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CROI Resistance Report 5: the K65R Mutation, its relevance, prevalence, & mechanism of action
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Written for NATAP by Andrew Zolopa, MD
Stanford University
K65R: Prevalence, Mechanism of Resistance and Mutational Interactions
In reviewing the VIRCO database, Parikh in collaboration with Bacheler showed that the prevalence of 65R is increasing although it still remains a relatively uncommon mutation in clinical samples sent for resistance testing (presumably from patients on failing ARV regimens). [abstract 54] The prevalence of 65R increased from 0.4% in 1998 to 3.6% in 2003. This trend may in fact be related to the increased use of nucleoside/tide analogs that select for this mutation (which includes ABC, DDI, DDC and TDF) and or the decreased use of drugs that might be antagonistic to it's development - namely AZT and possibly D4T.
Parikh went on to report that there is a strong negative correlation between the prevalence of TAM's and that of 65R in the VIRCO database. The investigators explored the potential molecular mechanisms that underlie this apparent antagonism. TAM's work by increasing the ability of the virus that possesses these mutations to remove nucleoside/tides that block reverse transcription through a process called pyrophosphorolysis. By more easily removing the analogs the virus is able to continue the process of reverse transcription and therefore, is "resistant' to the inhibitory effects of the drugs. The investigators showed that the presence of 65R reduced primer unblocking which resulted in a reduction in AZT resistance. Using two different clones with multiple TAM's (41L, 210W, 215Y and 67N,70R, 215F and 219Q) the investigators demonstrated that AZT resistance was reduced by 10 fold when the 65R mutation was added to the clone. Primer unblocking (pyrophosphorolyis) was reduced in these clones with TAM's and 65R. The investigators also demonstrated that TAM's reduced resistance mediated by 65R to ddC, ddI, ABC and TDF, although the mechanism of this antagonistic interaction was not defined.
In a second presentation examining the mechanisms of resistance of the 65R mutation, White and colleagues from Gilead described two important factors responsible for nucleoside/tide resistance. Mutants with 65R demonstrated decreased incorporation of ddI, TDF, ABC, d4T as well as AZT. This decrease in incorporation would be expected to result in resistance to these drugs. However, 65R, also was shown to decrease excision (pyrophosphorolysis) as was demonstrated in the study by Parikh et al. This would tend to decrease observed resistance seen with viruses that contained 65R (less excision equates to more inhibition, that is the inhibitor is better able to work -- therefore, less resistance, follow?). So when determining the impact of 65R on the resistance profile of a particular drug one needs to consider these two competing effects -- decreased incorporation, which increases resistance, and decreased primer unblocking or excision which should diminish resistance. In turns out for AZT, even though 65R decreases incorporation which should result in a virus that is resistant to AZT, the impact on decreasing excision of AZT is much greater and therefore the net effect is that viruses with 65R, tend to be hypersusceptible to AZT compared to wild-type viruses while for TDF, ddI and ABC the impact on incorporation outweighs the effect of decreased excision resulting in a virus that has reduced susceptibility to these drugs.
Why is it important for the practitioner to know K65R's mechanism of resistance? If we have an understanding the mechanism by which 65R causes resistance we will be better able to interpret the resistance resulting for patients in whom this mutation develops. Clinical questions that remain to be answered and will in part be informed by this knowledge include: How much virologic activity remains in the presence of 65R for drugs like TDF and ABC? Does the 65R associated hypersusceptibility of AZT have clinical implications? What impact does 65R have on the virologic activity of d4T?
K65R remains a relatively rare mutation as does the multi-nucleoside complex of Q151M. French investigators reported finding 24 clinical isolates with 65R out of a database that contained over 3,000 samples (prevalence 0.8%) but what was interesting is the investigators found that Q151M complex was associated with the 65R. [abstract 627] In the 33 samples in which a Q151M complex was found 9 also had the 65R mutation. It appeared that most of these cases where in TDF naïve patients. The evolution of these "double" mutants is unclear from the cross sectional survey of the database but it does raise the possibility of multi-nucleoside resistance pathways in which highly resistant viruses develop both 65R and Q151M. Further evaluation of other databases is required.
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