The HIV life cycle entry reverse transcription transcription integration virus production nuclear import Hazuda 2012
Integration Insertion of the viral DNA into host chromosomal DNA, essential step in cycle Point of no return: cell becomes a permanent carrier Causes persistence and is the barrier to cure ( eg HCV)
Integration (six steps) Binding of integrase to DNA Processing of the 3 ends Translocation of the pre-integration complex to cell nucleus Binding of pre-integration complex to host DNA Transfer of viral DNA into host DNA ( strand transfer) Repair of the gaps formed during strand transfer by host cell enzymes
Integration (two reactions) 3 processing: 2/3 nucleotides are removed from the 3 ends from the DNA strand at both sides of the proviral DNA Strand transfer reaction: the processed 3 ends of viral DNA are covalently bound to the host chromosomal DNA
Craigie and Bushman 2012 The mechanics of integration
Integration
Integrase structure Zinc Finger Catalytic Core DNA Binding H H C C D64 D116 E152 SH3 1 50 212 288 Asp-Asp-Glu (DDE) motif binds Mg2+ Motif is well conserved in evolution 8
Zinc finger Function is to assist in forming multimers of integrase and binding of cellular factors
Real is beautiful Craigie and Bushman 2012
Access to chromatin
Pre-integration complex (PIC) Several host cellular proteins have been shown to interact with Integrase to facilitate the integration proces ( pre-integration complex PIC) Human chromatin-associated protein LEDGF tightly binds HIV Integrase and directs the pre-integration complex towards highly expressed host genes
HIV integrates in gene-dense/transciption active regions Schröder 2002
Maldarelli et al. 2014 HIV reservoir integration sites
Wagner et al. 2014 HIV integration in cancer genes?
Integrase (is not alone) Nuclear traffic and core proteins Nuclear entry factors Histones DNA repair proteins
Integrase (additional role) ) Integrase is also required during viral production from the integrated provirus during particle maturation Two dimers combine resulting in four integrase molecules
Disturbing intergrase entry reverse transcription transcription integration virus production nuclear import Hazuda 2012
Jurado et al. 2013 Integrase role after integration
Integrase inhibition
strand transfer inhibitors Dead end, two LTR circles unintegrated linear DNA Hazuda 2012
Integrase strand-transfer inhibitors (INSTIs) Métifiot, Marchand and Pommier 2013
HIV integration
Resistance maps to the active site Zinc Finger Catalytic Core DNA Binding H H C C D64 D116-(35)-E152 SH3 1 50 212 288 Resistance Mutations: Q148K/R N155H resistance positions are proximal to the DDE residues that coordinate the essential active site metals, the changes diminish the binding of the integrase to complex. 24
HIV plasma RNA decay during ART initiation Perelson et al. 1996
Murray et al. 2010 INIs and HIV plasma RNA decay
Protease and RT inhibitors virus production unintegrated linear DNA Hazuda 2012
Preintegration latency How long can it last? Hazuda 2012
strand transfer inhibitors unintegrated linear DNA Hazuda 2012
Buzon et al. 2010 Survival time preintegration complex?
Murray et al. 2010 Inhibition of virus production from cells harbouring the pre-integration complex
strand transfer inhibition unintegrated linear DNA Hazuda 2012
Allosteric Integrase Inhibitors (ALLINIs)
Pre-integration complex (PIC) Several host cellular proteins have been shown to interact with Integrase to facilitate the integration proces Human chromatin-associated protein LEDGF tightly binds HIV Integrase and directs the preintegration complex towards highly expressed genes Allinis bins intergase at the LEDGF binding site
Allinis and multimerisation
Christ and Debyzer 2013 Lens EpiDermal Growth Factor LEDGF p75
Krishnan and Engelman 2012 ALLINIs
Slaughter et al. 2014 ALLINIs : resistance
Take it home Integration of the DNA provirus, catalyzed by the integrase, is an essential step of HIV replication HIV DNA preferentially integrates in active cellular genes, some of which may favor amplification of HIV clones in the reservoir Integrase strand transfer inhibitors (INSTIs) produce a unique HIV plasma RNA response The ever-surprising allosteric integrase inhibitors (ALLINIs) have contributed into understanding of an additional role of integrase
Acknowledgements Francois Clavel (Paris, France) for slides