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Reconstitution and Characterization of Human Endogenous Retrovirus-K Young Nam Lee

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Reconstitution and Characterization of Human Endogenous Retrovirus-K Young Nam Lee Rockefeller University Digital Commons @ RU Student Theses and Dissertations 2010 Reconstitution and Characterization of Human Endogenous Retrovirus-K Young Nam Lee Follow this and additional works at: http://digitalcommons.rockefeller.edu/ student_theses_and_dissertations Part of the Life Sciences Commons Recommended Citation Lee, Young Nam, "Reconstitution and Characterization of Human Endogenous Retrovirus-K" (2010). Student Theses and Dissertations. Paper 78. This Thesis is brought to you for free and open access by Digital Commons @ RU. It has been accepted for inclusion in Student Theses and Dissertations by an authorized administrator of Digital Commons @ RU. For more information, please contact [email protected]. RECONSTITUTION AND CHARACTERIZATION OF HUMAN ENDOGENOUS RETROVIRUS-K A Thesis Presented to the Faculty of The Rockefeller University in Partial Fulfillment of the Requirements for the degree of Doctor of Philosophy by Young Nam Lee June 2010 © Copyright by Young Nam Lee 2010 RECONSTITUTION AND CHARACTERIZATION OF HUMAN ENDOGENOUS RETROVIRUS-K Young Nam Lee, Ph.D. The Rockefeller University 2010 Retroviruses are a family of clinically significant and scientifically fascinating viruses that infect a wide array of organisms from all vertebrate classes. The two hallmark events in the life cycle of retroviruses are the reverse transcription of the single stranded RNA (ssRNA) genome generating a double stranded DNA (dsDNA) and the integration of this dsDNA into the host genome. Because integration is irreversible and the infected cells are usually difficult to target for elimination in the host, the infection is generally permanent. HIV-1, the most important and well-studied member of all retroviruses, is the causative agent of acquired immune deficiency syndrome (AIDS) for which no vaccine or cure is known. Since recognition of the AIDS epidemic, around 25 million people have died from HIV-1 related causes, including 2 million in 2007. Currently, 33 million people are believed to be living with the virus, with most of these people living in sub-Saharan Africa, where 67% of all infected people reside and 75% of AIDS deaths occurred in 2007. When retroviruses infect germ cells or germ cell progenitors, the virus can become endogenized. These viruses, called endogenous retroviruses (ERV), make up more than 8% of the human genome. The integrated virus will be present in the genome of all cells of the individual derived from the infected germ cell, and be passed on to progeny in a Mendelian manner to following generations. Both chance and the insertion’s effect on the fitness of the host can determine the allelic frequency in the population. Hence, elements which produce large quantities of viral proteins and progeny or elements that insert into a necessary gene will likely reduce the fitness of the host and as an allele will be negatively selected in the host population. Currently, there is no known replication competent HERV, as most proviruses are filled with deletions and premature stop codons. However, one family of Class II HERV, HERV-K(HML-2), seems to have been replicating until recently. The HERV-K(HML-2) family includes human specific members and elements that are polymorphic in the human population, suggesting replication since the divergence of humans from chimpanzees 6 million years ago and potentially more recently as well. In this body of work, the problem of the lack of a replication competent virus sequence is circumvented by deducing a consensus sequence from the youngest set of HERV proviruses. Named HERV-KCON, we find that many of its components are functional individually and together enable infection of target cells in a single-cycle infection system. Using this system, we have characterized the previously unknown aspects of HERV-K(HML-2) life cycle, such as location of assembly and budding, dependency on cell replication, and more extensively, its integration site preference. HERV-KCON’s interaction with current anti-retroviral host proteins is accessed, and evidence of the same interaction occurring in vivo is presented in the context of APOBEC3G. I would like to dedicate this work to my family. My parents, who started with very little, have always provided me with a supportive and loving environment, and instilled in me the value of education and hard work. My sister has always been kind, and despite our divergent professional and personal paths, still represents in many ways what I strive toward. They enabled everything I have done. iii ACKNOWLEDGMENTS I would like to acknowledge the people my laboratories, past and present, for their scientific and personal support. Thank you for