Search tips
Search criteria 


Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
Cell Mol Biol (Noisy-le-grand). Author manuscript; available in PMC 2013 January 23.
Published in final edited form as:
Cell Mol Biol (Noisy-le-grand). 2012 December 22; 58(1): 187–195.
PMCID: PMC3551986



4′-ethynyl-2-fluoro-2′-deoxyadenosine (EFdA) is a highly potent inhibitor of HIV-1 reverse transcriptase (RT). We have previously shown that its exceptional antiviral activity stems from a unique mechanism of action that is based primarily on blocking translocation of RT; therefore we named EFdA a Translocation Defective RT Inhibitor (TDRTI). The N348I mutation at the connection subdomain (CS) of HIV-1 RT confers clinically significant resistance to both nucleoside (NRTIs) and non-nucleoside RT inhibitors (NNRTIs). In this study we tested EFdA-triphosphate (TP) together with a related compound, ENdA-TP (4′-ethynyl-2-amino-2′-deoxyadenosine triphosphate) against HIV-1 RTs that carry clinically relevant drug resistance mutations: N348I, D67N/K70R/L210Q/T215F, D67N/K70R/L210Q/T215F/N348I, and A62V/V75I/F77L/F116Y/Q151M. We demonstrate that these enzymes remain susceptible to TDRTIs. Similar to WT RT, the N348I RT is inhibited by EFdA mainly at the point of incorporation through decreased translocation. In addition, the N348I substitution decreases the RNase H cleavage of DNA terminated with EFdA-MP (T/PEFdA-MP). Moreover, N348I RT unblocks EFdA-terminated primers with similar efficiency as the WT enzyme, and further enhances EFdA unblocking in the background of AZT-resistance mutations. This study provides biochemical insights into the mechanism of inhibition of N348I RT by TDRTIs and highlights the excellent efficacy of this class of inhibitors against WT and drug-resistant HIV-1 RTs.

Keywords: EFdA, ENdA, N348I, Translocation Defective Reverse Transcriptase Inhibitors, Reverse Transcriptase, HIV-1, Antivirals


Human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT) is a key enzyme that converts single-stranded genomic RNA into double-stranded DNA, which in turn is transported to the nucleus and integrated into the host cell genome. The HIV-1 RT catalyzes both the RNA- and DNA-dependent DNA polymerase, and RNase H activities (11). The functional form of HIV-1 RT is a heterodimer consisting of p66 and p51 polypeptides (16). The p66 subunit has both enzymatic activities and includes the polymerase and the RNase H domains. The polymerase domain consists of the fingers, palm and thumb subdomains which are analogous to a right hand connected to the RNase H domain through the connection subdomain (42) (Fig. 1).

Figure 1
HIV-1 RT structure with highlighted residues of drug-resistance

The p51 subunit lacks the RNase H domain and has been proposed to play a structural role (42), although recent work from our laboratory has also shown that mutations in the p51 subunit affect the polymerase and RNase H enzymatic functions of RT (38).

Highly active antiretroviral therapies (HAART) have been very effective in suppressing viral loads and having a significant impact on the life expectancy of HIV patients. Key components of HAART are drugs that target HIV RT. The two classes of RT inhibitors currently used in the clinic are nucleoside/nucleotide RT inhibitors (NRTI) and non-nucleoside RT inhibitors (NNRTI). NRTIs inhibit RT by acting as chain-terminators after they are incorporated into the nascent DNA chain. NNRTIs act non-competitively by binding to a hydrophobic pocket adjacent to, but distinct from the polymerase active site of RT and by imposing rigidity to the movements of thumb subdomain required for efficient polymerase function (20, 22, 35, 37, 39, 41).

Despite the phenomenal success of HAART regimens, continuous use of antivirals leads to the emergence of viruses that are resistant to all known anti-AIDS drugs. The mutations associated with NNRTI resistance are generally located at the NNRTI binding pocket (NNIBP). However, the mutations that cause resistance to NRTIs have been noted to be scattered in the polymerase domain (22, 39). While most NNRTI and NRTI resistance mutations are at the palm and fingers subdomains of HIV-1 RT, it has recently been shown that some mutations associated with NNRTI and NRTI resistance are at the connection and RNase H regions of RT (6, 8, 13, 15, 17, 29, 43). The most significant of these mutations is N348I, which confers moderate resistance to both NRTIs and NNRTIs, and is present in a significant number of clinical isolates, especially in the presence of other NRTI mutations.

