The HIV life cycle. integration. virus production. entry. transcription. reverse transcription. nuclear import
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2 The HIV life cycle entry reverse transcription transcription integration virus production nuclear import Hazuda 2012
3 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)
4 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
5 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
6 Craigie and Bushman 2012 The mechanics of integration
7 Integration
8 Integrase structure Zinc Finger Catalytic Core DNA Binding H H C C D64 D116 E152 SH Asp-Asp-Glu (DDE) motif binds Mg2+ Motif is well conserved in evolution 8
9 Zinc finger Function is to assist in forming multimers of integrase and binding of cellular factors
10 Real is beautiful Craigie and Bushman 2012
11 Access to chromatin
12 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
13 HIV integrates in gene-dense/transciption active regions Schröder 2002
14 Maldarelli et al HIV reservoir integration sites
15 Wagner et al HIV integration in cancer genes?
16 Integrase (is not alone) Nuclear traffic and core proteins Nuclear entry factors Histones DNA repair proteins
17 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
18 Disturbing intergrase entry reverse transcription transcription integration virus production nuclear import Hazuda 2012
19 Jurado et al Integrase role after integration
20 Integrase inhibition
21 strand transfer inhibitors Dead end, two LTR circles unintegrated linear DNA Hazuda 2012
22 Integrase strand-transfer inhibitors (INSTIs) Métifiot, Marchand and Pommier 2013
23 HIV integration
24 Resistance maps to the active site Zinc Finger Catalytic Core DNA Binding H H C C D64 D116-(35)-E152 SH 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
25 HIV plasma RNA decay during ART initiation Perelson et al. 1996
26 Murray et al INIs and HIV plasma RNA decay
27 Protease and RT inhibitors virus production unintegrated linear DNA Hazuda 2012
28 Preintegration latency How long can it last? Hazuda 2012
29 strand transfer inhibitors unintegrated linear DNA Hazuda 2012
30 Buzon et al Survival time preintegration complex?
31 Murray et al Inhibition of virus production from cells harbouring the pre-integration complex
32 strand transfer inhibition unintegrated linear DNA Hazuda 2012
33 Allosteric Integrase Inhibitors (ALLINIs)
34 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
35 Allinis and multimerisation
36 Christ and Debyzer 2013 Lens EpiDermal Growth Factor LEDGF p75
37 Krishnan and Engelman 2012 ALLINIs
38 Slaughter et al ALLINIs : resistance
39 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
40 Acknowledgements Francois Clavel (Paris, France) for slides
41
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