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Interactions Between APOBEC3 and Murine Retroviruses: Mechanisms of Restriction and Drug Resistance

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Interactions Between APOBEC3 and Murine Retroviruses: Mechanisms of Restriction and Drug Resistance University of Pennsylvania ScholarlyCommons Publicly Accessible Penn Dissertations 2013 Interactions Between APOBEC3 and Murine Retroviruses: Mechanisms of Restriction and Drug Resistance Alyssa Lea MacMillan University of Pennsylvania, [email protected] Follow this and additional works at: https://repository.upenn.edu/edissertations Part of the Virology Commons Recommended Citation MacMillan, Alyssa Lea, "Interactions Between APOBEC3 and Murine Retroviruses: Mechanisms of Restriction and Drug Resistance" (2013). Publicly Accessible Penn Dissertations. 894. https://repository.upenn.edu/edissertations/894 This paper is posted at ScholarlyCommons. https://repository.upenn.edu/edissertations/894 For more information, please contact [email protected]. Interactions Between APOBEC3 and Murine Retroviruses: Mechanisms of Restriction and Drug Resistance Abstract APOBEC3 proteins are important for antiretroviral defense in mammals. The activity of these factors has been well characterized in vitro, identifying cytidine deamination as an active source of viral restriction leading to hypermutation of viral DNA synthesized during reverse transcription. These mutations can result in viral lethality via disruption of critical genes, but in some cases is insufficiento t completely obstruct viral replication. This sublethal level of mutagenesis could aid in viral evolution. A cytidine deaminase-independent mechanism of restriction has also been identified, as catalytically inactive proteins are still able to inhibit infection in vitro. Murine retroviruses do not exhibit characteristics of hypermutation by mouse APOBEC3 in vivo. However, human APOBEC3G protein expressed in transgenic mice maintains antiviral restriction and actively deaminates viral genomes. The mechanism by which endogenous APOBEC3 proteins function is unclear. The mouse provides a system amenable to studying the interaction of APOBEC3 and retroviral targets in vivo. Virions packaging endogenous protein were isolated from mice for analysis of APOBEC3 without a need for protein overexpression. Biochemical and molecular studies are possible using endogenous protein and viral nucleic acids. Additionally, the effect of APOBEC3-mediated viral mutagenesis and subsequent drug resistance can be modeled in this system. Human APOBEC3G transgenic mice infected with murine retroviruses and treated with an antiretroviral drug allows examination of natural levels of viral replication, APOBEC3 induced hypermutation, and potential viral escape. Studies described herein explore mechanisms of APOBEC3-mediated restriction and drug resistance in vivo. We show that endogenous APOBEC3 protein is efficiently packaged into viral cores, and this protein maintains catalytic activity against artificial substrates. We recovered low levels of G-to-A mutations from natural reverse transcription products, although approximately five to ten fold lower than that thought to be necessary for efficient viral restriction. We show that inhibition of reverse transcription is the main mechanism of restriction in vivo, and can be targeted through virion-packaged or cell-associated protein. Transgenically-expressed human APOBEC3G is instead able to heavily deaminate viral DNA, although frequently to sublethal levels. We assessed the effect of both murine APOBEC3 and APOBEC3G on viral replication in the presence and absence of an antiretroviral drug, and examined viruses for drug resistance mutations. APOBEC3G has a clear effect on the rate of viral mutagenesis in vivo, with the potential to induce drug resistance mutations. Degree Type Dissertation Degree Name Doctor of Philosophy (PhD) Graduate Group Cell & Molecular Biology First Advisor Susan R. Ross Keywords cytidine deaminase, HIV, intrinsic immunity, restriction factor Subject Categories Virology This dissertation is available at ScholarlyCommons: https://repository.upenn.edu/edissertations/894 INTERACTIONS BETWEEN APOBEC3 AND MURINE RETROVIRUSES: MECHANISMS OF RESTRICTION AND DRUG RESISTANCE Alyssa L. MacMillan A DISSERTATION in Cell and Molecular Biology Presented to the Faculties of the University of Pennsylvania in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy 2013 Supervisor of Dissertation _____________________ Susan R. Ross, Ph.D. Professor of Microbiology Graduate Group Chairperson _____________________ Daniel S. Kessler, Ph.D., Associate Professor of Cell and Developmental Biology Dissertation Committee Paul Bates, Ph.D., Professor of Microbiology Rahul Kohli, M.D., Ph.D., Assistant Professor of Medicine Sara Cherry, Ph.D., Associate Professor of Microbiology James Hoxie, M.D., Professor