Universitat Autònoma de Barcelona Faculty of Sciences, Department of Biochemistry And Molecular Biology Effect of the HIV-1 Integrase Inhibitor Raltegravir on Drug Susceptibility, Replication Capacity and Residual Viremia in HIV-infected Subjects Maria José Buzón Gómez Retrovirology Laboratory Institut de Recerca de la SIDA, Fundació IrsiCaixa Hospital Germans Trias i Pujol 2010 Thesis to obtain the PhD degree of the Universidad Autònoma de Barcelona Director: Dr. Javier Martínez-Picado Tutor: Xavier Avilés Puigvert This thesis has been supported by the Spanish AIDS network “Red Temática Cooperativa de Investigación en SIDA” (RD06/0006) and by funding from the European Community's Seventh Framework Program (FP7/2007-2013) under the project "Collaborative HIV and Anti-HIV Drug Resistance Network (CHAIN)" (grant agreement no. 223131) and by an unrestricted grant from Merck Sharp & Dohme (MSD). MJ Buzón was supported by Agència de Gestió d’Ajuts Universitaris i de Recerca from Generalitat de Catalunya (grant 2009FI_B 00368 and the European Social Fund. Additional support was provided by the Spanish AIDS Network “Red Temática Cooperativa de Investigación en SIDA” (RIS) through grants RD06/0006/0020 and the Fundación para la investigación y Prevención del SIDA en España (FIPSE) through grant 36630/07. The printing of this thesis was made possible by the financial aid of the UAB Cover design: Marta Massanella, Maria Carmen Puertas, Nuria Izquierdo-Useros and Gerard Minuesa. El doctor Javier Martínez-Picado, investigador principal en el laboratorio de Retrovirologia del Instituto de Investigación del SIDA, Fundación IrsiCaixa, certifica: Que el trabajo experimental y la tesis titulada “Effect of the HIV-1 integrase inhibitor raltegravir on drug susceptibility, replication capacity and residual viremia in HIV-infected subjects” han sido realizados por Maria José Buzón Gómez bajo su dirección y que considera que son aptos para su lectura y defensa con el objecto de optar al título de Doctora por la Universidad Autònoma de Barcelona. Badalona, 23 de Septiembre de 2010 Dr. Javier Martínez-Picado El doctor Xavier Avilés Puigvert, coordinador del programa de doctorado en Bioquímica y Biología Molecular de la Universidad Autònoma de Barcelona, certifica: Que el trabajo experimental y la tesis titulada “Effect of the HIV-1 integrase inhibitor raltegravir on drug susceptibility, replication capacity and residual viremia in HIV-infected subjects” han sido realizados por Maria José Buzón Gómez bajo su tutela y que considera que son aptos para su lectura y defensa con el objecto de optar al título de Doctora por la Universidad Autònoma de Barcelona. Badalona, 23 de Septiembre de 2010 Dr. Xavier Avilés Puigvert “La ignorancia afirma o niega rotundamente; la Ciencia duda” Voltaire CONTENTS RESUMEN............................................................................................................ 5 SUMMARY........................................................................................................... 8 Chapter 1 General Introduction Human Immunodeficiency Virus ..................................................................13 Classification and Structure ..........................................................................13 Replication cycle of HIV and antiretroviral drugs ....................................16 Viral entry........................................................................................................................................... 16 Transcription .................................................................................................................................... 17 Integration.......................................................................................................................................... 19 Transcription and translation.................................................................................................... 21 New virion production and budding....................................................................................... 22 Virion maturation ........................................................................................................................... 22 HIV-1 quasispecies and viral fitness ............................................................25 Fitness and development of resistance mutations ...................................26 Fitness and development of protease resistance mutations........................................ 26 Fitness and development of reverse-transcriptase resistance mutations............. 27 Fitness and development of integrase resistance mutations....................................... 28 Dynamics of persistent viremia ....................................................................29 Mechanisms of viral latency ....................................................................................................... 30 Ongoing viral replication ............................................................................................................. 31 Therapeutic strategies for viral eradication ..............................................32 Treatment intensification............................................................................................................ 32 Immunosuppressants.................................................................................................................... 32 Reactivation of the latent reservoir ........................................................................................ 33 HYPOTHESIS AND OBJECTIVES.................................................................35 Chapter 2 Materials and Methods Study subjects ...................................................................................................41 Subjects included in “Raltegravir susceptibility and the fitness progression of HIV-1 integrase in subjects on long-term integrase-sparing antiretroviral therapy” study .................................................................................................................................. 41 1 Subjects included in “The HIV-1 integrase genotype strongly predicts Raltegravir susceptibility but not viral fitness of primary virus isolates” study......................... 41 Subjects included in “HIV-1 replication and immune dynamics are affected by raltegravir intensification of HAART-suppressed subjects” study............................ 42 Population-based sequencing of the HIV-1................................................43 Calculation of the evolution rate ..................................................................45 Construction of cloning vectors ....................................................................45 Construction of HIV-1 integrase-defective hemiplasmid vector (pJM30∆integrase) ......................................................................................................................... 45 Construction of HIV-1 integrase-defective plasmid vector (pNL4-3∆integrase) 46 Construction of HIV-1 polymerase -defective plasmid vector (pJM31∆Polymerase) .................................................................................................................... 47 Generation of recombinant viruses .............................................................48 Generation of labelled integrase-recombinant viruses .................................................. 48 Generation of recombinant viruses......................................................................................... 49 Generation of integrase-recombinant viruses as controls............................................ 51 Recovery of primary virus isolates ..............................................................51 Raltegravir susceptibility assay ....................................................................52 Growth rate assay ............................................................................................53 Competition assay ...........................................................................................53 Nucleic acid purification for quantification of HIV-1 DNA forms ..........55 Quantification of HIV-1 2–long terminal repeat circles ...........................55 Quantification of total HIV-1 DNA ................................................................56 Quantification of integrated HIV-1 DNA .....................................................57 Single-copy assay for HIV-1 RNA concentrations in plasma ..................57 Analysis of lymphocyte subsets and immune activation ........................58 Soluble CD14 (sCD14) ....................................................................................58 Statistical analysis ...........................................................................................59 Chapter 3. Results I Raltegravir Susceptibility and the Fitness Progression of HIV-1 Integrase in Subjects on Long-Term Integrase-Sparing Antiretroviral Therapy INTRODUCTION ...............................................................................................63 RESULTS ............................................................................................................65 2 Subjects characteristics................................................................................................................ 65 The integrase gene remains highly conserved after long-term antiretroviral therapy................................................................................................................................................