DOCSLIB.ORG

Role of HIV-1 Nef Dimerization in SERINC5 Restriction Factor Antagonism And

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Role of HIV-1 Nef Dimerization in SERINC5 Restriction Factor Antagonism And Title Page Role of HIV-1 Nef dimerization in SERINC5 restriction factor antagonism and Src-family kinase recruitment by Ryan Staudt B.S., Pennsylvania State University, 2014 Submitted to the Graduate Faculty of the School of Medicine in partial fulfillment of the requirements for the degree of Doctor of Philosophy University of Pittsburgh 2020 Committee Page UNIVERSITY OF PITTSBURGH SCHOOL OF MEDICINE This dissertation was presented by Ryan Staudt It was defended on July 17, 2020 and approved by Robert Binder, Ph.D., Associate Professor, Department of Immunology, School of Medicine Lori Emert-Sedlak Ph.D., Research Assistant Professor, Department of Microbiology and Molecular Genetics, School of Medicine Lawrence Kane, Ph.D., Professor, Department of Immunology, School of Medicine Nicolas Sluis-Cremer, Ph.D., Professor, Department of Medicine, Division of Infectious Diseases Dissertation Director: Thomas E. Smithgall, Ph.D., Professor and Chair of Microbiology and Molecular Genetics, School of Medicine ii Copyright © by Ryan Staudt 2020 iii For Shirley Staudt iv Abstract Role of HIV-1 Nef dimerization in SERINC5 restriction factor antagonism and Src- family kinase recruitment Ryan Staudt, PhD University of Pittsburgh, 2020 The HIV-1 Nef accessory protein plays a prominent role in viral pathogenesis, immune evasion of HIV-infected cells, and AIDS progression. Nef lacks intrinsic biochemical activity, and instead functions through interactions with multiple host cell effector proteins. Previous studies have shown that Nef homo-dimerizes at a hydrophobic interface between two Nef molecules. As Nef dimerization has been implicated in several Nef functions, it may represent a novel and effective target for antiretroviral drug development. Research that further expands our understanding of Nef dimerization may aid in the development of future therapeutics. Therefore, for the first part of my dissertation, I explored the role of Nef dimerization in Nef antagonism of the host restriction factor SERINC5. We showed that Nef homodimers are required for the AP-2 dependent downregulation of SERINC5, trafficking to late endosomes, and exclusion from newly synthesized viral particles. Disruption of Nef dimerization impaired antagonism of SERINC5, rescuing SERINC5 inhibition of viral infectivity. In addition, dimerization-defective Nef mutants retained interaction with both SERINC5 and AP-2 by fluorescence complementation assay, suggesting that the Nef dimer bridges SERINC5 with AP-2 to drive endocytosis of cell-surface SERINC5. For the second part of my dissertation, I examined the dynamics and individual contribution of predicted dimer-stabilizing residues towards dimerization and complex formation in solution. Crystal structures have revealed distinct orientations of the Nef dimer interface within v complexes of Nef bound to Src family kinase SH3 domain vs. Nef complexes bound to SH3-SH2 domain. I was able to validate the orientation of the dimer interface captured in each structure using biophysical and biochemical studies. As part of a collaborative study, I was able to confirm that Nef Asp123 contributes to Nef dimerization within the SH3-bound but not the SH3-SH2 bound structure of Nef. I also identified a Nef residue (Tyr115) that stabilizes Nef dimerization within both complexes, suggesting that it is a consistent feature of Nef dimerization across two potential stages of kinase domain engagement. Taken together, this dissertation helps to further illuminate the mechanics of Nef dimerization, and implicates Nef dimerization in one of the more recently discovered Nef functions: Nef antagonism of SERINC5. vi Table of Contents Acknowledgements .................................................................................................................. xv 1.0 Introduction .........................................................................................................................1 1.1 Human Immunodeficiency Virus (HIV-1)..................................................................1 1.1.1 History and Global Burden of HIV/AIDS ....................................................... 1 1.1.2 Background on human and simian retroviruses ............................................. 2 1.1.3 HIV-1 Genome.................................................................................................. 5 1.1.4 HIV-1 Structure ............................................................................................... 7 1.1.5 HIV Life Cycle .................................................................................................. 9 1.1.6 HIV-1 Transmission and Pathogenesis. ......................................................... 12 1.1.7 HIV-1 Treatments and Curative Strategies .................................................. 14 1.2 HIV-1 Nef .................................................................................................................. 17 1.2.1 Role of HIV Nef in viral pathogenesis ........................................................... 17 1.2.2 General Structure of Nef ................................................................................ 17 1.2.3 Key Functions of Nef ...................................................................................... 18 1.2.3.1 Promotion of viral infectivity .............................................................. 18 1.2.3.2 Alteration of host cell protein trafficking ........................................... 19 1.2.3.3 Nef-dependent kinase activation ......................................................... 22 1.2.4 Nef Binding Partners- Description and Structure......................................... 