In light of the new emerging drug resistance mutations, it is essential to identify inhibitors that are very potent and effective against viral strains that are resistant to all approved therapeutics. One such inhibitor is 4′-ethynyl-2-fluoro-2′-deoxyadenosine triphosphate (EFdA-TP) (18, 19). We have recently reported the mechanism of HIV inhibition by EFdA (26). In contrast to other approved NRTIs, which have a modification at 3′OH, EFdA contains a 3′OH moiety and blocks DNA synthesis by locking the primer terminus at the pre-translocation site of HIV-1 RT. In addition to EFdA, we have recently shown that ENdA also inhibits HIV RT potently acting as a TDRTI (data not shown).

Recently, using transient-state kinetic experiments we established the mechanism of NNRTI resistance of HIV-1 RT containing the N348I mutation at the connection subdomain of the enzyme (38). We showed that the resistance to the NNRTI nevirapine (NEV) is primarily the result of changes distant from the NNRTI binding pocket, which decrease inhibitor binding (increase Kd-NVP) by primarily decreasing the association rate of the inhibitor (kon-NVP). Moreover, the N348I mutation increased nucleic acid binding affinity, enhanced processivity and lowered the catalytic turnover rate of the natural substrate. In this study we determine the ability of TDRTIs to block reverse transcription by the multi-drug resistant N348I HIV-1 RT as well as other NRTI resistant RTs, D67N/K70R/L210Q/T215F (resistant to AZT by the excision mechanism) D67N/K70R/L210Q/T215F/N348I, and A62V/V75I/F77L/F116Y/Q151M (multidrug resistant to AZT and dideoxynucleotide RT inhibitors).


Enzymes and Nucleic acids

The RT genes coding for p66 and p51 subunits of BH10 HIV-1 were cloned in the pETDuet-1 vector (Novagen) using restriction sites NcoI and SacI for the p51 subunit, and SacII and AvrII for the p66 subunit (2, 38). The sequences coding for a hexa-histidine tag and the 3C protease recognition sequence were added at the N-terminus of the p51 subunit. RT was expressed in BL21 (Invitrogen) and purified by nickel affinity chromatography and monoQ anion exchange chromatography (33). Oligonucleotides used in this study were chemically synthesized and purchased from Integrated DNA Technologies (Coralville, IA). Sequences of the DNA substrates are shown in Table 1. Deoxynucleotide triphosphates and dideoxynucleotide triphosphates were purchased from Fermentas (Glen Burnie, MD). EFdA and ENdA were synthesized by Yamasa Corporation (Chiba, Japan) as described before (30). Using EFdA and ENdA as starting material the triphosphate forms EFdA-TP and ENdA-TP were synthesized by TriLink BioTechnologies (San Diego, CA). Concentrations of nucleotides, EFdA-TP and ENdA-TP were calculated spectrophotometrically on the basis of absorption at 260 nm and their extinction coefficients. All nucleotides were treated with inorganic pyrophosphatase (Roche Diagnostics) as described previously (24) to remove traces of PPi contamination that might interfere with the rescue assay.

Table 1
DNA and RNA sequences used in this study.

Primer extension assays

Inhibition of HIV-1 RT by TDRTIs

DNA template (Td31) was annealed to 5′-Cy3 labeled DNA primer (Pd18). To monitor primer extension, the Td31/5′Cy3-Pd18 hybrid (20 nM) was incubated at 37°C with WT or drug-resistant HIV-1 RTs (20 nM) in a buffer containing 50 mM Tris (pH 7.8) and 50 mM NaCl (RT buffer). Varying amounts of EFdA-TP or ENdA-TP were added and the reactions were initiated by the addition of 6 mM MgCl2 to a final volume of 20 μl. All dNTPs were present at a final concentration of 1 μM. The reactions were terminated after 15 minutes by adding equal volume of 100% formamide containing traces of bromophenol blue. The products were resolved on a 15% polyacrylamide 7 M urea gel. In this and in subsequent assays, the gels were scanned with a PhosphorImager (FujiFilm FLA 5000), the bands for fully extended product were quantified using Multi Gauge (FujiFilm) and results were plotted using one site competition equation on GraphPad Prism 4 to determine the IC50 for EFdA-TP and ENdA-TP.

