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J Virol. 2009 September; 83(17): 8502–8513.
Published online 2009 June 24. doi:  10.1128/JVI.00859-09
PMCID: PMC2738196

Subtype-Specific Differences in the Human Immunodeficiency Virus Type 1 Reverse Transcriptase Connection Subdomain of CRF01_AE Are Associated with Higher Levels of Resistance to 3′-Azido-3′-Deoxythymidine [down-pointing small open triangle]

Abstract

We previously shown that mutations in the connection (CN) subdomain of human immunodeficiency virus type 1 (HIV-1) subtype B reverse transcriptase (RT) increase 3′-azido-3′-deoxythymidine (AZT) resistance in the context of thymidine analog mutations (TAMs) by affecting the balance between polymerization and RNase H activity. To determine whether this balance affects drug resistance in other HIV-1 subtypes, recombinant subtype CRF01_AE was analyzed. Interestingly, CRF01_AE containing TAMs exhibited 64-fold higher AZT resistance relative to wild-type B, whereas AZT resistance of subtype B containing the same TAMs was 13-fold higher, which in turn correlated with higher levels of AZT-monophosphate (AZTMP) excision on both RNA and DNA templates. The high level of AZT resistance exhibited by CRF01_AE was primarily associated with the T400 residue in wild-type subtype AE CN subdomain. An A400T substitution in subtype B enhanced AZT resistance, increased AZTMP excision on both RNA and DNA templates, and reduced RNase H cleavage. Replacing the T400 residue in CRF01_AE with alanine restored AZT sensitivity and reduced AZTMP excision on both RNA and DNA templates, suggesting that the T400 residue increases AZT resistance in CRF01_AE at least in part by directly increasing the efficiency of AZTMP excision. These results show for the first time that CRF01_AE exhibits higher levels of AZT resistance in the presence of TAMs and that this resistance is primarily associated with T400. Our results also show that mixing the RT polymerase, CN, and RNase H domains from different subtypes can underestimate AZT resistance levels, and they emphasize the need to develop subtype-specific genotypic and phenotypic assays to provide more accurate estimates of clinical drug resistance.

Phylogenetic analysis of human immunodeficiency virus type 1 (HIV-1) indicates that HIV-1 is composed of three separate genetic groups named M (main), O (outlier), and N (non-M/non-O). Group M, which worldwide accounts for greater than 90% of all HIV-1 infections, is further subdivided into nine subtypes: A, B, C, D, F, G, H, J, and K. More than 50% of HIV-1 infections worldwide are of subtype C and are predominantly located in sub-Saharan Africa and India, whereas subtype B, which accounts for 10% of HIV-1 infections, is predominantly found in North and South America, Western Europe, Japan, and Australia (8).

Cocirculation of different HIV-1 subtypes in the same geographic location has led to co- or superinfections of HIV-1, leading to the generation of recombinants (18, 22, 39). Recombinant viruses that have become established within the human population and that have the capacity to spread are referred to as circulating recombinant forms (CRFs), whereas viral recombinants that have been isolated only from a single patient are referred to as unique recombinant forms. Currently there are 43 recognized CRFs (http://www.hiv.lanl.gov), accounting for 18% of HIV-1 infections (3).

One prominent CRF is CRF01_AE, which is a recombinant between HIV-1 subtype A and a yet to be identified “subtype E.” CRF01_AE was originally identified in Thailand (17, 21) and remains responsible for 83% of all infections in South and Southeast Asia (8). Previously, a unique CRF01_AE isolate (99JP-NH3, also referred to as 99JP-NH3-II) was reported that was highly resistant to nucleoside reverse transcriptase inhibitors (NRTIs) including 3′-azido-3′-deoxythymidine (AZT) (>400-fold). This patient's virus contained numerous substitutions in the RT polymerase (POL) domain in addition to a long insertion between amino acids 67 and 68 (28, 29, 32).

Recently, we reported that mutations conferring NRTI resistance in subtype B lie within not only the POL domain but also the connection (CN) subdomain of RT (5, 19). Analysis of HIV-1 variants from treatment-experienced patients identified CN mutations E312Q, G335(C/D), N348I, A360(I/V), V365I, and A376S in subtype B that significantly enhanced AZT resistance in the presence of thymidine analog mutations (TAMs; D67N, K70R, T215Y, and K219Q). We determined that the CN subdomain mutations increased AZT resistance largely by reducing RNase H cleavage, which in turn reduced template RNA degradation, providing more time for RT to excise the incorporated AZT monophosphate (AZTMP). These observations strongly supported our model for a mechanism of NRTI drug resistance, which proposes that a balance between RT polymerization and RNase H activity is an important determinant for NRTI resistance. In this model, reducing RNase H activity allows the enzyme to remain associated with the template-primer for a longer time, leading to more efficient excision of the NRTI from the terminated primer and thereby resulting in a higher level of drug resistance (5, 19, 20). Other studies have also supported our resistance model and further established the role of CN subdomain mutations N348I and G333D in NRTI resistance (2, 6, 12, 43, 45). In addition, prolonged in vitro selection for AZT resistance produced an AZT-resistant HIV-1 strain containing CN subdomain mutation A371V, supporting the role of CN subdomain mutations in NRTI resistance (2).

