Antiviral Therapy 14:

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1 Antiviral Therapy 14: Original article Mutations in the thumb connection and RNase H domain of HIV type-1 reverse transcriptase of antiretroviral treatment-experienced patients Joshua M Waters 1 *, Wesley O Neal 2, Kirsten L White 3, Charles Wakeford 1, Eric B Lansdon 3, Jeanette Harris 1, Evguenia S Svarovskaia 1, Michael D Miller 3 and Katyna Borroto-Esoda 1 1 Gilead Sciences, Inc., Durham, NC, USA 2 East Carolina University, Greenville, NC, USA 3 Gilead Sciences, Inc., Foster City, CA, USA *Corresponding author: Joshua.Waters@gilead.com Background: Antiretroviral therapy that targets HIV type-1 (HIV-1) reverse transcriptase (RT) can be linked to mutations in the thumb connection (amino acids [AA] ) and RNase H (AA ) domains, which could affect drug resistance. Methods: Genotypical and statistical analyses were performed on HIV-1 RT from 100 antiretroviral treatmentnaive and 248 antiretroviral treatment-experienced patients, the majority of whom were infected with HIV-1 subtype B. The RT region was analysed in three parts: the polymerase (AA 1 240), thumb connection (AA ) and RNase H (AA ) domains. Results: The polymerase domain had statistically significant changes between the two groups at 24 AA positions that are known resistance sites. Within the thumb connection domain, R284 and N348 had statistically significant changes between the groups (P=0.007 and P 0.001, respectively). In treatment-experienced patients, 17.3% had R284K, whereas 24.5% had N348I substitutions. Both R284 and N348 were 100% conserved in treatment-naive patients. Within the RNase H domain, only K451 showed a statistically significant change (P 0.001), with K451R present in 11% of treatment-experienced patients but remaining 100% conserved among treatment-naive patients. Conclusions: RT mutations at three positions outside of the polymerase region were associated with antiretroviral therapy: R284K, N348I and K451R. Both R284K and K451R interact with the phosphate backbone of the template or primer in HIV-1 RT crystal structures and could potentially influence the positioning of the primer strand, thus affecting polymerization, the efficiency of nucleoside reverse transcriptase inhibitor excision and/or RNase H activity. Introduction The HIV type-1 (HIV-1) reverse transcriptase (RT) converts the viral genome from single-stranded RNA into double-stranded DNA [1,2]. This process involves two distinct types of enzymatic activities: polymerization of DNA from either an RNA or DNA template and cleavage of RNA by RNase H as part of an RNA DNA duplex [3 5]. The RNase H region is encoded by amino acids (AA) of the RT [6]. Many antiretroviral therapy (ART) regimens contain RT-targeted drug classes of nucleoside reverse transcriptase inhibitors (NRTIs) and non-nrtis (NNRTIs) to suppress viral replication. Use of these RT inhibitor drugs can lead to the emergence of drug-resistant HIV-1 variants. Current HIV-1 genotypical analyses generally focus on AA of the RT gene and therefore miss potential ART-related mutations in the thumb connection (AA ) and RNase H domains of RT that might impart resistance to antiretroviral drugs either individually or in concert with other RT mutations [7,8]. Recently, several studies have reported that mutations in the thumb connection domain at positions E312, G335, N348, G359, A360, V365, A376, K390, K395 and E396 increase resistance to NRTIs and NNRTIs in vitro, particularly to zidovudine (AZT), in the presence of the thymidine analogue mutations (TAMs) M41L, D67N, K70R, L210W, T215Y/F and K219Q/ E/N [8 12]. A subsequent study demonstrated that in treatment-experienced patients, positions R358, G359, A360, K366, A371 and A400 showed an increased prevalence of changes, whereas changes at position I International Medical Press (print) (online) 231

