHD1 protein in maintaining normal cellular function, such as nucleic acid metabolism and the proliferation of cells.

Download Full-text