23 1.2.4.1 Src family kinases ................................................................................ 24 1.2.4.2 Adaptor protein complexes ................................................................. 25 1.2.4.3 CD4 ...................................................................................................... 27 vii 1.2.4.4 MHC-I.................................................................................................. 28 1.2.5 Critical Nef Motifs and features .................................................................... 29 1.2.5.1 N-terminal Flexible Arm and Myristoylation Site ............................. 29 1.2.5.2 PxxP Motif: Structural Basis of Kinase Engagement ........................ 31 1.2.5.3 Large Internal Loop: Dileucine and Diacidic motifs ......................... 37 1.2.5.4 Dimerization Interface ........................................................................ 41 1.2.6 Nef as a pharmacologic target........................................................................ 45 1.3 SERINC5 ................................................................................................................... 47 1.3.1 Initial discovery and identification as a retroviral restriction factor ........... 47 1.3.2 Structure and critical features of SERINC5 ................................................. 49 1.3.3 SERINC5 restriction of viral infectivity ........................................................ 52 1.3.4 Retroviral antagonism of SERINC5 .............................................................. 53 1.4 Hypotheses and Specific Aims .................................................................................. 55 1.4.1 Hypothesis 1: Nef homodimerization is essential for downregulation of SERINC5 ..................................................................................................... 55 1.4.2 Specific Aims for Hypothesis 1 ...................................................................... 56 1.4.2.1 Aim 1: Explore the impact of dimerization-defective Nef mutants on HIV-1 infectivity and virion exclusion of SERINC5. ......................... 56 1.4.2.2 Aim 2: Examine whether disruption of Nef dimerization impairs Nef downregulation and intracellular retargeting of SERINC5. ............. 57 1.4.2.3 Aim 3: Evaluate whether dimerization interface mutations impair Nef association with SERINC5, AP-2, or Nef within cells. ....................... 58 1.4.3 Hypothesis 2 .................................................................................................... 58 viii 1.4.4 Specific Aims for Hypothesis 2 ...................................................................... 60 1.4.4.1 Aim 1: Determine the binding affinity of Nef dimer mutants towards Hck SH3 and SH3-SH2 regulatory domain proteins ......................... 60 1.4.4.2 Aim 2: Evaluate whether Nef dimer mutants form 2:2 heterocomplexes with SH3 and SH3-SH2 in solution ..................................................... 60 1.4.4.3 Aim 3: Explore whether Nef dimer interface mutants display functional activation of Hck in vitro ................................................... 61 2.0 Nef homodimers couple SERINC5 to AP-2 for Downregulation in HIV-1 Producer Cells .................................................................................................................................. 62 2.1 Chapter 2 summary................................................................................................... 62 2.2 Introduction ............................................................................................................... 63 2.3 Results ........................................................................................................................ 65 2.3.1 Structural basis of HIV-1 Nef homodimerization ......................................... 65 2.3.2 Dimerization-defective
Load more

Recommended publications
  • Impact of Natural HIV-1 Nef Alleles and Polymorphisms on SERINC3/5 Downregulation
    Impact of natural HIV-1 Nef alleles and polymorphisms on SERINC3/5 downregulation by Steven W. Jin B.Sc., Simon Fraser University, 2016 Thesis Submitted in Partial Fulfillment of the Requirements for the Degree of Master of Science in the Master of Science Program Faculty of Health Sciences © Steven W. Jin 2019 SIMON FRASER UNIVERSITY Spring 2019 Copyright in this work rests with the author. Please ensure that any reproduction or re-use is done in accordance with the relevant national copyright legislation. Approval Name: Steven W. Jin Degree: Master of Science Title: Impact of natural HIV-1 Nef alleles and polymorphisms on SERINC3/5 downregulation Examining Committee: Chair: Kanna Hayashi Assistant Professor Mark Brockman Senior Supervisor Associate Professor Masahiro Niikura Supervisor Associate Professor Ralph Pantophlet Supervisor Associate Professor Lisa Craig Examiner Professor Department of Molecular Biology and Biochemistry Date Defended/Approved: April 25, 2019 ii Ethics Statement iii Abstract HIV-1 Nef is a multifunctional accessory protein required for efficient viral pathogenesis. It was recently identified that the serine incorporators (SERINC) 3 and 5 are host restriction factors that decrease the infectivity of HIV-1 when incorporated into newly formed virions. However, Nef counteracts these effects by downregulating SERINC from the cell surface. Currently, there lacks a comprehensive study investigating the impact of primary Nef alleles on SERINC downregulation, as most studies to date utilize lab- adapted or reference HIV strains. In this thesis, I characterized and compared SERINC downregulation from >400 Nef alleles isolated from patients with distinct clinical outcomes and subtypes. I found that primary Nef alleles displayed a dynamic range of SERINC downregulation abilities, thus allowing naturally-occurring polymorphisms that modulate this activity to be identified.
