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:
Co-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
Drug resistant virus (DRV)
carrying Drug resistant
mutation (DRM)
Host
immune
response
LatencyArchived
resistance virus
Recombination
???
Viral fitness??
HIV Drug resistance Classification
Major (Primary)
Resistance
Discriminatory
mutations e.g.
K65R, M184V,
Q151M
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/3TC or FTC/EFV or NVP ----> 164 patients
A62V co-occurs significantly more (p <0.01, fisher exact test) with M184V
and K65R
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 cooccurring 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.
Methodology
Mutated construct
Plasmid Extraction
NL4.3 eGFP derived vector
MJ4_HIV-1C pol gene
Schematic of Plasmid map
Plasmid constructs (n=8)
• 62V+65R+184V
• 62V
• Wild type
• 184V
• 65R
• 62V + 65R
• 62V +184V
• 65R+184V
Overall workflow for Growth experiment setup
Mutant
Constructs
Transfect
293T 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 qPCR experiment setup
Results - Growth curves
Results - Statistical analysis
Discussion
• 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??
• Lit. suggests secondary mutations would contribute to higher levels of
TDF resistance or fitness compensation.
Conclusion and Recommendations
• 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
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