Site-specific Fe2+ Footprinting Assay

Site-specific Fe2+ footprints were monitored on 5′-Cy3-labeled DNA templates. 100 nM of 5′Cy3-Td43/Pd20 was incubated with 600 nM WT or N348I RT in a buffer containing 120 mM sodium cacodylate (pH 7), 20 mM NaCl, 6 mM MgCl2, and either of 5 μM ddATP or 1 μM EFdA-TP, to allow quantitative chain-termination. Prior to the treatment with Fe2+, complexes were pre-incubated for 7 min with increasing concentrations of the next incoming nucleotide (dTTP). The complexes were treated with ammonium iron sulfate (1 mM) as previously described (21). This reaction relies on autoxidation of Fe2+ (3) to create a local concentration of hydroxyl radical which cleaves the DNA at the nucleotide closest to the Fe2+ specifically bound to the RNase H active site.

ATP-dependent Excision and Rescue assay

20 nM of purified Td31/Pd18-EFdA-MP or Tr31/Pd18-EFdA-MP were incubated with 60 nM WT, N348I, D67N/K70R/L210Q/T215F or D67N/K70R/L210Q/T215F/N348I RT in the presence of 3.5 mM ATP, 100 μM dATP, 0.5 μM dTTP, and 10 μM ddGTP in RT buffer and 10 mM MgCl2. Aliquots of the reaction were stopped at different time points (0–90 min) and analyzed as described above.

RNase H Assays

RNase H assays were performed by incubating the RNA/DNA duplex5′Cy3-Tr35/Pd25 or 5′Cy3-Tr35/Pd25-ddAMP or 5′Cy3-Tr35/Pd25-EFdA-MP (50 nM) with WT or N348I RT (50 nM) in RT buffer at 37 °C with MgCl2 (6 mM). Reactions were quenched after incubation (1–5 min) with equal volumes of formamide containing trace amounts of bromophenol blue. Reaction products were analyzed as before. The primary RNase H cleavage product is mainly 18 nucleotides from the 3′-end of the DNA primer (18 nucleotides), and the secondary cleavage product is mainly 12 nucleotides from the 3′-end of the primer (12 nucleotides) as reported previously (10, 12, 38).


The inhibitors used here to characterize the susceptibility of N348I to various drugs are adenosine analogs. The structures of these analogs are shown in Fig. 2. The normal deoxynucleotide dA is shown in Fig. 2A. EFdA and ENdA are shown in Figs. 2B and 2C, respectively. It can be seen in these figures that unlike other anti-HIV NRTIs both EFdA and ENdA have a 3′OH. These compounds also contain an ethynyl group at the 4′ position. EFdA and ENdA differ in their substitutions at the 2 position of the purine ring. EFdA at this position has fluorine whereas ENdA has an amino group.

Figure 2
Chemical structures of dA, EFdA and ENdA

Inhibition of WT and N348I mutant of HIV-1 RT

The inhibition of WT, N348I, D67N/K70R/L210Q/T215F, D67N/K70R/L210Q/T215F/N348I mutants of HIV-1 RT by EFdA-TP and ENdA-TP was assessed by a primer extension assay. As shown in Fig. 3A–3D, EFdA-TP and ENdA-TP suppressed RT-catalyzed DNA synthesis in a dose-dependent manner. The IC50 values for both analogs are shown in Table 2. N348I, D67N/K70R/L210Q/T215F and D67N/K70R/L210Q/T215F/N348I RTs were inhibited by EFdA-TP and ENdA-TP with similar efficiency compared to the WT enzyme. In addition, another mutant HIV-1 RT (A62V/V75I/F77L/F116Y/Q151M) was included in drug susceptibility assays (Fig. 3E).

Figure 3
EFdA-TP and ENdA-TP inhibit WT and drug-resistant HIV-1 RTs
Table 2
IC50 values of EFdA-TP and ENdA-TP against WT and drug-resistant HIV-1 RTs

We have previously shown that EFdA inhibits DNA synthesis at the point of incorporation. Thus, we examined here the stopping patterns after incorporation products of the primer extension assay for the stopping patterns (Fig. 3). The primer synthesis shown in Fig. 3 clearly demonstrates that the stopping pattern follows the incorporation of adenosine analogs. Three distinct bands at positions 1, 6 and 10 indicate that both analogs inhibit RT mainly at the point of incorporation. Therefore, these compounds act primarily as obligate chain terminators. There is also an additional band at position 7, suggesting that in some instances EFdA may allow addition of one nucleotide after its incorporation, thus acting as a delayed chain terminator (Fig. 3). This type of inhibition is far less common and is sequence-dependent. These finding agrees with our previous studies on WT RT (26).