    [Show full text]
  • SERINC5 (NM 178276) Human Untagged Clone Product Data
    OriGene Technologies, Inc. 9620 Medical Center Drive, Ste 200 Rockville, MD 20850, US Phone: +1-888-267-4436 [email protected] EU: [email protected] CN: [email protected] Product datasheet for SC313396 SERINC5 (NM_178276) Human Untagged Clone Product data: Product Type: Expression Plasmids Product Name: SERINC5 (NM_178276) Human Untagged Clone Tag: Tag Free Symbol: SERINC5 Synonyms: C5orf12; TPO1 Vector: pCMV6-Entry (PS100001) E. coli Selection: Kanamycin (25 ug/mL) Cell Selection: Neomycin Fully Sequenced ORF: >NCBI ORF sequence for NM_178276, the custom clone sequence may differ by one or more nucleotides ATGTCAGCTCAGTGCTGTGCGGGCCAGCTGGCCTGCTGCTGTGGGTCTGCAGGCTGCTCTCTCTGCTGTG ATTGCTGCCCCAGGATTCGGCAGTCCCTCAGCACCCGCTTCATGTACGCCCTCTACTTCATTCTGGTCGT CGTCCTCTGCTGCATCATGATGTCAACAACCGTGGCTCACAAGATGAAAGAGCACATTCCTTTTTTTGAA GATATGTGTAAAGGCATTAAAGCTGGTGACACCTGTGAGAAGCTGGTGGGATATTCTGCCGTGTATAGAG TCTGTTTTGGAATGGCTTGTTTCTTCTTTATCTTCTGTCTACTGACCTTGAAAATCAACAACAGCAAAAG TTGTAGAGCTCATATTCACAATGGCTTTTGGTTCTTTAAACTTCTGCTGTTGGGGGCCATGTGCTCAGGA GCTTTCTTCATTCCAGATCAGGACACCTTTCTGAACGCCTGGCGCTATGTGGGAGCCGTCGGAGGCTTCC TCTTCATTGGCATCCAGCTCCTCCTGCTCGTGGAGTTTGCACATAAGTGGAACAAGAACTGGACAGCAGG CACAGCCAGTAACAAGCTGTGGTACGCCTCCCTGGCCCTGGTGACGCTCATCATGTATTCCATTGCCACT GGAGGCTTGGTTTTGATGGCAGTGTTTTATACACAGAAAGACAGCTGCATGGAAAACAAAATTCTGCTGG GAGTAAATGGAGGCCTGTGCCTGCTTATATCATTGGTAGCCATCTCACCCTGGGTCCAAAATCGACAGCC ACACTCGGGGCTCTTACAATCAGGGGTCATAAGCTGCTATGTCACCTACCTCACCTTCTCAGCTCTGTCC AGCAAACCTGCAGAAGTAGTTCTAGATGAACATGGGAAAAATGTTACAATCTGTGTGCCTGACTTTGGTC AAGACCTGTACAGAGATGAAAACTTGGTGACTATACTGGGGACCAGCCTCTTAATCGGATGTATCTTGTA
    [Show full text]
  • Coevolution of Retroviruses with Serincs Following Whole-Genome
    bioRxiv preprint doi: https://doi.org/10.1101/2020.02.24.962506; this version posted February 24, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. Ramdas et al. 1 Coevolution of retroviruses with SERINCs following whole-genome duplication 2 divergence 3 4 Pavitra Ramdas1, Vipin Bhardwaj1, Aman Singh1, Nagarjun Vijay2, Ajit Chande1* 5 1Molecular Virology Laboratory & 2Computational Evolutionary Genomics Lab from the 6 Department of Biological Sciences, Indian Institute of Science Education and Research (IISER) 7 Bhopal, India. 8 9 Abstract 10 The SERINC gene family comprises of five paralogs in humans of which SERINC3 and 11 SERINC5 restrict HIV-1 in a Nef-dependent manner. The origin of this anti-retroviral activity, its 12 prevalence among the remaining human paralogs, and its ability to target retroviruses remain 13 largely unknown. Here we show that despite their early divergence, the anti-retroviral activity is 14 functionally conserved among four human SERINC paralogs with SERINC2 being an 15 exception. The lack of activity in human SERINC2 is associated with its post-whole genome 16 duplication (WGD) divergence, as evidenced by the ability of pre-WGD orthologs from yeast, 17 fly, and a post-WGD-proximate SERINC2 from coelacanth to inhibit HIV-1. Intriguingly, potent 18 retroviral factors from HIV-1 and MLV are not able to relieve the SERINC2-mediated particle 19 infectivity inhibition, indicating that such activity was directed towards other retroviruses that 20 are found in coelacanth (like foamy viruses).
    [Show full text]
  • UNIVERSITY of CALIFORNIA, SAN DIEGO Investigating Transmitted