In the present study, we sought to extend our analysis of CN subdomain mutations and the effect of balance between polymerization and RNase H activity on NRTI resistance to include additional HIV-1 subtypes, specifically CRF01_AE. Surprisingly, the results showed that in the context of TAMs, the wild-type AE CN subdomain conferred a higher level of resistance to AZT than the CN subdomain from subtype B, suggesting that CRF01_AE virus is predisposed to higher levels of AZT resistance. Additionally, we identified the CN subdomain amino acid T400 in CRF01_AE as being primarily responsible for the increase in AZT resistance and analyzed its effects on nucleotide excision and RNase H activity.

MATERIALS AND METHODS

Plasmids, cloning, and mutagenesis.

pHCMV-G expresses the G glycoprotein of vesicular stomatitis virus (44). Construction of vector pHL(WT) was previously described (19) and will be referred to as pHL(B-WT) in this paper to specify its wild-type subtype B genotype. pHL(AE-WT) and pHL(AET1) contain the POL, CN, and RNase H (RH) domains from wild-type CRF01_AE virus CM235-2 (17) and treatment-experienced CRF01_AE patient virus 99JP-NH3 (28, 29), respectively, subcloned into pHL(B-WT). All three constructs express the firefly luciferase reporter gene and all of the HIV-1 proteins except Nef and Env. It should be noted that the POL domain cloned into pHL(AET1) is a version of the patient's POL domain that includes the following selected amino acid mutations: the 11-amino-acid insert between RT position 67 and 68; T69I; and NRTI resistance mutations M41L, L210W, and T215Y (M41L/L210W/T215Y) (see reference 28 for vector ERT-mt6). Vectors pHL(B-TAMs) and pHL(AE-TAMs) were created by introducing NRTI resistance mutations D67N/K70R/T215Y/K219Q into pHL(B-WT) and pHL(AE-WT), respectively. Site-directed mutagenesis was carried out using a QuikChange Site-Directed Mutagenesis Kit (Stratagene), and the presence or absence of each mutation was verified by DNA sequencing.

Antiviral drugs.

NRTIs AZT and 2′,3′-didehydro-3′-dideoxythymidine (d4T) were obtained from Sigma-Aldrich. 2′,3′-Dideoxy-3′-thiacytidine (3TC) was obtained from Moravek Biochemicals. Non-NRTIs (4S)-6-chloro-4-(cyclopropylethynyl)-1,4-dihydro-4-(trifluoromethyl)-2H-3,1-benzoxazin-2-one (efavirenz) and 11-cyclopropyl-5,11-dihydro-4-methyl-6H-dipyrido[3,2-b:2′,3′-e][1,4]diazepin-6-one (nevirapine) were obtained from the NIH AIDS Research and Reference Reagent Program.

Cells, transfection, virus production, and drug susceptibility assays.

Human 293T cells (American Type Culture Collection) were maintained at 5% CO2 and 37°C in Dulbecco's modified Eagle's medium (CellGro) supplemented with 10% fetal calf serum (HyClone), penicillin (50 U/ml; Gibco), and streptomycin (50 μg/ml; Gibco). To produce virus, 293T cells were transfected with each vector using calcium phosphate precipitation or GeneJet (SignaGen Laboratories) in the presence of pHCMV-G. Forty-eight hours later, virus was harvested, spun to remove cellular debris, filtered through a Millex GS 0.45-μm-pore-size filter (Nalgene), concentrated 20- or 100-fold by centrifugation through a sucrose gradient, and stored at −80°C. Single-cycle drug susceptibility assays were performed as previously described (20).

Replicative capacity assay.

By utilizing a single-cycle replication assay, virus containing the vector of interest was harvested from 293T cells, normalized by using p24 capsid (CA) amounts (HIV-1 p24 ELISA Kit; Perkin Elmer), and used to infect target 293T cells plated at 4,000 cells/well of a 96-well plate. Normalized luciferase light units measured 48 h postinfection were used to determine replicative capacity.

RNase H and ATP-mediated excision and/or extension assays.