Effect of EFdA-MP on Translocation of WT and N348I mutant of HIV-1 RT

The connection subdomain mutation N348I has been related to the altered DNA binding affinity and processivity of the mutant enzyme compared to the WT RT (4, 38). Since EFdA is a TDRTI and its incorporation is assumed to affect the translocation and thereby DNA binding and processivity, we investigated the translocation of EFdA-containing template-primers using the hydroxyl radical site-specific footprinting assay (21). The results of the footprinting assay shown in Fig. 4 demonstrate that the presence of EFdA-MP at the 3′ end of the DNA primer blocks translocation and prevents incorporation of the next incoming dNTP. Therefore, similar to WT RT, the mutant N348I RT is also inhibited by EFdA-TP via the same mechanism.

Figure 4
Determination of the translocation state of WT or N348I HIV-1 RT bound to Td43/P20-EFdA-MP

Effect of EFdA-MP on RNase H activity of WT and N348I RTs

The template/primers containing EFdA-MP, ddAMP, or without inhibitor incorporated at the 3′ end of the primer were used in RNase H assays with WT and N348I RTs in a time dependent manner. As previously noted, Fig. 5 shows that N348I mutant RT has decreased RNase H activity for all substrates used in this assay. The RNase H assays carried out in presence of T/P trap showed the disappearance of the secondary cuts for both enzymes used here. This is likely due to a defect in translocation that EFdA imposes on the enzyme. Interestingly, the primary cut of EFdA-terminated primers is a single band when the T/P has EFdA, but not ddA at the 3′ primer terminus. Moreover, the RNA cleavage of Tr35/Pd25 – EFdA-MP was less than that of Tr35/Pd25-ddAMP or Tr35/Pd25 possibly because of less favorable positioning at the RNase H of T/P with EFdA at the 3′ terminus.

Figure 5
Effect of EFdA on RNase H activity of WT and N348I HIV-1 RTs

ATP-dependent unblocking of EFdA-MP terminated primers by WT and N348I RTs

Since EFdA-MP-terminated primers bind predominantly in a pre-translocation mode we expected that EFdA-MP will be efficiently unblocked by both WT and N348I RTs. The ATP-dependent excision and subsequent rescue of EFdA-MP primers is shown in Fig. 6. The bands marked as ‘Rescued Primer” have comparable product for the WT and N348I mutant enzyme for both DNA (Fig. 6A) and RNA (Fig. 6B) templates suggesting that resistance mutant N348I does not have any significant effect on the unblocking of EFdA-MP containing primers (RNA vs. DNA) (Fig. 6). However, the N348I mutation in the background of AZT resistance mutations D67N, K70R, L210Q and T215F showed a 2-fold increase in unblocking EFdA-MP containing primers both with DNA and RNA templates (Fig. 6).

Figure 6
ATP-dependent unblocking of EFdA-MP terminated primers. ATP-dependent rescue of Td31/Pd18-EFdA-MP (A) and Tr31/Pd18-EFdA-MP (B). Purified T/PEFdA-MP was incubated with WT, N348I, D67N/K70R/L210Q/T215F or D67N/K70R/L210Q/T215F/N348I HIV-1 RT in the presence ...


There are currently more than 20 antiretrovirals that have been approved by the US Food and Drug Administration for the treatment of HIV infection. They fall into four categories, targeting HIV RT, protease, integrase, the entry step, and the fusion of the viral and cell membranes. RT inhibitors are either NRTIs or NNRTIs. The NRTIs such zidovudine (AZT) and lamivudine compete with the natural substrates and get incorporated into the nascent DNA chain, blocking further polymerization because they lack a 3′OH group required for DNA synthesis. NNRTIs such as nevirapine and efavirenz inhibit the polymerase activity of RT by binding at a hydrophobic pocket nearly 10 Å away from the polymerase active site (Fig. 1). This pocket is created after the binding of NNRTIs. The highly active antiretroviral therapy (HAART) introduced in the mid-90s contains the combination of antivirals (generally a protease inhibitor and two NRTIs or an NNRTI and two NRTIs) targets the replication of the resistant virus.

Extended or incomplete treatments with antiretrovirals result in the emergence of drug resistance mutations. In the case of drugs that target RT, most of the resistance mutations were found to be present in the polymerase domain of RT. These resistance mutations against NRTIs function primarily with two mechanisms: (i) they reduce the binding affinity/incorporation of NRTI (34, 40) or (ii) enhance the selective excision of incorporated NRTI from a chain-terminated primer terminus (9, 2325, 36). The resistance against NNRTIs is primarily through the mutations that reduce the binding affinity of NNRTIs (7, 31, 32, 35).