2 JM Waters et al. showed a decreased prevalence [13]. Positions G359, A360, H361, K390, K395 and E396 contact the DNA primer and RNA template near the RNase H active site and are part of the RNase H primer grip [14]. The most well characterized mutation in the connection domain, N348I, confers resistance to AZT in the absence and presence of TAMs and decreases susceptibility to NNRTIs in vitro [8,10,11,15]. There are eight AA residues that are well conserved in all known bacterial and retroviral RNase H enzymes (D443, G444, E478, D498, S499, H539, Q545 and D549); the four acidic residues (D443, E478, D498 and D549) play an important role in forming two metalbinding sites [6,16]. Positions T473, Q475, K476, Y501 and I505 are part of the RNase H primer grip that is important for the proper binding and positioning of the nucleic acid to the RT [14]. It has been suggested that mutations at these positions will considerably increase resistance to AZT in the presence of TAMs [9]. A recent study supports a mechanism in which the equilibrium of NRTI incorporation, nucleotide excision and RNase H activity is crucial for NRTI- mediated abrogation of HIV-1 replication as well as in NRTI resistance [7]. In vitro experiments have shown that mutations at highly conserved AA positions within the RNase H region (D475E, N494D, H539N and D549N) can alter the RNase H enzymatic activity (processive activity and binding affinity), thereby affecting the replication cycle of HIV-1 [17 20]. In vitro experimental data also suggest that the Q509L mutation in the RNase H region, when combined with other mutations (particularly A371V and TAMs in the polymerase region), increases resistance to AZT, lamivudine (3TC) and abacavir [21]. Three recent studies [13,22,23] compared the RNase H domain of treatment-naive patients to NRTIexperienced patients. In one study, several positions (L469, T470, A554 and K558) within the RNase H domain were found to mutate more frequently in treatment-experienced than in treatment-naive patients [22]. Only changes at positions A554 and K558 were considered as statistically more prevalent. All four positions were highly polymorphic with 3 AA substitution possibilities and only position K558 could be associated with any specific resistance pattern in the RT, where it was associated with TAMs [22]. Another study found that mutations in RNase H at positions D460, P468, H483, K512 and S519 were polymorphic in both wildtype and treatment-experienced patients, that mutations at L469, L491 and K527 occurred more frequently in patients with TAMs than patients in the control group and that the mutation at Q524 was seen only within viruses with complex TAMs and nucleoside analogue mutations [23]. A third study found an increased prevalence of mutations at positions I506, K527, K530 and Q547, and a decreased prevalence of mutations at positions T470 and K512 of the RNase H domain of treatment- experienced patients [13]. In the current study, we performed comprehensive sequencing and statistical analyses of a large group of treatment-naive and treatment-experienced HIV-1- infected patients to determine if any mutations in the thumb connection or RNase H domains are associated with the current ART. We found that the R284K and N348I mutations in the thumb connection domain and the K451R mutation of the RNase H domain were significantly enriched in the ART-experienced population. Methods Study patients ART-naive patient samples (n=100) were randomly selected from a pool of 598 baseline samples from patients enrolled in the Gilead Study 903. Study 903 was a randomized double-blind study comparing a treatment regimen of tenofovir disoproxil fumarate (TDF), 3TC and efavirenz (EFV) with a treatment regimen of stavudine (d4t), 3TC and EFV, which enrolled patients in the US and Europe in late Patients in Study 903 had a mean plasma HIV-1 RNA level of 4.61 log 10 copies/ml, a mean CD4 + T-cell count of 279 cells/mm 3 and most of the patients were infected with HIV-1 subtype B [24]. All 100 patients from Study 903 included in this analysis were infected with HIV-1 subtype B. ART-experienced patient samples (n=110) were randomly selected from a pool of 274 baseline samples from patients enrolled in Gilead Study 907 during early Study 907 was a Phase III randomized double-blind placebo-controlled multicentre study of the safety and efficacy of TDF administered orally to HIV-1-infected patients with plasma HIV-1 RNA levels ,000 copies/ml. Study 907 patients were on stable ART that contained 4 active agents for 8 weeks at study entry (mean of 65 months for entire study population), had a mean plasma HIV-1 RNA level of 3.36 log 10 copies/ml and a mean CD4 + T-cell count of 427 cells/mm 3. Approximately 70% of patients were infected with HIV-1 that expressed TAMs (mean 2.8 TAMs) [25]. All 110 patients from Study 907 used for this analysis were infected with HIV-1 subtype B. An additional 138 ART-experienced patient samples from the baseline time point of Gilead Study GS were also used for this analysis. GS was a recent Phase II randomized study of ritonavir-boosted elvitegravir (GS-9137) versus a comparator ritonavirboosted protease inhibitor (PI) in combination with optimized background therapy in ART- experienced patients, which enrolled patients in Study patients were required to be on an ART regimen for 30 days prior to baseline, with no prior treatment experience with an integrase inhibitor and had 1 protease gene International Medical Press