    UNIVERSITY OF CALIFORNIA, SAN DIEGO Investigating transmitted/founder HIV-1 nef and env effects on SERINC5 inhibition of infectivity A thesis submitted in partial satisfaction of the requirements for the degree Master of Science in Biology by Jasmine Jane Chau Committee in charge: John Guatelli, Chair Michael David, Co-Chair Lisa McDonnell 2017 Copyright Jasmine Jane Chau, 2017 All rights reserved. The Thesis of Jasmine Jane Chau is approved, and it is acceptable in quality and form for publication on microfilm and electronically: Co-Chair Chair University of California, San Diego 2017 iii DEDICATION This thesis is in dedication to my parents, who have always supported me unconditionally in all my endeavors. I would also like to thank my two sisters for all the love and laughter they bring into my life. I would not be where I am today without all their love and support. iv TABLE OF CONTENTS Signature Page…………………………………………………………………….. iii Dedication…………………………………………………………………………. iv Table of Contents………………………………………………………………….. v List of Figures……………………………………………………………………... vi List of Tables……………………………………………………………………… viii Acknowledgements……………………………………………………………….. ix Abstract of the Thesis……………………………………...……………………… x Introduction……………………………………………………………………….. 1 Materials and Methods……………………………………………………………. 6 Results…………………………………………………………………………….. 13 Discussion………………………………………………………………………… 18 Figures and Tables………………………………………………………………… 24 References………………………………………………………………………… 39 v LIST OF FIGURES Figure 1: Schematic of plans for making Env
    [Show full text]
  • Tumour-Stroma Signalling in Cancer Cell Motility and Metastasis
    Tumour-Stroma Signalling in Cancer Cell Motility and Metastasis by Valbona Luga A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy, Department of Molecular Genetics, University of Toronto © Copyright by Valbona Luga, 2013 Tumour-Stroma Signalling in Cancer Cell Motility and Metastasis Valbona Luga Doctor of Philosophy Department of Molecular Genetics University of Toronto 2013 Abstract The tumour-associated stroma, consisting of fibroblasts, inflammatory cells, vasculature and extracellular matrix proteins, plays a critical role in tumour growth, but how it regulates cancer cell migration and metastasis is poorly understood. The Wnt-planar cell polarity (PCP) pathway regulates convergent extension movements in vertebrate development. However, it is unclear whether this pathway also functions in cancer cell migration. In addition, the factors that mobilize long-range signalling of Wnt morphogens, which are tightly associated with the plasma membrane, have yet to be completely characterized. Here, I show that fibroblasts secrete membrane microvesicles of endocytic origin, termed exosomes, which promote tumour cell protrusive activity, motility and metastasis via the exosome component Cd81. In addition, I demonstrate that fibroblast exosomes activate autocrine Wnt-PCP signalling in breast cancer cells as detected by the association of Wnt with Fzd receptors and the asymmetric distribution of Fzd-Dvl and Vangl-Pk complexes in exosome-stimulated cancer cell protrusive structures. Moreover, I show that Pk expression in breast cancer cells is essential for fibroblast-stimulated cancer cell metastasis. Lastly, I reveal that trafficking in cancer cells promotes tethering of autocrine Wnt11 to fibroblast exosomes. These studies further our understanding of the role of ii the tumour-associated stroma in cancer metastasis and bring us closer to a more targeted approach for the treatment of cancer spread.
