Neotypification of Pleurocapsa fuliginosa and epitypification of P. minor (Pleurocapsales): resolving a polyphyletic cyanobacterial genus

Strains with complete morphological match to Pleurocapsa fuliginosa and P. minor were isolated from Oahu (Hawaii, USA), with another strain matching P. minor isolated from a wet rock face in Utah (USA). Phylogenetically these baeocyte and pseudofilament producing strains fell in a single well-supported clade among a number of pleurocapsalean strains. They were sister to a clade of baeocyte-producing strains that lack the ability to form pseudofilaments and likely belong in an as-yet-to- be-described genus. Strains putatively named Pleurocapsa are scattered throughout the Pleurocapsales and Chroococcales, indicating a need for clear definition of the genus so that revisionary work and alpha-level taxonomy can move forward. To satisfy this need, P. fuliginosa HA4302-MV1 and P. minor HA4230-MV1 were chosen as neotype and epitype, respectively, establishing the genus based on molecular sequence data. In addition to the distinctive morphology of the genus, all Pleuro- capsa species for which 16S-23S ITS regions are available have an unusually long, branched D5 helix at the termination of the ITS region. The sister clade of strains that lack the ability to form pseudofilaments also possess an unusually long and branched D5 helix as well, suggesting that this feature of the ITS region may be a family-level synapomorphy

Chemoproteomics reveals Toll-like receptor fatty acylation

Partial funding for Open Access provided by The Ohio State University Open Access Fund.Background: Palmitoylation is a 16-carbon lipid post-translational modification that increases protein hydrophobicity. This form of protein fatty acylation is emerging as a critical regulatory modification for multiple aspects of cellular interactions and signaling. Despite recent advances in the development of chemical tools for the rapid identification and visualization of palmitoylated proteins, the palmitoyl proteome has not been fully defined. Here we sought to identify and compare the palmitoylated proteins in murine fibroblasts and dendritic cells. Results: A total of 563 putative palmitoylation substrates were identified, more than 200 of which have not been previously suggested to be palmitoylated in past proteomic studies. Here we validate the palmitoylation of several new proteins including Toll-like receptors (TLRs) 2, 5 and 10, CD80, CD86, and NEDD4. Palmitoylation of TLR2, which was uniquely identified in dendritic cells, was mapped to a transmembrane domain-proximal cysteine. Inhibition of TLR2 S-palmitoylation pharmacologically or by cysteine mutagenesis led to decreased cell surface expression and a decreased inflammatory response to microbial ligands. Conclusions: This work identifies many fatty acylated proteins involved in fundamental cellular processes as well as cell type-specific functions, highlighting the value of examining the palmitoyl proteomes of multiple cell types. Spalmitoylation of TLR2 is a previously unknown immunoregulatory mechanism that represents an entirely novel avenue for modulation of TLR2 inflammatory activity.This work was supported by funding from the NIH/NIAID (grant R00AI095348 to J.S.Y.), the NIH/NIGMS (R01GM087544 to HCH), and the Ohio State University Public Health Preparedness for Infectious Diseases (PHPID) program. NMC is supported by the Ohio State University Systems and Integrative Biology Training Program (NIH/NIGMS grant T32GM068412). BWZ is a fellow of the National Science Foundation Graduate Research Fellowship Program (DGE-0937362)

E3 Ubiquitin Ligase NEDD4 Promotes Influenza Virus Infection by Decreasing Levels of the Antiviral Protein IFITM3.

Interferon (IFN)-induced transmembrane protein 3 (IFITM3) is a cell-intrinsic factor that limits influenza virus infections. We previously showed that IFITM3 degradation is increased by its ubiquitination, though the ubiquitin ligase responsible for this modification remained elusive. Here, we demonstrate that the E3 ubiquitin ligase NEDD4 ubiquitinates IFITM3 in cells and in vitro. This IFITM3 ubiquitination is dependent upon the presence of a PPxY motif within IFITM3 and the WW domain-containing region of NEDD4. In NEDD4 knockout mouse embryonic fibroblasts, we observed defective IFITM3 ubiquitination and accumulation of high levels of basal IFITM3 as compared to wild type cells. Heightened IFITM3 levels significantly protected NEDD4 knockout cells from infection by influenza A and B viruses. Similarly, knockdown of NEDD4 in human lung cells resulted in an increase in steady state IFITM3 and a decrease in influenza virus infection, demonstrating a conservation of this NEDD4-dependent IFITM3 regulatory mechanism in mouse and human cells. Consistent with the known association of NEDD4 with lysosomes, we demonstrate for the first time that steady state turnover of IFITM3 occurs through the lysosomal degradation pathway. Overall, this work identifies the enzyme NEDD4 as a new therapeutic target for the prevention of influenza virus infections, and introduces a new paradigm for up-regulating cellular levels of IFITM3 independently of IFN or infection

Structural Basis for a Species-Specific Determinant of an SIV Vif Protein toward Hominid APOBEC3G Antagonism

