We have discovered a novel HIV mutation that causes high-level resistance to TFV-DF. We have also determined the biochemical mechanism of this resistance. TFV-DF is a valuable NRTI therapeutic option for patients infected with multi-drug resistant Q151Mc HIV-1 
. We demonstrate here that Q151Mc can acquire an additional mutation, K70Q, which expands the multi-drug resistance to include high-level resistance to TFV-DF. We identified this mutant during genotypic analysis of clinical isolates from an HIV-infected patient who was not responding to TFV-DF. The K70Q/Q151Mc set of mutations is currently rare among HIV-infected patients. However, we believe that similar to K65R, its prevalence will increase, as tenofovir use continues to rise. Our virological studies with recombinant viruses confirmed that the observed enhancement and expansion of multi-drug resistance is the consequence of the addition of K70Q to Q151Mc HIV. Recently, the concept of clinical cut-offs (CCOs) has been introduced to improve the prediction of drug resistance during antiretroviral therapies. CCOs are better correlated with virologic response than biological cut-offs 
. Importantly, K70Q/Q151Mc is 10 times less susceptible to TFV-DF than WT HIV-1, whereas the CCOs for TFV-DF is defined as a 2.1-fold reduction in virologic response to this inhibitor. Moreover, K70Q/Q151Mc is at least twice as resistant to TFV as the well-known TFV-resistant K65R in the background of Q151Mc (as reported in the Stanford HIV Drug Resistance Database).
Previous studies have offered insights into the drug resistance mechanism of similar mutations (K70E, K70G, K70R, and K70T). Specifically, K70E was selected in patients with virological failure after TFV-DF-based antiviral therapy 
. K70T emerged in the background of Q151Mc during in vitro
selection by TFV-DF 
. K70R is a key mutation involved in resistance to AZT and appears in the background of other excision enhancement mutations 
. In our case, a new mutation (K70Q) was identified in a patient infected with Q151Mc HIV-1 during the course of TFV-DF-based antiviral therapy. The International AIDS Society-USA publishes 
every year a list of HIV-1 drug resistance mutations compiled by a panel of experts charged with the goal of delivering accurate, unbiased, and evidence-based information for use by HIV clinical practitioners. In order for a novel mutation to be accepted in the list it should meet at least one
of the following criteria: 1) in vitro
passage experiments or validation of contribution to resistance by using site-directed mutagenesis; 2) susceptibility testing of laboratory or clinical isolates; 3) nucleotide sequencing of viruses from patients in whom the drug is failing; 4) correlation studies between genotype at baseline and virologic response in patients exposed to a drug. Our study has unambiguously demonstrated that K70Q meets at least the first three criteria: evidence for criterion #1 is shown in ; for criterion #2 in and ; and for criterion #3 in and Figure S1
. Therefore, the K70Q mutation meets the criteria of a clinically relevant mutation.
In addition to the clinical and virological studies, we used biochemical techniques to determine the mechanism of TFV resistance imparted by the K70Q mutation to Q151Mc RTs. We used primer extension assays to show that K70Q/Q151Mc RT is less susceptible to TFV-DP than WT and Q151Mc RTs. We demonstrated that the mechanism of this resistance is not based on excision. On the contrary, we showed that the ATP-based excision of the mutant enzymes was slightly decreased with respect to WT RT, possibly because of decreased affinity of the mutant enzymes for the ATP excision substrate, incurred by changes in the binding environment of ATP, such as the loss of lysine at position 70.
Using transient-state kinetics we unambiguously established that the overall mechanism of K70Q/Q151Mc resistance to TFV is due to enhanced discrimination between the natural dATP substrate and TFV-DP. While all mutant enzymes had comparable efficiency of dATP incorporation, they displayed varying affinity and turnover rates of incorporation. It appears that the stronger effect of the enhanced discrimination overcomes the slight increase in sensitivity due to the small increase in excision. As a result, the mutant enzymes are resistant to the inhibitor.
Mutations at position 70 of RT have been known to confer NRTI resistance by two distinct mechanisms: K70R combined with at least two excision enhancing mutations, D67N and T215Y, enhances ATP-mediated excision of AZT and d4T 
). On the other hand, K70E causes resistance to 3TC, TFV, and ABC by lowering the maximum rate of inhibitor incorporation by RT (kpol-dependent exclusion mechanism
. Our results establish that in the background of Q151Mc, K70Q causes TFV resistance through a third mechanism: by decreasing the binding affinity of the inhibitor (Kd
-dependent exclusion mechanism
). Taken together, these findings highlight the remarkable ability of RT to use separate mutations at a single position to acquire NRTI resistance through three different mechanisms.
Our cell-based assays with infectious HIV-1 show that Q151Mc remains susceptible to TFV-DF, a finding consistent with previous reports 
. Similarly, clinical isolates deposited at the Stanford HIV resistance database and carrying the Q151Mc mutation were also susceptible to TFV-DF, unless they also had the K65R mutation. However, pre-steady state characterization of TFV-DP incorporation by Q151Mc in this work () and by others 
showed that Q151Mc is less susceptible to TFV-DP than WT RT. This small discrepancy may be the result of potential differences in DNA-dependent and RNA-dependent DNA synthesis, or the result of the slightly increased excision of Q151Mc RT compared to WT RT ().
To gain insights into the possible structural changes caused by the addition of K70Q to Q151Mc, we compared the molecular model of K70Q/Q151Mc RT/DNA/TFV-DP with the crystal structure of WT RT/DNA/TFV-DP 
(). The network of hydrogen bonds involving the side-chains of K65, R72, and Q151 in the WT structure 
, is disrupted in the mutant structure. Also, Q151M and associated mutations A62V, V75I, and F77L are likely to modify the hydrophobic core of the fingers. We and others have previously shown that the side-chains of residues 72 and 65 interact with each other 
and with Q151 and the α- and γ-phosphates of the incoming dNTP 
or TFV-DP 
. The functions of these residues have been established by several biochemical studies 
. The reduction in polymerase rate (kpol
) and in binding affinity for TFV-DP (increased Kd.TFV-DP
) may be the consequence of one or more such structural changes. Our molecular dynamics simulation experiments suggested a re-arrangement in the position of the side chain of K65, which is a catalytically important residue. While the precise effect of this change is not clear at this point, such changes could influence the overall binding of the substrate and/or the rate of nucleotide incorporation. Moreover, such movement of K65 in the presence of a mutation at position 70 is consistent with our previously reported crystallographic data, which established that there is an interplay between the positioning of the side chains at positions 70 and 65 
. Ongoing crystallographic studies are expected to provide more detailed structural insights into the role of K70Q in drug resistance.
In summary, we report here clinical data showing that addition of the K70Q mutation to the Q151Mc background confers high-level HIV resistance to TFV-DF and enhances resistance to other NRTIs. The biochemical mechanism of the TFV resistance is based on reduced binding affinity and incorporation of TFV-DP. Detection of this novel pattern of TFV-DF resistance may help adjust therapeutic regimens for the treatment of patients infected with multi-drug resistant HIV-1.