3 Mutations in AA of HIV-1 RT mutation as defined by International AIDS Society (IAS) USA guidelines [26]. Patients enrolled in this study were highly treatment-experienced with a median of 3 TAMs and 11 PI resistance mutations [27]. Of the 138 samples, 136 were HIV-1 subtype B, 1 was subtype C and 1 was subtype E. HIV-1 subtype analyses HIV-1 subtypes were determined through sequence cluster analysis of RT and protease using the Stanford HIV database [28]. Subtypes were assigned according to the RT sequence. In cases where the RT and protease sequences defined different HIV-1 subtypes, the subtype for the RT sequence was reported. Genotypical analyses HIV-1 RNA was extracted from patient plasma using the QIAamp Viral RNA Mini Kit (QIAGEN Services, Inc., Germantown, MD, USA). Extracted RNA was then reverse transcribed in two separate assays (one targeting RT AA and the other targeting RT AA of the RT) using custom primers (available upon request) to produce complementary DNA for PCR amplification. In each assay, two rounds of amplification using custom primers (available upon request) were performed resulting in amplified product that was then purified, quantified and normalized for use in cycle sequencing reactions using Big Dye Terminator version 3.1 (Applied Biosystems, Foster City, CA, USA). Samples were analysed by population-based sequencing using a 3100 ABI Prism Genetic Analyzer (Applied Biosystems). The RT sequence corresponding with AA was assessed using Sequencher (Gene Codes, Corp., Ann Arbor, MI, USA) and SeqAlign (Gilead Sciences, Durham, NC, USA) editing software. The detection limit for characterization of minor sequence species with this assay was approximately 25%. Sequences were compared with an HIV-1 subtype B consensus reference strand. Nucleotide positions that indicated a mixture of species (wild-type/mutant[s] and/or mutant/mutant[s]) were characterized as not conserved. For the purposes of our study, we divided the RT into three regions: the polymerase region (AA 1 240), the thumb connection domain (AA ) and the RNase H domain (AA ). Analysis of the thumb connection domain was on the basis of data obtained from Study 903 and Study 907 samples (100 treatment-naive versus 110 treatment-experienced). The RNase H analysis included all 248 treatment- experienced patient samples (Studies 907 and GS ) versus 100 treatment-naive samples (Study 903). Statistical analyses Sequences for each patient were compared with a reference to determine the percentage of conserved AA positions for each group (that is, ART-naive and ARTexperienced); the two groups were then compared with each other using a Fisher s exact test [29]. An analysis of the differences between the groups within the polymerase region was used in conjunction with well characterized NRTI and NNRTI resistance mutation profiles to establish a meaningful significance criterion for comparisons between groups in the RNase H and thumb connection domains. The threshold for significance was established as a P-value 0.01 at a conserved AA position (defined as 95% homology in the ART-naive group or 100% conserved in either group). For positions at which a mutation was determined to be statistically different between the ART treatment groups, the proportion of patients with TAMs (M41L, D67N, K70R, L210W, T215F/Y and K219Q/E/N) with the given mutation was compared with the proportion with TAMs without the given mutation. For these analyses, a Fisher s exact test was used at a significance level of Similar analyses were conducted with respect to M184V/I and NNRTI resistance mutations (K101E, K103N, V108I, Y181C, Y188L and G190A/S). Results Analysis of the polymerase region Mutations within the first 240 AA of polymeraseassociated RT inhibitors have been previously characterized [26,30,31]. Following recommendations by the IAS, we considered RT mutations at positions M41, E44, K65, D67, T69, K70, L74, V75, V118, Q151, M184, L210, T215 and K219 as known NRTI resistance mutations, and mutations at positions K101, K103, V108, V179, Y181, Y188, G190 