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Elucidating the Mechanisms of HIV-1 Antiviral Activity by SERINC5

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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. Lastly, the SERINC5 region or motif required for antiviral activity against HIV-1 was investigated through the use of two sets of SERINC5 and non-antiviral SERINC1 region-swapped chimeras. Through these experiments, a central region of SERINC5 encompassing amino acids 176-311 was found to be required for its anti-HIV-1 activity. These observations pave the way for future studies to find specific motifs required for SERINC5-HIV-1 and SERINC5-Nef interaction and elucidate a more detailed antiviral mechanism. Abstract (French) Les incorporateurs de sérine (SERINC) constituent une famille de protéines transmembranaires hautement conservée chez les eucaryotes et qui sont connues pour jouer un rôle important dans la stimulation de la biosynthèse des lipides dans de nombreux types cellulaires. Comme leur nom le suggère, ils activent la phosphatidylsérine synthase et la palmitoyltransférase via l’incorporation de l’acide aminé sérine afin de faciliter la synthèse de la phosphatidylsérine et des sphingolipides, respectivement. En 2015, deux groupes ont indépendamment découvert qu’en l’absence de Nef du VIH-1, SERINC5 et, dans une moindre mesure, SERINC3, sont alors incorporés dans le virion et préviennent l’expansion adéquate des pores viraux, permettant donc de prévenir la déposition du noyau viral et réduire l’infectiosité. Ces résultats ont identifié SERINC5 comme étant un facteur de restriction de l’hôte et a finalement résolu le mystère se cachant derrière le mécanisme de la régulation positive de l’infectiosité du VIH-1 médiée par Nef. Peu de choses sont connues à propos de la protéine SERINC5 elle-même et les spécificités de sa régulation négative du VIH-1 et de sa régulation négative par Nef. Dans notre étude, des modifications post-traductionnelles de SERINC5 par de l’ubiquitine ont été explorées via co-immunoprécipitation. Nous avons découvert que SERINC5 était en effet ubiquitinée, et que cette ubiquitination ne correspond probablement pas à sa régulation négative par Nef ou à la dégradation protéasomale. Nous avons ensuite recherché un motif de liaison de Nef sur SERINC5 grâce à l’utilisation d’un nouveau test d’internalisation de CD4-SERINC5 chimérique. Finalement, le motif ou la région SERINC5 requise pour l’activité antivirale contre le VIH-1 a été examinée en utilisant deux ensembles de protéines chimériques par échange de régions entre SERINC5 et la protéine non-antivirale SERINC1. À travers ces expériences, une région centrale de SERINC5, englobant les acides aminés 176 et 311, a été découverte comme étant requise pour ses activités anti-VIH-1.Ces observations permettent de préparer la voie pour de futures études ayant pour but de trouver des motifs spécifiques requis pour l’interaction entre SERINC5-HIV-1 et SERINC5-Nef et élucider un mécanisme antiviral plus détaillé. Table of Contents ABSTRACT (ENGLISH) ............................................................................................................. 1 ABSTRACT (FRENCH) .............................................................................................................. 2 TABLE OF CONTENTS ............................................................................................................. 3 ACKNOWLEDGEMENTS ......................................................................................................... 5 ABBREVIATIONS ....................................................................................................................... 6 CHAPTER 1: INTRODUCTION .............................................................................................. 10 1.1 HISTORY AND EPIDEMIOLOGY OF HIV-1 ............................................................................. 10 1.1.1 Discovery ..................................................................................................................... 10 1.1.2 Epidemiology ............................................................................................................... 10 1.1.3 Classification and Evolution of HIV ............................................................................ 11 1.2 HIV-1 PATHOGENESIS ......................................................................................................... 15 1.2.1 HIV-1 Transmission ..................................................................................................... 15 1.2.2 Disease Progression .................................................................................................... 15 1.3 HIV-1 TREATMENTS ............................................................................................................ 17 1.4 VIROLOGY ............................................................................................................................ 21 1.4.1 HIV-1 Genome and Accessory Proteins ...................................................................... 21 1.4.2 HIV-1 Particle Structure .............................................................................................. 23 1.4.3 HIV Replication ........................................................................................................... 24 1.4.3.1 Virion Attachment and Fusion .............................................................................. 24 1.4.3.2 Reverse transcription and integration ................................................................... 25 1.4.3.3 Sequential Production of Viral Proteins ............................................................... 28 1.4.4.4 Viral Particle Assembly, Release, and Maturation ............................................... 30 1.5 HOST RESTRICTION AGAINST HIV-I ..................................................................................... 32 1.5.1 Properties of Host Restriction Factors ........................................................................ 32 1.5.2 APOBEC3G ................................................................................................................. 33 1.5.3 TRIM5α ........................................................................................................................ 35 1.5.4 Tetherin ........................................................................................................................ 36 1.5.5 SAMHD1 ...................................................................................................................... 38 1.5.6 SLFN11 ........................................................................................................................ 39 1.5.7 MxB .............................................................................................................................. 40 1.5.8 IFITM ........................................................................................................................... 40 1.5.9 Recently Identified Restriction Factors ....................................................................... 41 1.5.10 Nef and SERINC5 ...................................................................................................... 43 1.6 PROJECT OBJECTIVES ........................................................................................................... 45 CHAPTER 2: METHODS ......................................................................................................... 47 2.1 CELL LINES AND CULTURING SYSTEM ................................................................................. 47 2.2 PLASMIDS AND CHIMERIC CONSTRUCTS .............................................................................. 47 2.3 TRANSFECTION .................................................................................................................... 50 2.4 CO-IMMUNOPRECIPITATION ................................................................................................
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