Primate lentiviruses encode a Vif protein that counteracts the host antiviral APOBEC3 (A3) family members. The adaptation of Vif to species-specific A3 determinants is a critical event that allowed the spillover of a lentivirus from monkey reservoirs to chimpanzees and subsequently to humans, which gave rise to HIV-1 and the acquired immune deficiency syndrome (AIDS) pandemic. How Vif-A3 protein interactions are remodeled during evolution is unclear. Here, we report a 2.94 Å crystal structure of the Vif substrate receptor complex from simian immunodeficiency virus isolated from red-capped mangabey (SIVrcm). The structure of the SIVrcm Vif complex illuminates the stage of lentiviral Vif evolution that is immediately prior to entering hominid primates. Structure-function studies reveal the adaptations that allowed SIVrcm Vif to antagonize hominid A3G. These studies show a partitioning between an evolutionarily dynamic specificity determinant and a conserved protein interacting surface on Vif that enables adaptation while maintaining protein interactions required for potent A3 antagonism

NEDD4 regulates cellular susceptibility to influenza virus infection by controlling IFITM3 levels.

<p>A,B) NEDD4 WT and KO MEFs were transfected for 24 h with control siRNA (siCont) or siRNA targeting IFITM3 (siIFITM3). A) Cells were collected just prior to infection for confirmation of IFITM3 knockdown by anti-IFITM3 Western blotting, with anti-actin blotting serving as a protein loading control. B) Following siRNA treatment, cells were infected with influenza virus strain PR8 at an MOI of 10 for 24 h. Cells were then fixed and stained with anti-influenza virus NP to measure the percentage of cells infected using flow cytometry. Results shown are representative of three independent experiments, each with samples run in triplicate. Error bars represent standard deviation. * Indicates p-value less than 0.001 calculated with Student’s t-test for comparison of samples denoted by horizontal lines.</p

The IFITM3 PPxY motif is required for ubiquitination by NEDD4.

<p>A-C) HEK293T cells were co-transfected with plasmids expressing myc-hIFITM3 or FLAG-NEDD4 as indicated. A-B) Cell lysates were immunoprecipitated with anti-myc resin, and examined by Western blotting with anti-myc and anti-ubiquitin (Ub). Western blotting of cell lysate with anti-FLAG antibodies was performed to confirm expression of NEDD4. Western blotting with anti-GAPDH antibodies was performed to confirm comparable protein loading. C) Cell lysates were immunoprecipitated with anti-myc or anti-FLAG resin, and co-immunoprecipitation was examined by Western blotting with both anti-myc and anti-FLAG antibodies for each immunoprecipitate. Western blots of cell lysates with anti-myc and anti-FLAG antibodies were performed to confirm expression of IFITM3 and NEDD4, respectively. Anti-GAPDH Western blotting was performed to confirm comparable protein loading.</p

NEDD4 knockout decreases IFITM3 ubiquitination and protects cells from virus infection.

<p>A) Cell lysates from NEDD4 WT and KO MEFs were subjected to anti-IFITM3, anti-NEDD4, and anti-actin immunoblotting to evaluate NEDD4 levels in each cell line and the effect of NEDD4 on endogenous IFITM3 levels. Retroviral reconstitution of the indicated cells with an empty retrovirus control or retrovirus expressing NEDD4 is denoted by + Vector and + NEDD4, respectively. B) 2 mg of protein from NEDD4 WT and KO cell lysates were immunoprecipitated for endogenous IFITM3 and examined by Western blotting with anti-ubiquitin and anti-IFITM3 antibodies. C) The indicated cell lines were infected for 24 h with influenza A virus (IAV) PR8 and X-31 strains at an MOI of 5. Cells were then fixed and stained with anti-influenza virus NP to measure the percentage of cells infected using flow cytometry. D) NEDD4 WT and KO MEFs were infected with influenza virus 2011 isolates (IAV or influenza B virus (IBV)) or Sendai virus (SeV) at an MOI of 5 for 24 h, or were infected with VSV G-pseudotyped retrovirus (VSV G-pseudo) for 48 h. Cells were then fixed and stained with anti-influenza NP in the case of influenza virus infections or were examined for GFP positivity in the case of VSV G-pseudo or SeV to measure the percentage of cells infected using flow cytometry. C,D) Non-infected samples were used as a baseline for gating of infected cells. Results shown are representative of at least three independent experiments, each performed with triplicate samples. The average percent infection of WT NEDD4 cells was set to 1 for the calculation of relative percent infection. Error bars represent standard deviation of triplicate samples. * Indicates a p-value less than 0.001 calculated by Student’s t-test in comparison to values for NEDD4 WT cells.</p

NEDD4 ubiquitinates IFITM3 in vitro.

<p>HA-hIFITM3 was added to reactions containing NEDD4-compatible E1 and E2 ubiquitin ligases, ubiquitin (WT or mutants in which only K48 or K63 were not mutated), and reaction buffer containing ATP in the presence or absence of NEDD4. The reaction was allowed to proceed for 1 h at 37°C, and IFITM3 was re-immunoprecipitated and subjected to Western blotting with anti-ubiquitin (Ub) and anti-HA antibodies. IgG H.C. indicates detection of the heavy chain of the immunoglobulin used for immunoprecipitation.</p