INVESTIGATING THE FITNESS BENEFIT OF REVERSE TRANSCRIPTASE (RT) MUTATION A 62 V CO-OCCURRING WITH M 184 V AND K 65 R IN HIV-1 SUBTYPE C Duncan T Njenda (MSc) Division of Medical Virology Supervisor: Co-supervisor: Professor G. U. Van Zyl Professor S. Engelbrecht Dr G. B. Jacobs
OVERVIEW * * * * HIV-1 viral evolutionary dynamics Preliminary data Research hypothesis, Aims and objectives Methodology Results Discussion Conclusion and recommendations Acknowledgements
HIV viral evolutionary Dynamics ARV therapy Host immune response Latency. Archived resistance virus Recombination Drug resistant virus (DRV) carrying Drug resistant mutation (DRM) ? ? ? Viral fitness? ?
HIV Drug resistance Classification Major (Primary) Resistance Discriminatory mutations e. g. K 65 R, M 184 V, Q 151 M Primer Unblocking mutations e. g. TAMs I & II Acquired DRMs Accessory mutations Secondary resistance DRM Compensatory mutations Transmitted resistance MTCT -Heterosexual transmission
Preliminary Data Tygerberg Hospital HIV-1 cohort on TDF/3 TC or FTC/EFV or NVP ----> 164 patients A 62 V co-occurs significantly more (p <0. 01, fisher exact test) with M 184 V and K 65 R
Research hypothesis & objectives Research hypothesis * The A 62 V mutation is selected as a compensatory mutation to restore viral fitness in patients harboring the K 65 R and M 184 V reverse transcriptase mutations. Study aim: * to investigate the relative fitness interaction of A 62 V when cooccurring with K 65 R and M 184 V in HIV-1 subtype C Objectives: • Synthesis of A 62 V; M 184 V, K 65 R, A 62 V+M 184 V; K 65 R+ M 184 V; A 62 V+ K 65 R and A 62 V+K 65 R+M 184 V full length genome (FLG) plasmid constructs harbouring these mutations • Testing the relative fitness of A 62 V+K 65 R+M 184 V vs M 184 V+K 65 R clones in a head to head in-house allele-specific q. PCR based growth competition assay.
Methodology Plasmid Extraction NL 4. 3 e. GFP derived vector Mutated construct MJ 4_HIV-1 C pol gene
Schematic of Plasmid map Plasmid constructs (n=8) • 62 V+65 R+184 V • 62 V • Wild type • 184 V • 65 R • 62 V +184 V • 65 R+184 V
Overall workflow for Growth experiment setup Mutant Constructs Transfect 293 T cells to generate viral stocks Inoculate virus in TZMbl cells grown in 6 well culture plates flask harvest every 0, 48, 72 hrs
Allele-specific q. PCR experiment setup
Results - Growth curves
Results - Statistical analysis
Discussion • A 62 V mutation does not have a significant effect on viral fitness when it co-occurs with M 184 V and K 65 R. • Unexpected! • Previous work suggested A 62 V restored fitness loss conferred by K 65 R in the presence of S 68 G (Svarovskaia et al. , 2008) • A 62 V restored fitness when it occurred in concert with Q 151 M, F 116 Y, F 77 L and V 75 L mutations (Maeda et al. , 1998). • Stanford HIVDB indicates A 62 V as a resistance mutation http: //hivdb. stanford. edu/ but no peer reviewed publication that provides evidence of the effect of A 62 V on TDF resistance? ? • Lit. suggests secondary mutations would contribute to higher levels of TDF resistance or fitness compensation.
Conclusion and Recommendations • A 62 V reverse transcriptase mutation in HIV-1 has no significant fitness compensation effect when it co-occurs with M 184 V and K 65 R • 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
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: Ø Poliomyelitis Research Foundation (PRF) Ø Harry Crossley Foundation
Thank you for your Attention!
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