and K238 as known NNRTI resistance mutations [26]. AA positions K43, E203, H208, H221, K223 and L228 have also been associated with ART in previous studies and were also considered as known resistance mutations [30,31]. To determine a criterion for statistical significance that produces only results that can be attributed to mutations caused by selective drug pressure, we compared the polymerase region from AA in the treatmentnaive study population with the same region in the treatment-experienced study population. Statistical significance was observed only at positions M41, K43, E44, D67, T69, K70, L74, K101, K103, V108, V118, V179, Y181, M184, Y188, G190, E203, H208, L210, T215, K219, H221, K223 and L228 (all with P-values <0.001), V75 (P<0.007) and K238 (P<0.008). All of these positions are associated with NRTI and NNRTI resistance [26,30,31]. As expected, there was a high level of conservation in the treatment-naive group (>95% conserved) at each of the previously mentioned positions. A high level of conservation in the treatmentnaive group or 100% in either group was necessary to Antiviral Therapy

4 JM Waters et al. Table 1. Comparison of the thumb connection domains (amino acids ) of HIV type-1 polymerase between treatmentnaive and treatment-experienced patients Consensus amino acid Sequence conservation in Sequence conservation in at position treatment-naive patients, % (n=100) treatment-experienced patients, % (n=110) P-value R E312 a I326 b G335 a N348 a <0.001 R358 c G359 ac A360 ac V365 a K366 c A371 c d A376 a K390 ac K395 a K396 a NC A400 c d T409 e <0.001 d a Previously reported mutations that might confer or increase resistance to nucleoside reverse transcriptase inhibitors and non-nucleoside reverse transcriptase inhibitors [9,10,13,34]. b Previously reported mutations associated with a decreased prevalence as a result of treatment [13]. c Previously reported mutations associated with an increased prevalence as a result of treatment [13]. d These positions did not meet all criteria set forth to establish significance for our analysis. e This position was compared in 100 treatment-naive versus 248 treatment-experienced patients. Statistical significance (P 0.01) is indicated in bold. NC, not calculated because of 100% conservation in both groups. avoid classifying naturally polymorphic positions as significant as a result of a low P-value between groups. Other known but low-prevalence resistance mutations associated with NRTI treatment (for example, K65R and Q151M) were observed in low frequencies, but were not statistically significant in this analysis. These results indicate that applying the strict criteria of a P-value 0.01 and 95% homology in the ART-naive group or 100% conserved in either group to other regions on the HIV-1 genome would result in the determination of only those AA positions with mutations that could be attributed to ART. Analysis of the thumb connection domain The majority of AA positions, (138/186, 74.2%), in the thumb connection domain (AA ) were highly conserved ( 95%) in both the treatment-naive and treatment-experienced study populations. Our analysis confirmed a difference in the frequency of mutations at position N348 between the treatment-naive and treatment-experienced groups. In the treatment-naive group, the N348 position was 100% conserved as compared with the treatment-experienced group, where 24.5% of samples had N348I (Table 1). In addition to position N348, R284 was also statistically significantly different between the treatment-naive and treatmentexperienced groups (Table 1). AA position R284 was 100% conserved in the treatment-naive group, but only 92.7% conserved in the treatment-experienced group (P=0.007). All eight patients with a mutation at R284 had an R284K substitution. We compared the genotypical profile of viruses with the R284K or N348I mutation to those without in the treatment-experienced group in order to determine whether R284K or N348I mutations were associated with any specific ART. In lieu of prior treatment history, which was not always available, we classified the polymerase mutations into three classes that have been associated with specific ART: mutations associated with the thymidine analogues AZT and d4t (TAMs), the M184V/I mutations associated with cytidine analogues 3TC and emtricitabine and mutations associated with NNRTIs, such as EFV and nevaripine. Numerically, viruses with either R284K or N348I mutations in their RT more frequently expressed the M184V/I mutation and NNRTI resistance mutations than viruses without R284K or N348I. Conversely, viruses without R284K or N348I more frequently expressed 1 TAMs; however, these associations did not achieve statistical significance in our data set (data not shown). Other AA positions in the thumb connection domain described in the literature were not statistically significant in our analyses (Table 1). Analysis of the RNase H domain The majority of AA positions (98/134, 73%), in the RNase H domain (AA ) were highly conserved International Medical Press