Recent studies showed that connection subdomain mutations can confer resistance to NRTIs. Nikolenko et al. suggested that some of these mutations increase AZT resistance by reducing template RNA degradation, thereby preserving the RNA template and providing additional time for RT to excise AZT monophosphate (27, 28). Hachiya et al., (13) as well as another research group (43) identified a clinical isolate with phenotypic resistance to nevirapine (NVP) in the absence of known NNRTI mutations. This resistance was shown to be caused by N348I, a mutation at the connection subdomain of HIV-1 RT. This mutation is not a polymorphism, as it exists in more than 10% of drug-treated, but not drug-naïve HIV patients. The connection subdomain mutation N348I has been related to the altered DNA binding affinity and processivity of the mutant enzyme compared to the WT RT (4, 38). Ehteshami et al. showed that N348I enhances resistance to AZT through both RNase H-dependent and -independent mechanisms (10). Since EFdA is a TDRTI and its incorporation is assumed to affect the translocation and thereby DNA binding and processivity, we investigated the susceptibility of two highly potent antiretrovirals EFdA and ENdA.

We report that both EFdA-TP and ENdA-TP are very potent inhibitors of N348I, D67N/K70R/L210Q/T215F, D67N/K70R/L210Q/T215F/N348I, and A62V/V75I/F77L/F116Y/Q151M RTs. They inhibit RT primarily at the point of incorporation and since they prevent enzyme translocation they both belong to the TDRTI class of NRTIs. The D67N/K70R/L210Q/T215F set of mutations are the classical thymidine-associated mutations (TAMs), which are known to cause resistance to AZT by enhancing excision of AZT-terminated primers (1, 5, 23). The A62V/V75I/F77L/F116Y/Q151M set of mutations is known as the “Q151M” complex RT, and has been known as a multidrug-resistance mutation, since the latter mutations are known to be involved in resistant variants with reduced susceptibility to dideoxynucleotides and to AZT. Unlike D67N/K70R/L210Q/T215F RT, the Q151M complex decreases susceptibility to NRTIs by decreasing incorporation efficiency of the inhibitors rather than increasing excision and unblocking of chain-terminated primers (14). Finally, N348I is known to cause resistance to both NRTIs and NNRTIs. Hence, collectively, these mutants represent all mechanisms by which RT becomes resistant to available antivirals. Importantly, we find that they are all susceptible to the EFdA and ENdA TDRTIs. Hence, this new class of RT inhibitors should be able to efficiently block viruses that carry clinically relevant mutations, including the new connection domain mutation N348I.


This work was supported by the Ministry of Knowledge and Economy, Bilateral International Collaborative Research and Development Program, Republic of Korea, National Institutes of Health Grants AI076119, AI074389, and AI100890 (to S. G. S.) and AI079801 (to M.A.P.).


Nucleoside reverse transcriptase inhibitor
Reverse transcriptase
Template/Primer terminated with 4′-ethynyl-2-fluoro-2′-deoxyadenosine
Translocation defective reverse transcriptase inhibitor


