ALLINIs modulate the interaction between HIV-1 Rev and integrase

Transcription

1 ALLINIs modulate the interaction between HIV-1 Rev and integrase Date: 13 June 2017 Author Shaakira Abrahams Salerwe Mosebi, Qasim Fish, Raymond Hewer, Maria Papathanasopoulos

2 Outline Background Results and discussion In vitro selection studies selected by ALLINIs The interaction between Rev and integrase in the presence of ALLINNIs The effect of ALLINIs and Rev on multimerization of integrase Conclusion Acknowledgements

3 Background HIV-1 Rev regulates the integration step by inhibiting integration Integrase (IN) forms part of the pre-integration complex that comprise LEDGF/p75, viral DNA and viral accessory proteins Highly dependent on the equilibrium state of IN dimer LEDGF/p75 binds to the hydrophobic pocket of the IN dimer interface Rev Binds to the LEDGF/p75 interface - Interplay between IN, LEDGF/p75 and Rev for the IN dimer interface Rev inhibits integration through multimerization

4 Background: ALLINIs Allosteric IN inhibitors (ALLINIs): Allosterically inhibit the catalytic activities of IN Interplay between ALLINIs and LEDGF/p75 for dimerization interface of IN Mechanism of action: Shifts oligomerization of IN into an inactive multimeric form Produces defective and less infectious virions ALLINIs used in this study: CX05168, Christ et al, 2010 and Mut 101, Le Rouzic, 2013

5 Background ALLINIs and Rev effect the HIV-1 integration step in a similar manner Interplay between Rev and LEDGF/p75; Interplay between ALLINIs and LEDGF/p75 Multimerization of IN Prevents IN from binding to host DNA Hypothesis: ALLINIs modulate Rev s regulatory role in HIV-1 integration Objectives 1. Select mutations in the pol and rev gene using ALLINIs 2. Determine the binding interaction of Rev and IN in the presence of ALLINIs 3. Determine whether the Rev-driven multimerization is modulated by ALLINIs

6 Results and discussion 1. Select mutations in the pol gene using ALLINIs 2. Determine the binding interaction of Rev and IN in the presence of ALLINIs 3. Determine whether the Rev-driven multimerization is modulated by ALLINIs

7 In vitro selection studies Figure 1: Viral fitness of FV 26 and FV 3 in vitro selected by Mut 101 and CX05168

8 EC 50 of CX05168 against HIV-1 primary Subtype C EC 50 of CX05168 against FV 3 and FV 26 higher than Subtype B NL4-3 EC 50 = 2.35µM (Christ et al, 2010) FV 3 EC 50 = 4.3 ± 0.4µM FV 26 EC 50 = 40.7 ± 0.8µM Polymorphic mutations found in drug naïve FV3 and FV 26 FV 3 FV 26 L101F T218I A265V I84M L101Y K215N * *

9 Phenotypic susceptibility associated with mutations selected by Mut 101 Conducted an antiviral assay using Mut 101 FV Passage EC 50 ± SD Fold change Possible (µm) mutation Drug 0.48 ± 0.15 naïve 8 ND H171L Drug 0.53 ± 0.01 naïve ± fold reduction L172I ± fold increase L172I K215D 41 ND H171Q I84M; L101Y; K215N

10 Results and discussion 1. Select mutations in the pol and rev gene using ALLINIs 2. Determine the binding interaction of Rev and IN in the presence of ALLINIs 3. Determine whether the Rev-driven multimerization is modulated by ALLINIs

11 Incubation of IN with ALLINIs then Rev * IN + Rev IN + Mut 101/CX Rev IN + Rev + Mut 101/CX05168 K d Rev IN is significantly lower than the K d of Rev IN in the presence of Mut 101 and CX05168 when incubating IN with ALLINI first then incubating with Rev Suggest that Mut 101 and CX05168 decreases the interaction between Rev and IN CX05168 displaces Ala128 on IN to which Rev binds Mut 101 alters alpha helices of IN (res and ) rev is unable to bind

12 Interaction of IN with Rev then ALLINIs * K d of Rev IN not significantly different to K d of Rev- IN when incubating IN and Rev followed by ALLINIs ALLINIs do not interfere with the Rev IN interaction once Rev is bound to IN Rev masks the interaction site of Mut 101(res 174) and CX05168 (res 128) to IN

13 Results and discussion 1. Select mutations in the pol and rev gene using ALLINIs 2. Determine the binding interaction of Rev and IN in the presence of ALLINIs 3. Determine whether the Rev-driven multimerization is modulated by ALLINIs

14 Multimerization of IN induced by ALLINIs and Rev Multimerization of IN was determined through AlphaScreen dimerization assay Multimerization of IN: Mut 101 > CX05168 > Rev

15 Rev decreases ALLINI induced multimerization of IN Rev reverse multimerization of IN induced by ALLINIs Phenomenon is observed by LEDGF/p75 and ALLINIs (Feng at al, 2016) LEDGF/p75 and ALLINIs have different multimerization patterns LEDGF/p75: NTD-CCD ALINIs: CCD-CCD Propose that Rev induces multimerization in a similar manner as LEDGF/p75 consequently reversing multimerization of IN induced by Mut 101 and CX05168

16 Conclusion Mut 101 resistant phenotype was observed through an EC 50 shift while CX05168 was not as effective against HIV-1 Subtype C FV 3 and FV 26 strains as Subtype B The behaviour of Rev and ALLINIs in the integration step are modulated when combined

17 Acknowledgements Dr Salerwe Mosebi Mintek Dr Raymond Hewer (UKZN) Prof Maria Papathanasopoulos (Wits - HIV Pathogenesis Research Unit) Mr. Qasim Fish Ms. Angela Harrison Mintek Centre of Metal Based Drug Discovery (CMDD) NRF PDP Scholarship

18 Thank You