5 Mutations in AA of HIV-1 RT Table 2. Comparison of the RNase H domain (amino acids ) of HIV type-1 reverse transcriptase between treatment-naive and treatment-experienced patients Consensus amino acid Sequence conservation in Sequence conservation in at position treatment-naive patients, % (n=100) treatment-experienced patients, % (n=248) P-value K <0.001 D460 a P468 a L469 a T470 ab T473 a NC Q475 a NC K476 a H483 a c L491 a c N494 a NC Y501 a NC I505 a I506 d Q509 a c K512 ab S519 a c Q524 a K527 ad c K530 d H539 a Q547 d D549 a NC A554 a K558 a a Previously reported mutations that might confer or increase resistance to nucleoside reverse transcriptase inhibitors and non-nucleoside reverse transcriptase inhibitors [6,9,16 23]. b Previously reported mutations associated with a decreased prevalence as a result of treatment [13]. c These positions did not meet all criteria set forth to establish significance for our analysis. d Previously reported mutations associated with an increased prevalence as a result of treatment [13]. Statistical significance (P 0.01) is indicated in bold. NC, not calculated because of 100% conservation in both groups. ( 95% conservation in both study populations). We identified only one change at AA position K451 that was statistically significantly different between the treatment-naive and treatment-experienced patients within the RNase H domain (Table 2). The K451 position was 100% conserved in the treatment-naive group (n=100), but only 89.1% (n=248) conserved in the treatmentexperienced group (P<0.001) with all 27 treatmentexperienced patients having the K451R substitution. To determine if the K451R substitution was associated with any other known resistance patterns, specifically TAMs, M184V/I and NNRTI resistance mutations, we compared the genotypical profile of patients with and without the K451R mutation within the treatmentexperienced group (data not shown). There was a trend towards a higher incidence of NNRTI mutations in patients with the K451R mutation as compared with patients without K451R (74% with K451R versus 59% without); however, this difference did not achieve statistical significance (P=0.15). All other AA positions within the RNase H domain that were described previously in the literature did not meet all statistical significance criteria in this analysis (Table 2). Discussion Our analyses of RT beyond the polymerase domain using our stringent assessment criteria to define statistical significance resulted in the identification of R284K and N348I of the thumb connection domain and a novel mutation K451R of the RNase H domain as statistically associated with NRTI and/or NNRTI therapy. A P-value of 0.05 with no conservation criteria is the typical industry standard that is used to assess statistical significance, but was found in our study to be too broad of an acceptance value that resulted in numerous naturally polymorphic positions being classified as statistically significant. An RT mutation profile analysis of the Stanford database [28] also showed an increased prevalence of R284K, N348I and K451R in treatment-experienced patients (P<0.001 for each), whereas the R284K frequencies were 1.5% (65/4,211) versus 3.7% Antiviral Therapy