1. Arion D, Kaushik N, McCormick S, Borkow G, Parniak MA. Phenotypic mechanism of HIV-1 resistance to 3′-azido-3′-deoxythymidine AZT: increased polymerization processivity and enhanced sensitivity to pyrophosphate of the mutant viral reverse transcriptase. Biochemistry. 1998;37:15908–15917. [PubMed]
2. Bauman JD, Das K, Ho WC, Baweja M, Himmel DM, Clark AD, Jr, Oren DA, Boyer PL, Hughes SH, Shatkin AJ, Arnold E. Crystal engineering of HIV-1 reverse transcriptase for structure-based drug design. Nucleic Acids Res. 2008;36:5083–5092. [PMC free article] [PubMed]
3. Biaglow JE, Kachur AV. The generation of hydroxyl radicals in the reaction of molecular oxygen with polyphosphate complexes of ferrous ion. Radiat Res. 1997;148:181–187. [PubMed]
4. Biondi MJ, Beilhartz GL, McCormick S, Gotte M. N348I in HIV-1 reverse transcriptase can counteract the nevirapine-mediated bias toward RNase H cleavage during plus-strand initiation. J Biol Chem. 285:26966–26975. [PMC free article] [PubMed]
5. Boyer PL, Sarafianos SG, Arnold E, Hughes SH. Selective excision of AZTMP by drug-resistant human immunodeficiency virus reverse transcriptase. J Virol. 2001;75:4832–4842. [PMC free article] [PubMed]
6. Brehm JH, Koontz D, Meteer JD, Pathak V, Sluis-Cremer N, Mellors JW. Selection of mutations in the connection and RNase H domains of human immunodeficiency virus type 1 reverse transcriptase that increase resistance to 3′-azido-3′-dideoxythymidine. J Virol. 2007;81:7852–7859. [PMC free article] [PubMed]
7. Das K, Ding J, Hsiou Y, Clark AD, Jr, Moereels H, Koymans L, Andries K, Pauwels R, Janssen PA, Boyer PL, Clark P, Smith RH, Jr, Kroeger Smith MB, Michejda CJ, Hughes SH, Arnold E. Crystal structures of 8-Cl and 9-Cl TIBO complexed with wild-type HIV-1 RT and 8-Cl TIBO complexed with the Tyr181Cys HIV-1 RT drug-resistant mutant. J Mol Biol. 1996;264:1085–1100. [PubMed]
8. Delviks-Frankenberry KA, Nikolenko GN, Barr R, Pathak VK. Mutations in human immunodeficiency virus type 1 RNase H primer grip enhance 3′-azido-3′-deoxythymidine resistance. J Virol. 2007;81:6837–6845. [PMC free article] [PubMed]
9. Dharmasena S, Pongracz Z, Arnold E, Sarafianos SG, Parniak MA. 3′-Azido-3′-deoxythymidine-(5′)-tetraphospho-(5′)-adenosine, the product of ATP-mediated excision of chain-terminating AZTMP, is a potent chain-terminating substrate for HIV-1 reverse transcriptase. Biochemistry. 2007;46:828–836. [PubMed]
10. Ehteshami M, Beilhartz GL, Scarth BJ, Tchesnokov EP, McCormick S, Wynhoven B, Harrigan PR, Gotte M. Connection domain mutations N348I and A360V in HIV-1 reverse transcriptase enhance resistance to 3′-azido-3′-deoxythymidine through both RNase H-dependent and -independent mechanisms. J Biol Chem. 2008;283:22222–22232. [PMC free article] [PubMed]
11. Goff SP. Retroviral reverse transcriptase: synthesis, structure, and function. J Acquir Immune Defic Syndr. 1990;3:817–831. [PubMed]
12. Gotte M, Rausch JW, Marchand B, Sarafianos S, Le Grice SF. Reverse transcriptase in motion: conformational dynamics of enzyme-substrate interactions. Biochim Biophys Acta. 1804:1202–1212. [PMC free article] [PubMed]
13. Hachiya A, Kodama EN, Sarafianos SG, Schuckmann MM, Sakagami Y, Matsuoka M, Takiguchi M, Gatanaga H, Oka S. Amino acid mutation N348I in the connection subdomain of human immunodeficiency virus type 1 reverse transcriptase confers multiclass resistance to nucleoside and nonnucleoside reverse transcriptase inhibitors. J Virol. 2008;82:3261–3270. [PMC free article] [PubMed]
14. Hachiya A, Kodama EN, Schuckmann MM, Kirby KA, Michailidis E, Sakagami Y, Oka S, Singh K, Sarafianos SG. K70Q adds high-level tenofovir resistance to “Q151M complex” HIV reverse transcriptase through the enhanced discrimination mechanism. PloS one. 2011;6:e16242. [PMC free article] [PubMed]