    [Show full text]
  • Functional Proteomic Atlas of HIV Infection in Primary Human CD4+ T
    TOOLS AND RESOURCES Functional proteomic atlas of HIV infection in primary human CD4+ T cells Adi Naamati1, James C Williamson1,2, Edward JD Greenwood1,2, Sara Marelli1, Paul J Lehner2, Nicholas J Matheson1* 1Department of Medicine, University of Cambridge, Cambridge, United Kingdom; 2Cambridge Institute for Medical Research, University of Cambridge, Cambridge, United Kingdom Abstract Viruses manipulate host cells to enhance their replication, and the identification of cellular factors targeted by viruses has led to key insights into both viral pathogenesis and cell biology. In this study, we develop an HIV reporter virus (HIV-AFMACS) displaying a streptavidin- binding affinity tag at the surface of infected cells, allowing facile one-step selection with streptavidin-conjugated magnetic beads. We use this system to obtain pure populations of HIV- infected primary human CD4+ T cells for detailed proteomic analysis, and quantitate approximately 9000 proteins across multiple donors on a dynamic background of T cell activation. Amongst 650 HIV-dependent changes (q < 0.05), we describe novel Vif-dependent targets FMR1 and DPH7, and 192 proteins not identified and/or regulated in T cell lines, such as ARID5A and PTPN22. We therefore provide a high-coverage functional proteomic atlas of HIV infection, and a mechanistic account of host factors subverted by the virus in its natural target cell. DOI: https://doi.org/10.7554/eLife.41431.001 Introduction *For correspondence: Remodelling of the host proteome during viral infection may reflect direct effects of viral proteins, [email protected] secondary effects or cytopathicity accompanying viral replication, or host countermeasures such as the interferon (IFN) response.
    [Show full text]
  • Coevolution of Retroviruses with Serincs Following Whole-Genome
    bioRxiv preprint doi: https://doi.org/10.1101/2020.02.24.962506; this version posted February 27, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. Ramdas et al. 1 Coevolution of retroviruses with SERINCs following whole-genome duplication 2 divergence 3 4 Pavitra Ramdas1, Vipin Bhardwaj1, Aman Singh1, Nagarjun Vijay2, Ajit Chande1* 5 1Molecular Virology Laboratory & 2Computational Evolutionary Genomics Lab from the 6 Department of Biological Sciences, Indian Institute of Science Education and Research (IISER) 7 Bhopal, India. 8 9 Abstract 10 The SERINC gene family comprises of five paralogs in humans of which SERINC3 and 11 SERINC5 inhibit HIV-1 infectivity and are counteracted by Nef. The origin of this anti-retroviral 12 activity, its prevalence among the remaining human paralogs, and its ability to target 13 retroviruses remain largely unknown. Here we show that despite their early divergence, the 14 anti-retroviral activity is functionally conserved among four human SERINC paralogs with 15 SERINC2 being an exception. The lack of activity in human SERINC2 is associated with its 16 post-whole genome duplication (WGD) divergence, as evidenced by the ability of pre-WGD 17 orthologs from yeast, fly, and a post-WGD-proximate SERINC2 from coelacanth to inhibit nef- 18 defective HIV-1. Intriguingly, potent retroviral factors from HIV-1 and MLV are not able to relieve 19 the SERINC2-mediated particle infectivity inhibition, indicating that such activity was directed 20 towards other retroviruses that are found in coelacanth (like foamy viruses).