6 JM Waters et al. (80/2,126), N348I were 0.1% (2/1546) versus 4.2% (24/562) and R451K were 2.1% (21/990) versus 6.2% (24/386) in treatment-naive versus treatmentexperienced patients, respectively. Residue R284 is located in the thumb domain of RT and exhibits sequence homology with other nucleic acid polymerases, and is part of a group of residues that is key in RNA RT interactions termed the helix clamp (Figure 1) [30,32]. This AA motif has been identified in the crystal structure model as an element of the enzyme s nucleic-acid-binding apparatus [32]. By virtue of its location and interaction with the template, R284K is likely to affect aspects of DNA polymerization, processivity, excision or even RNase H activity. Another function of RT that might be affected, albeit indirectly, is fidelity, via altering efficiency of either misincorporation or mismatch extension, both of which are polymerase activity-associated functions. Although crystal structures have shown that R284 directly interacts with the substrate in the p66 subunit, the interaction of R284 in the p51 unit might also contribute to an altered interaction of RT with nucleic acid substrates [32]. Previous analysis of subclonal samples derived from treatment-experienced patients has shown that N348I in the thumb connection domain affects resistance to AZT when associated with TAMs [10]. Our observations are in agreement as we found a statistically significant link between ART and the development of the N348I mutation in the thumb connection domain. A recent study demonstrated that N348I was associated with TAMs, M184V/I and NNRTI resistance mutations Figure 1. HIV-1 polymerase with bound primer and template p66 pol R284 (p66) p66 RNase H D185 K451 D443 R284 (p51) Template Primer p51 HIV type-1 (HIV-1) reverse transcriptase (RT) thumb connection and RNase H domain amino acid residues that were increased in prevalence in this study. The RT polymerase (pol) and thumb connection domains are shown in green with one of the catalytic residues indicated by the D185 residue (red). The RNase H domain is shown in cyan with its catalytic site shown by one of its catalytic residues D443 (red). The R284 (p66 and p51 subunits) and K451 are shown in yellow. The contact template is shown in orange and primer strand in pink. Alterations in the positioning of the primer and template could potentially affect pol processivity and translocation, RNase H activity or nucleoside reverse transciptase inhibitor excision. Position R284 (p51) could also be involved in the primer or template positioning. Position N348 in the thumb connection domain is located on the opposite side of RT in this model (not shown). The structural p51 subunit is shown in grey. This figure was generated from the crystal structure (Protein Data Bank code IRTD) by Huang et al. [33] International Medical Press

7 Mutations in AA of HIV-1 RT in vivo, and contributed to an increase in viral load with the potential to show dual resistance to both NRTI and NNRTI drug classes [8]. Although N348I and TAMs frequently occurred together, we were unable to confirm a significant association between development of the N348I and any specific pattern of NRTI or NNRTI mutations in our population. Using the crystal structure of HIV-1 RT bound to primer and template (Protein Data Bank code IRTD), one can see that K451 in the RNase H domain contacts the primer strand (Figure 1). It is conceivable that the K451R substitution could alter the positioning of the primer strand, which could potentially affect polymerization, RNase H activity or the efficiency of NRTI excision [33]. In vitro, Q509L has been selected as a resistance mutation to AZT in the context of TAMs [21]. In our analyses, Q509 was 96% conserved in the treatment-naive group with three possible AA changes (histidine, leucine and lysine), whereas in the treatment-experienced group it was 90% conserved with five possible AA changes (lysine, histidine, isoleucine, leucine and arginine; Table 2). Therefore, our analysis showed that Q509L occurs in both treatment-naive and treatment-experienced patients at very low frequencies. Although in vitro virological and biochemical data have shown that this mutation can increase AZT resistance, our analysis of clinical samples from heavily treatment-experienced patients suggests that this mutation is infrequent in the population despite the presence of TAMs in approximately 70% of patients within our analysis group. Positions D460, P468, L469, T470, H483, I506, K512, S519, Q524, K530, Q547, A554 and K558 (having previously been reported to be associated with ART, showing an increased prevalence in treatmentexperienced patients or affecting the functionality of enzymatic processes [13,19 23]), did not meet our criteria for statistical significance (Table 2). In treatmentexperienced patients, there was an increased prevalence of mutations at positions K527, K530 and Q547 in our analyses, as was found in previous studies; however, position I506 did not show an increased prevalence as it had before [13]. None of the positions were well conserved in either the