of Medicine ii ACKNOWLEDGMENT A PhD degree is awarded to a single person, but the effort and support come from many. I owe significant thanks to my advisor, Susan Ross, for supporting me through many years in her laboratory. Her patience, expertise, and constructive criticism are much appreciated. Former lab member Chioma Okeoma was critical in the early stages of this project, and her kindness and intelligence continue to be extraordinarily valuable to me. I also owe much thanks to current postdoctoral lab members Christian Cuevas and Spiros Stavrou for answering my endless questions and for helpful discussions. Transgenic animal models used in this work would also not have been available without the incredibly hard work of Spiros. Kristin Blouch offered not only animal expertise and assistance, but also constant support and friendship, for which I am most grateful. Of course non-lab support was also critical for my overall sanity and well-being. My parents, Marty and Carol Huegel, have been extremely supportive of my extended education, and I would not have made it this far without them. I owe the most to my two strongest pillars of support, my twin sister Julianne Huegel and my husband Matt MacMillan. I cannot thank them enough for their never ending devotion and care. iii ABSTRACT INTERACTIONS BETWEEN APOBEC3 AND MURINE RETROVIRUSES: MECHANISMS OF RESTRICTION AND DRUG RESISTANCE Alyssa L. MacMillan Susan R. Ross, Ph.D. APOBEC3 proteins are important for antiretroviral defense in mammals. The activity of these factors has been well characterized in vitro, identifying cytidine deamination as an active source of viral restriction leading to hypermutation of viral DNA synthesized during reverse transcription. These mutations can result in viral lethality via disruption of critical genes, but in some cases is insufficient to completely obstruct viral replication. This sublethal level of mutagenesis could aid in viral evolution. A cytidine deaminase-independent mechanism of restriction has also been identified, as catalytically inactive proteins are still able to inhibit infection in vitro. Murine retroviruses do not exhibit characteristics of hypermutation by mouse APOBEC3 in vivo. However, human APOBEC3G protein expressed in transgenic mice maintains antiviral restriction and actively deaminates viral genomes. The mechanism by which endogenous APOBEC3 proteins function is unclear. The mouse provides a system amenable to studying the interaction of APOBEC3 and retroviral targets in vivo. Virions packaging endogenous protein were isolated from mice for analysis of APOBEC3 without a need for protein overexpression. Biochemical and molecular studies are possible using endogenous protein and viral nucleic acids. Additionally, the effect of APOBEC3-mediated viral mutagenesis and subsequent drug resistance can be modeled in this system. Human APOBEC3G transgenic mice infected with murine retroviruses and treated with an antiretroviral drug allows examination of natural levels of viral replication, APOBEC3 induced hypermutation, and potential viral escape. Studies described herein explore mechanisms of APOBEC3-mediated restriction and drug resistance in vivo. We show that endogenous APOBEC3 protein is efficiently packaged into iv viral cores, and this protein maintains catalytic activity against artificial substrates. We recovered low levels of G-to-A mutations from natural reverse transcription products, although approximately five to ten fold lower than that thought to be necessary for efficient viral restriction. We show that inhibition of reverse transcription is the main mechanism of restriction in vivo, and can be targeted through virion-packaged or cell-associated protein. Transgenically-expressed human APOBEC3G is instead able to heavily deaminate viral DNA, although frequently to sublethal levels. We assessed the effect of both murine APOBEC3 and APOBEC3G on viral replication in the presence and absence of an antiretroviral drug, and examined viruses for drug resistance mutations. APOBEC3G has a clear effect on the rate of viral mutagenesis in vivo, with the potential to induce drug resistance mutations. v TABLE OF CONTENTS Abstract…………………………………………………………………………………………….. iii List of Tables…………….………………………………………………………………………… ix List of Figures……………………………………………………………………………………… x Chapter 1: Introduction……………………………………………………………………………. 1 Chapter 1.1: Retroviruses……………………………………………………………….. 1 Chapter 1.2: The RNA Genome and Virus Structure………………………………… 2 1.2.1 The viral gene gag……………………………………………………….. 2 1.2.2 The viral genes pro and pol…………………………………………….. 4 1.2.3 The viral gene env……………………………………………………….. 5 1.2.4 The long terminal repeats……………………………………………….. 6 1.2.5 Virus Structure……………………………………………………………. 7 Chapter 1.3: Viral Replication Cycle…………………………………………………… 8 1.3.1. Viral Entry…………………………………………………………………8 1.3.2. Reverse
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