    [Show full text]
  • By IL-4 in Memory CD8 T Cells Negative Regulation of NKG2D
    Negative Regulation of NKG2D Expression by IL-4 in Memory CD8 T Cells Erwan Ventre, Lilia Brinza, Stephane Schicklin, Julien Mafille, Charles-Antoine Coupet, Antoine Marçais, Sophia This information is current as Djebali, Virginie Jubin, Thierry Walzer and Jacqueline of October 2, 2021. Marvel J Immunol published online 31 August 2012 http://www.jimmunol.org/content/early/2012/08/31/jimmun ol.1102954 Downloaded from Supplementary http://www.jimmunol.org/content/suppl/2012/09/04/jimmunol.110295 Material 4.DC1 http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication by guest on October 2, 2021 *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2012 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published August 31, 2012, doi:10.4049/jimmunol.1102954 The Journal of Immunology Negative Regulation of NKG2D Expression by IL-4 in Memory CD8 T Cells Erwan Ventre, Lilia Brinza,1 Stephane Schicklin,1 Julien Mafille, Charles-Antoine Coupet, Antoine Marc¸ais, Sophia Djebali, Virginie Jubin, Thierry Walzer, and Jacqueline Marvel IL-4 is one of the main cytokines produced during Th2-inducing pathologies.
    [Show full text]
  • Elucidating the Mechanisms of HIV-1 Antiviral Activity by SERINC5
    Elucidating the Mechanisms of HIV-1 Antiviral Activity by SERINC5 Master’s Thesis Khaled Moumneh Division of Experimental Medicine McGill University, Montreal Date of submission: April 2017 Supervisor: Dr. Chen Liang A thesis submitted to McGill University in partial fulfillment of the requirements of the degree of Master of Science. ©Khaled Moumneh, 2017 Abstract (English) The serine incorporators (SERINC) are a highly conserved transmembrane protein family in eukaryotes that are known to play an important role in stimulating lipid biosynthesis in a variety of cells. As their name suggests, they activate phosphatidylserine synthase and palmitoyltransferase via the incorporation of the amino acid serine to drive the synthesis of phosphatidylserine and sphingolipids, respectively. In 2015, two groups independently discovered that in the absence of HIV-1 Nef, SERINC5 and to a lesser extent SERINC3 incorporated into the virion and prevented proper viral pore expansion thus preventing viral core deposition and decreasing infectivity. These findings identified SERINC5 as a host restriction factor and finally solved the mystery behind the mechanism of Nef-mediated up-regulation of HIV-1 infectivity. Little is known about the SERINC5 protein itself and the specifics of its downregulation of HIV-1 and its downregulation by Nef. In our study, SERINC5 post-translational modification by ubiquitin was explored via co-immunoprecipitation. We discovered that SERINC5 is ubiquitinated, and that this ubiquitination most likely does not correspond to its counteraction either by Nef or proteasomal degradation. Next, we set out to find a Nef binding motif on SERINC5 through the use of a novel CD4-SERINC5 chimera internalization assay.
    [Show full text]
  • Product Datasheet Qprest
    Product Datasheet QPrEST PRODUCT SPECIFICATION Product Name QPrEST SERC5 Mass Spectrometry Protein Standard Product Number QPrEST30515 Protein Name Serine incorporator 5 Uniprot ID Q86VE9 Gene SERINC5 Product Description Stable isotope-labeled standard for absolute protein quantification of Serine incorporator 5. Lys (13C and 15N) and Arg (13C and 15N) metabolically labeled recombinant human protein fragment. Application Absolute protein quantification using mass spectrometry Sequence (excluding RSSSDALQGRYAAPELEIARCCFCFSPGGEDTEEQQPGKEGPRVIYDEKK fusion tag) G Theoretical MW 23416 Da including N-terminal His6ABP fusion tag Fusion Tag A purification and quantification tag (QTag) consisting of a hexahistidine sequence followed by an Albumin Binding Protein (ABP) domain derived from Streptococcal Protein G. Expression Host Escherichia coli LysA ArgA BL21(DE3) Purification IMAC purification Purity >90% as determined by Bioanalyzer Protein 230 Purity Assay Isotopic Incorporation >99% Concentration >5 μM after reconstitution in 100 μl H20 Concentration Concentration determined by LC-MS/MS using a highly pure amino acid analyzed internal Determination reference (QTag), CV ≤10%. Amount >0.5 nmol per vial, two vials supplied. Formulation Lyophilized in 100 mM Tris-HCl 5% Trehalose, pH 8.0 Instructions for Spin vial before opening. Add 100 μL ultrapure H2O to the vial. Vortex thoroughly and spin Reconstitution down. For further dilution, see Application Protocol. Shipping Shipped at ambient temperature Storage Lyophilized product shall be stored at -20°C. See COA for expiry date. Reconstituted product can be stored at -20°C for up to 4 weeks. Avoid repeated freeze-thaw cycles. Notes For research use only Product of Sweden. For research use only. Not intended for pharmaceutical development, diagnostic, therapeutic or any in vivo use.