treatment-naive or treatmentexperienced group. Both positions L491 and K527 had P-values <0.01 (P=0.001 and P=0.005, respectively); however, these AAs were not well conserved in either the treatment-naive or treatment-experienced group with 3 different AA possibilities for each position (Table 2). The low percentage of conservation coupled with the various mutational possibilities at each of these positions indicates that these are polymorphic positions and are not likely to be associated with ART. Mutations at positions Q475, N494, H539 and D549 have been previously reported to affect RNase H enzymatic function [17 21]. In our analysis, however, a high conservation at these positions in both groups (in the treatment-naive group, Q475, N494 and D549 were 100% conserved and H539 was 99% conserved and in the treatment-experienced group, all four were 100% conserved; Table 2) indicates that although mutations at these positions affect enzymatic functions in vitro, their occurrence in vivo is highly infrequent. It has also been reported that positions L469, T470, A554 and K558 show a statistical prevalence toward mutations caused by ART as well as being associated with TAMs [22]. Our analyses showed that L469, T470 and A554 were all polymorphic ( 2 AA possibilities in the treatment-naive group and 5 AA possibilities in the treatment-experienced group with P>0.01) and changes at these positions were not statistically significant (Table 2). K558R was the only change in both the treatment-naive and treatment-experienced groups, but it was not highly conserved in either group nor statistically enriched (Table 2). In the thumb connection domain, mutations at positions E312, I326, G335, R358, G359, A360, V365, K366, A371, A376, K390, K395, E396 and A400 have been previously shown to increase resistance to NRTIs and NNRTIs in vitro or have shown increased or decreased prevalence in vivo as a result of therapy [9,10,13,34]. In our analyses, AA positions A371 and T409 had statistically significant P-values (0.007 and <0.001, respectively), but did not meet the acceptance criteria for conservation in either group (Table 1). Interestingly, T409 was polymorphic in the treatmentnaive group and was more conserved in the treatmentexperienced group (Table 1). Position E396, which is associated with the RNase H primer grip, was highly conserved in both groups (Table 1). Changes at positions R358, G359, A360, K366 and A371 showed an increased prevalence in treatment-experienced patients in our analyses as was seen in previous studies [13], but positions K390 and A400 did not (Table 1). These residues were not well conserved (<95%) in either group. Our analysis demonstrated that current ART, including NRTIs and NNRTIs, that target the HIV-1 polymerase function of RT cause mutations outside of AA of the polymerase region, specifically at positions R284 and N348 in the thumb connection domain and K451 in the RNase H domain. We used a conservative approach for defining these mutations with a P-value cutoff of 0.01 and a further requirement that the AA position in the treatment-naive group be 95% conserved or 100% conserved in either group. A limitation of our study relates to a possible sampling bias for patients enrolled in clinical trials. We pooled samples from two temporally distinct clinical trials to mitigate against such sampling bias. Another limitation of our study relates to showing Antiviral Therapy

8 JM Waters et al. significant associations between mutations found to be statistically significant in the thumb connection and RNase H domains as well as known RT resistance mutations (TAMs, NRTIs and NNRTIs) as a result of our small sample population. The clinical implications of the mutations described in this study are unknown; therefore, more studies need to be done to assess the potential effects of these mutations on the mechanism of resistance to antiretroviral drugs. Acknowledgements We would like thank Damian McColl, Hans Reiser, Mini Balakrishnan, Nicolas Margot and Rima Kulkarni for their contributions and critical reading of the manuscript. We would also like to thank Mick Hitchcock, Craig Gibbs and Allan Kutzenco for their review of this article. Part of this work was previously presented at the 20th International Conference on Antiviral Research on 30 April 3 May 2007 in Palm Springs, CA, USA. Disclosure statement This study was supported by Gilead Sciences, Inc. 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