15. Hachiya A, Shimane K, Sarafianos SG, Kodama EN, Sakagami Y, Negishi F, Koizumi H, Gatanaga H, Matsuoka M, Takiguchi M, Oka S. Clinical relevance of substitutions in the connection subdomain and RNase H domain of HIV-1 reverse transcriptase from a cohort of antiretroviral treatment-naive patients. Antiviral Res. 2009;82:115–121. [PMC free article] [PubMed]
16. Jacobo-Molina A, Ding J, Nanni RG, Clark AD, Jr, Lu X, Tantillo C, Williams RL, Kamer G, Ferris AL, Clark P, et al. Crystal structure of human immunodeficiency virus type 1 reverse transcriptase complexed with double-stranded DNA at 3.0 A resolution shows bent DNA. Proceedings of the National Academy of Sciences of the United States of America. 1993;90:6320–6324. [PubMed]
17. Julias JG, McWilliams MJ, Sarafianos SG, Alvord WG, Arnold E, Hughes SH. Mutation of amino acids in the connection domain of human immunodeficiency virus type 1 reverse transcriptase that contact the template-primer affects RNase H activity. J Virol. 2003;77:8548–8554. [PMC free article] [PubMed]
18. Kawamoto A, Kodama E, Sarafianos SG, Sakagami Y, Kohgo S, Kitano K, Ashida N, Iwai Y, Hayakawa H, Nakata H, Mitsuya H, Arnold E, Matsuoka M. 2′-deoxy-4′-C-ethynyl-2-halo-adenosines active against drug-resistant human immunodeficiency virus type 1 variants. Int J Biochem Cell Biol. 2008;40:2410–2420. [PubMed]
19. Kodama EI, Kohgo S, Kitano K, Machida H, Gatanaga H, Shigeta S, Matsuoka M, Ohrui H, Mitsuya H. 4′-Ethynyl nucleoside analogs: potent inhibitors of multidrug-resistant human immunodeficiency virus variants in vitro. Antimicrob Agents Chemother. 2001;45:1539–1546. [PMC free article] [PubMed]
20. Kohlstaedt LA, Wang J, Friedman JM, Rice PA, Steitz TA. Crystal structure at 3.5 A resolution of HIV-1 reverse transcriptase complexed with an inhibitor. Science. 1992;256:1783–1790. [PubMed]
21. Marchand B, Gotte M. Site-specific footprinting reveals differences in the translocation status of HIV-1 reverse transcriptase. Implications for polymerase translocation and drug resistance. J Biol Chem. 2003;278:35362–35372. [PubMed]
22. Menendez-Arias L, Berkhout B. Retroviral reverse transcription. Virus Res. 2008;134:1–3. [PubMed]
23. Meyer PR, Matsuura SE, Mian AM, So AG, Scott WA. A mechanism of AZT resistance: an increase in nucleotide-dependent primer unblocking by mutant HIV-1 reverse transcriptase. Mol Cell. 1999;4:35–43. [PubMed]
24. Meyer PR, Matsuura SE, So AG, Scott WA. Unblocking of chain-terminated primer by HIV-1 reverse transcriptase through a nucleotide-dependent mechanism. Proceedings of the National Academy of Sciences of the United States of America. 1998;95:13471–13476. [PubMed]
25. Meyer PR, Matsuura SE, Tolun AA, Pfeifer I, So AG, Mellors JW, Scott WA. Effects of specific zidovudine resistance mutations and substrate structure on nucleotide-dependent primer unblocking by human immunodeficiency virus type 1 reverse transcriptase. Antimicrob Agents Chemother. 2002;46:1540–1545. [PMC free article] [PubMed]
26. Michailidis E, Marchand B, Kodama EN, Singh K, Matsuoka M, Kirby KA, Ryan EM, Sawani AM, Nagy E, Ashida N, Mitsuya H, Parniak MA, Sarafianos SG. Mechanism of inhibition of HIV-1 reverse transcriptase by 4′-Ethynyl-2-fluoro-2′-deoxyadenosine triphosphate, a translocation-defective reverse transcriptase inhibitor. J Biol Chem. 2009;284:35681–35691. [PMC free article] [PubMed]
27. Nikolenko GN, Delviks-Frankenberry KA, Palmer S, Maldarelli F, Fivash MJ, Jr, Coffin JM, Pathak VK. Mutations in the connection domain of HIV-1 reverse transcriptase increase 3′-azido-3′-deoxythymidine resistance. Proceedings of the National Academy of Sciences of the United States of America. 2007;104:317–322. [PubMed]
28. Nikolenko GN, Delviks-Frankenberry KA, Pathak VK. A novel molecular mechanism of dual resistance to nucleoside and nonnucleoside reverse transcriptase inhibitors. J Virol. 2010;84:5238–5249. [PMC free article] [PubMed]