    [Show full text]
  • TIM-Mediated Inhibition of HIV-1 Release Is Antagonized by Nef but Potentiated by SERINC Proteins
    TIM-mediated inhibition of HIV-1 release is antagonized by Nef but potentiated by SERINC proteins Minghua Lia,b,1,2, Abdul A. Waheedc,1, Jingyou Yua,b,1,3, Cong Zenga,b, Hui-Yu Chend, Yi-Min Zhenga,b, Amin Feizpoure, Björn M. Reinharde, Suryaram Gummuluruf, Steven Lind, Eric O. Freedc, and Shan-Lu Liua,b,g,h,4 aCenter for Retrovirus Research, The Ohio State University, Columbus, OH 43210; bDepartment of Veterinary Biosciences, The Ohio State University, Columbus, OH 43210; cVirus–Cell Interaction Section, HIV Dynamics and Replication Program, National Cancer Institute, Frederick, MD 21702; dInstitute of Biological Chemistry, Academia Sinica, Taipei 115, Taiwan; eDepartment of Chemistry and The Photonics Center, Boston University, Boston, MA 02215; fDepartment of Microbiology, Boston University School of Medicine, Boston, MA 02118; gDepartment of Microbial Infection and Immunity, The Ohio State University, Columbus, OH 43210; and hViruses and Emerging Pathogens Thematic Program, Infectious Diseases Institute, The Ohio State University, Columbus, OH 43210 Edited by Michael Emerman, Fred Hutchinson Cancer Research Center, Seattle, WA, and accepted by Editorial Board Member Stephen P. Goff February 3, 2019 (received for review November 15, 2018) The T cell Ig and mucin domain (TIM) proteins inhibit release of HIV-1 Vif binds to apolipoprotein B mRNA-editing enzyme cata- HIV-1 and other enveloped viruses by interacting with cell- and lytic polypeptide-like 3 (APOBEC3), thereby inducing its proteaso- virion-associated phosphatidylserine (PS). Here, we show that the mal degradation and enhancing viral infectivity (14). To enable Nef proteins of HIV-1 and other lentiviruses antagonize TIM- efficient virus release, HIV-1 Vpu counteracts tetherin (also known mediated restriction.
    [Show full text]
  • S2 from Equine Infectious Anemia Virus Is an Infectivity Factor Which Counteracts the Retroviral Inhibitors SERINC5 and SERINC3
    S2 from equine infectious anemia virus is an infectivity factor which counteracts the retroviral inhibitors SERINC5 and SERINC3 Ajit Chandea,1, Emilia Cristiana Cuccurulloa,1, Annachiara Rosaa, Serena Ziglioa, Susan Carpenterb, and Massimo Pizzatoa,2 aCentre for Integrative Biology, University of Trento, 38123 Trento, Italy; and bDepartment of Animal Science, Iowa State University, Ames, IA 50011 Edited by Stephen P. Goff, Columbia University College of Physicians and Surgeons, New York, NY, and approved October 5, 2016 (received for review July 21, 2016) The lentivirus equine infectious anemia virus (EIAV) encodes the evolved the ability to counteract SERINC5 in primate lentivi- small protein S2, a pathogenic determinant that is important for ruses and gammaretroviruses independently, we sought to in- virus replication and disease progression in horses. No molecular vestigate whether the selective pressure imposed by this host function had been linked to this accessory protein. We report that factor has affected the evolution of other retroviruses. Because S2 can replace the activity of Negative factor (Nef) in HIV-1 infec- SERINC5 is highly expressed in blood-derived cells, we in- tivity, being required to antagonize the inhibitory activity of Ser- vestigated whether another blood-tropic retrovirus, Equine in- ine incorporator (SERINC) proteins on Nef-defective HIV-1. Like fectious anemia virus (EIAV), also evolved a Nef-like infectivity Nef, S2 excludes SERINC5 from virus particles and requires an ExxxLL motif predicted to recruit the clathrin adaptor, Adaptor factor. EIAV is a myeloid-tropic lentivirus that causes anemia protein 2 (AP2). Accordingly, functional endocytic machinery is and thrombocytopenia in horses worldwide and chronicizes into essential for S2-mediated infectivity enhancement, and S2-medi- an inapparent, asymptomatic carrier stage.
    [Show full text]