making late nights and weekends fun, in and out of lab. I would also like to acknowledge my friends. Thank you for your patience, your company, and your support. You made the unbearable moments pass and happier moments last. I also appreciate the help and support of my committee, Dr. Charles Rice, Dr. Jack Lenz, and Dr. Nina Papavasiliou, and my collaborators, Dr. Mike Malim (UCL), Dr. Fredrick Bushman (University of Pennsylvania), and Dr. Troy Brady (University of Pennsylvania). The Dean’s office members and the administrative staff of Rockefeller University and ADARC have been tremendously helpful over the years in multiple aspects of my life. Thank you. Lastly, I would like to acknowledge the city. I love New York. It is the only home I have known in my adult life. I pray that I will be back one day. iv TABLE OF CONTENTS Dedication iii Acknowledgements iv Table of Contents v Table of Figures vii Table of Tables ix List of Publications x I. INTRODUCTION 1 1. Retroviridae 1 2. Integration site preference of retroviruses 14 3. Endogenous retroviruses 15 4. History of the discovery of HERV-K superfamily 20 5. Activity and replication potential of HERV-K(HML-2) 23 6. Restriction factors 28 II. MATERIALS AND METHODS 37 1. Cloning of endogenous HERV-K proviruses 37 2. Derivation and synthesis of HERV-KCON 38 3. HERV-KCON–derived expression plasmids 40 4. Other expression plasmids 43 5. Cell lines 43 6. Transfection 44 7. Infection 44 8. Reverse Transcription Assay 45 9. Generation of anti-HERV-K CA antibody 45 10. HERV-K protein analysis 46 11. Analysis of de novo integrated HERV-KCON proviral DNA 47 12. HERV- K sequence analysis 48 13. HERV-K hypermutation assay 49 14. Aphidicolin induced cell arrest and infection 50 15. Infection and recovery of integration sites 50 16. Analysis of integration site distributions 52 III. RESULTS 53 Chapter 1. Endogenous HERV-K proviruses 53 1. Introduction to human endogenous retroviruses 53 2. HERV-K(HML-2) 54 v 3. HERV-K113 LTR 66 4. HERV-K113 and YY2 67 5. Replication capacity of K113 and K108 70 Chapter 2. Derivation of HERV-KCON and the single-cycle infection system 74 1. Derivation of HERV-KCON 74 2. HERV-KCON sequence 75 3. HERV-KCON proteins 89 4. Establishment of single-cycle infection system 96 Chapter 3. Restriction factors and HERV-K 109 1. Introduction to restriction factors 109 2. Effects of TRIM proteins on HERV-KCON infection 110 3. Effect of tetherin on HERV-KCON release 112 4. Effect of APOBEC proteins on HERV-KCON infection 115 5. Hypermutated HERV-K proviruses in modern human DNA 117 6. Flanking nucleotide characteristics of G-to-A changes in hypermutated HERV-K proviruses 120 7. Hypermutation of HERV-K by APOBEC3 proteins during in vitro replication 122 Chapter 4. Integration of HERV-KCON 129 1. Introduction to retrovirus integration 129 2. HERV-K integration preference relative to genomic markers 130 3. HERV-KCON integration preference relative to endogenous HERV-K integration sites 137 IV. DISCUSSION 143 V. REFERENCES 162 vi LIST OF FIGURES Figure 1 Phylogenetic analysis of Retrovirus family .......................................................... 2 Figure 2 Basic genomic layout of retroviruses ................................................................... 4 Figure 3 Life cycle of HIV-1 .............................................................................................. 5 Figure 4 Reverse transcription of retroviruses .................................................................... 7 Figure 5 Retrovirus integration ........................................................................................... 9 Figure 6 Location of restriction sites in HERV-K(HML-2) ............................................. 38 Figure 7 Map of pCRVI .................................................................................................... 42 Figure 8 Chromosomal distribution of HERV-K(HML-2) LTRs. ................................... 62 Figure 9 The activity of HERV-K(HML-2) LTR inducing factor or factors in NCCIT cells ................................................................................................................................... 68 Figure 10 The modest transcription enhancing activity of YY2 ....................................... 70 Figure 11 K113 and K108 derived proviral constructs ..................................................... 72 Figure 12 Undisrupted open reading frames in HERV-K proviruses ............................... 75 Figure 13 Phylogenetic analysis of HERV-K proviruses and HERV-KCON ..................... 76 Figure 14 Sequence of HERV-KCON ................................................................................. 88 Figure 15 Expression of HERV-K proteins and release of virus-like particles ................ 90 Figure 16 RT activity of HERV-KCON VLPs ...................................................................
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