29. Nikolenko GN, Palmer S, Maldarelli F, Mellors JW, Coffin JM, Pathak VK. Mechanism for nucleoside analog-mediated abrogation of HIV-1 replication: balance between RNase H activity and nucleotide excision. Proceedings of the National Academy of Sciences of the United States of America. 2005;102:2093–2098. [PubMed]
30. Ohrui H, Kohgo S, Hayakawa H, Kodama E, Matsuoka M, Nakata T, Mitsuya H. 2′-Deoxy-4′-C-ethynyl-2-fluoroadenosine: a nucleoside reverse transcriptase inhibitor with highly potent activity against all HIV-1 strains, favorable toxic profiles and stability in plasma. Nucleic Acids Symp Ser (Oxf) 2006:1–2. [PubMed]
31. Ren J, Esnouf R, Hopkins A, Ross C, Jones Y, Stammers D, Stuart D. The structure of HIV-1 reverse transcriptase complexed with 9-chloro-TIBO: lessons for inhibitor design. Structure. 1995;3:915–926. [PubMed]
32. Ren J, Nichols C, Bird L, Chamberlain P, Weaver K, Short S, Stuart DI, Stammers DK. Structural mechanisms of drug resistance for mutations at codons 181 and 188 in HIV-1 reverse transcriptase and the improved resilience of second generation non-nucleoside inhibitors. J Mol Biol. 2001;312:795–805. [PubMed]
33. Sarafianos SG, Clark AD, Jr, Tuske S, Squire CJ, Das K, Sheng D, Ilankumaran P, Ramesha AR, Kroth H, Sayer JM, Jerina DM, Boyer PL, Hughes SH, Arnold E. Trapping HIV-1 reverse transcriptase before and after translocation on DNA. J Biol Chem. 2003;278:16280–16288. [PubMed]
34. Sarafianos SG, Das K, Clark AD, Jr, Ding J, Boyer PL, Hughes SH, Arnold E. Lamivudine (3TC) resistance in HIV-1 reverse transcriptase involves steric hindrance with beta-branched amino acids. Proceedings of the National Academy of Sciences of the United States of America. 1999;96:10027–10032. [PubMed]
35. Sarafianos SG, Das K, Hughes SH, Arnold E. Taking aim at a moving target: designing drugs to inhibit drug-resistant HIV-1 reverse transcriptases. Curr Opin Struct Biol. 2004;14:716–730. [PubMed]
36. Sarafianos SG, Hughes SH, Arnold E. Designing anti-AIDS drugs targeting the major mechanism of HIV-1 RT resistance to nucleoside analog drugs. Int J Biochem Cell Biol. 2004;36:1706–1715. [PubMed]
37. Sarafianos SG, Marchand B, Das K, Himmel DM, Parniak MA, Hughes SH, Arnold E. Structure and Function of HIV-1 Reverse Transcriptase: Molecular Mechanisms of Polymerization and Inhibition. J Mol Biol. 2009;385:693–713. [PMC free article] [PubMed]
38. Schuckmann MM, Marchand B, Hachiya A, Kodama EN, Kirby KA, Singh K, Sarafianos SG. The N348I mutation at the connection subdomain of HIV-1 reverse transcriptase decreases binding to nevirapine. J Biol Chem. 285:38700–38709. [PMC free article] [PubMed]
39. Singh K, Marchand B, Kirby KA, Michailidis E, Sarafianos SG. Structural Aspects of Drug Resistance and Inhibition of HIV-1 Reverse Transcriptase. Viruses. 2010;2:606–638. [PMC free article] [PubMed]
40. Sluis-Cremer N, Arion D, Parniak MA. Molecular mechanisms of HIV-1 resistance to nucleoside reverse transcriptase inhibitors (NRTIs) Cell Mol Life Sci. 2000;57:1408–1422. [PubMed]
41. Smerdon SJ, Jager J, Wang J, Kohlstaedt LA, Chirino AJ, Friedman JM, Rice PA, Steitz TA. Structure of the binding site for nonnucleoside inhibitors of the reverse transcriptase of human immunodeficiency virus type 1. Proceedings of the National Academy of Sciences of the United States of America. 1994;91:3911–3915. [PubMed]
42. Wang J, Smerdon SJ, Jager J, Kohlstaedt LA, Rice PA, Friedman JM, Steitz TA. Structural basis of asymmetry in the human immunodeficiency virus type 1 reverse transcriptase heterodimer. Proceedings of the National Academy of Sciences of the United States of America. 1994;91:7242–7246. [PubMed]
43. Yap SH, Sheen CW, Fahey J, Zanin M, Tyssen D, Lima VD, Wynhoven B, Kuiper M, Sluis-Cremer N, Harrigan PR, Tachedjian G. N348I in the connection domain of HIV-1 reverse transcriptase confers zidovudine and nevirapine resistance. PLoS Med. 2007;4:e335. [PMC free article] [PubMed]