2. INVESTIGATING THE FITNESS BENEFIT OF REVERSE TRANSCRIPTASE (RT) MUTATION A62V CO-OCCURRING WITH M184V AND K65R IN HIV-1 SUBTYPE C Duncan T Njenda (MSc) Division of Medical Virology Supervisor: Professor G. U. Van Zyl Co-supervisor: Professor S. Engelbrecht Co-supervisor: Dr G. B. Jacobs

3. OVERVIEW * HIV-1 viral evolutionary dynamics * Preliminary data * Research hypothesis, Aims and objectives * Methodology * Results * Discussion * Conclusion and recommendations * Acknowledgements

4. HIV viral evolutionary Dynamics Host immune response Latency- Archived resistance virus Recombination Drug resistant virus (DRV) carrying Drug resistant mutation (DRM) Viral fitness?? ???

5. HIV Drug resistance Classification DRM Acquired DRMs Major (Primary) Resistance Discriminatory mutations e.g. K65R, M184V, Q151M Primer Unblocking mutations e.g. TAMs I & II Secondary resistance Accessory mutations Compensatory mutations Transmitted resistance MTCT -Heterosexual transmission

6. Preliminary Data Tygerberg Hospital HIV-1 cohort on TDF/3TC or FTC/EFV or NVP ----> 164 patients A62V co-occurs significantly more (p <0.01, fisher exact test) with M184V and K65R

7. Research hypothesis & objectives Research hypothesis * The A62V mutation is selected as a compensatory mutation to restore viral fitness in patients harboring the K65R and M184V reverse transcriptase mutations. Study aim: * to investigate the relative fitness interaction of A62V when co- occurring with K65R and M184V in HIV-1 subtype C Objectives: Synthesis of A62V; M184V,K65R, A62V+M184V; K65R+ M184V;A62V+ K65R and A62V+K65R+M184V full length genome (FLG) plasmid constructs harbouring these mutations Testing the relative fitness of A62V+K65R+M184V vs M184V+K65R clones in a head to head in-house allele-specific qPCR based growth competition assay.

8. Methodology Plasmid Extraction NL4.3 eGFP derived vector MJ4_HIV-1C pol gene Mutated construct

9. Schematic of Plasmid map Plasmid constructs (n=8) 62V+65R+184V 62V Wild type 184V 65R 62V + 65R 62V +184V 65R+184V

10. Mutant Constructs Transfect 293T cells to generate viral stocks Inoculate virus in TZM- bl cells grown in 6 well culture plates flask harvest every 0, 48,72 hrs Overall workflow for Growth experiment setup

11. Allele-specific qPCR experiment setup

12. Results - Growth curves

13. Results - Statistical analysis

14. A62V mutation does not have a significant effect on viral fitness when it co-occurs with M184V and K65R. Unexpected! Previous work suggested A62V restored fitness loss conferred by K65R in the presence of S68G (Svarovskaia et al., 2008) A62V restored fitness when it occurred in concert with Q151M, F116Y, F77L and V75L mutations (Maeda et al., 1998). Stanford HIVDB indicates A62V as a resistance mutation - http://hivdb.stanford.edu/ but no peer reviewed publication that provides evidence of the effect of A62V on TDF resistance?? http://hivdb.stanford.edu/ Lit. suggests secondary mutations would contribute to higher levels of TDF resistance or fitness compensation. Discussion

15. A62V reverse transcriptase mutation in HIV-1 has no significant fitness compensation effect when it co-occurs with M184V and K65R Further investigations would be required to investigate the fitness effect of individual mutations and all the possible mutation interactions Emphasis on patient monitoring and resistance testing Conclusion and Recommendations

16. ACKNOWLEDGEMENTS I am grateful and thankful to: * Professor G.U. van Zyl * Professor S. Engelbrecht * Dr G.B. Jacobs * Students and Staff of the Division of Medical Virology, Stellenbosch University I am grateful and thankful to the following Organisations for Funding:  Poliomyelitis Research Foundation (PRF)  Harry Crossley Foundation