RNase H cleavage and ATP-mediated excision and/or extension assays were preformed as previously described (5). Briefly, primary RNase H cleavages were assayed on an RNA/DNA hybrid consisting of an 18-nucleotide DNA (5′-AGCTCCCAGGCTCAGATC-3′) annealed to a 32P-labeled 18-nucleotide RNA (5′-GAUCUGAGCCUGGGAGCU-3′). Reactions were carried out in the presence of 50 nM hybrid and virus lysates containing 50 ng of p24 CA for 30 min at 37°C. Samples were run on a 15% denaturing polyacrylamide gel electrophoresis gel, band intensities were scanned using a phosphorimager (Bio-Rad), and density analysis was performed using Quantity One Software (Bio-Rad). ATP-mediated excision and/or extension assays were performed using a 32P-labeled 18-nucleotide DNA primer (5′-GTCACTGTTCGAGCACCA-3′) annealed to either a 42-nucleotide RNA (5′-AUCAGUGUAGACAAUCCCUAGCUAUGGUGCUCGAACAGUGAC-3′) or a 42-nucleotide DNA (5′-ATCAGTGTAGACAATCCCTAGCTATGGTGCTCGAACAGTGAC-3′) template and blocked with incorporation of AZT 5′-triphosphate (AZTTP) substrate. The excision and extension assays were carried out at 37°C with viral lysates containing 300 ng of p24 CA and reaction buffer containing 3.3 mM ATP, 10 mM MgCl2, 5 μM concentrations of the deoxynucleoside triphosphates, 2.5 μM AZTTP, and 5 nM substrate hybrid. The reactions were analyzed as described above. For assays in which extension (DNA polymerization) alone was measured, the reactions were carried out in the absence of AZTTP and ATP with an unblocked 32P-labeled 18-nucleotide DNA primer annealed to either the 42-nucleotide RNA or the DNA template.

Analysis of AE/B recombinants.

Boot-scanning was performed using SimPlot software, version 3.5.1 (15), for a 300-bp window moving at increments of 30 bp. As implemented in the software program, pairwise nucleotide evolutionary distances were calculated using the Kimura-2 parameter distance model with a transition/transversion ratio of 2.0. The neighbor-joining method was used to construct phylogenetic trees. RT recombinants were compared to a consensus parental CRF01_AE reference (GenBank accession numbers AF259954, U51189, AB032740, AB032741, and U51188) and a consensus parental subtype B′ reference (GenBank accession numbers U71182, AY180905, and AY713408), with subtype J (GenBank accession number AF082395) as an outlier. Bootstrap values of ≥70% were considered significant for assigning parenthood. GenBank accession numbers and references for the AE/B recombinants are as follows: AY167123 (31), EU031913 (14), EU031915 (14), DQ366665 (33), DQ366664 (33), DQ366663 (33), DQ366666 (33), EF495062 (13), AY358069 (36), AY358073 (36), and AF468970 (23). AY167123 was scanned with a 200-bp window and 20-bp step to remove one ambiguity with respect to a crossover junction. Recombinational breakpoints were also verified using the HIV subtyping tools REGA, version 2.0 (http://hivdb.stanford.edu/), and jpHMM (http://jphmm.gobics.de/).

Statistical analysis.

A Student's t test was used to determine statistically significant differences (SIGMAPLOT, version 8.0, software).

RESULTS

CRF01_AE from a treatment-experienced patient exhibits enhanced AZT resistance in the presence of TAMs.

We previously demonstrated that CN subdomains from subtype B treatment-experienced patients increased AZT resistance levels, indicating that mutations in the CN subdomain can influence NRTI resistance (5, 19). We therefore sought to determine whether other HIV-1 subtypes, specifically CRF01_AE, would also contain CN subdomain mutations that are associated with NRTI resistance. Figure Figure11 shows the RT alignment of wild-type subtype B, wild-type CRF01_AE, and treatment-experienced CRF01_AE patient isolate 99JP-NH3 (AET1). For the purposes of this study, the term “wild-type” refers to the prototype strain: pNL43 for subtype B and CM235-2 for CRF01_AE. The patient infected with AET1 initially received AZT and 2′,3′-dideoxyinosine treatment and was later switched to a regimen containing AZT, 3TC, and one protease inhibitor (nelfinavir or indinavir) (28). AET1 was reported to be highly resistant to NRTIs AZT (>400-fold), 3TC (>200-fold), d4T (10-fold), and 2′,3′-dideoxyinosine (26-fold) (28). Genotyping of the POL domain in RT from this virus revealed NRTI resistance mutations M41L/L210W/T215Y, an unusual 11-amino-acid insertion between amino acids 67 and 68 in RT, and a T69I mutation. The insertion was shown to be derived from human chromosome 17 through nonhomologous recombination (32).

FIG. 1.
Alignment of HIV-1 RT p66 protein sequences from subtype B and CRF01_AE. Shown are amino acids 1 to 560 for wild-type subtype B (B-WT; reference strain pNL4-3), wild-type CRF01_AE (AE-WT; reference strain CM235-2), and treatment-experienced CRF01_AE patient ...

To compare the levels of NRTI resistance between wild-type and treatment-experienced AE RTs, vectors were created by insertion into pHL(B-WT) (Fig. (Fig.2A)2A) the POL, CN, and RH domains from wild-type CRF01_AE [Fig. [Fig.2A,2A, pHL(AE-WT)] or treatment-experienced CRF01_AE [Fig. [Fig.2A,2A, pHL(AET1)]. Using these basic vectors, additional vectors that expressed chimeric subtype B and AE RTs were created to analyze NRTI resistance. All chimeric RT vectors that were constructed exhibited less than a threefold decrease in viral titers, suggesting that their replicative capacities were similar to the wild-type (data not shown). Furthermore, the replicative capacity of AET1 was previously shown to be decreased three- to fourfold (28).

FIG. 2.
AZT resistance associated with AE and subtype B RT chimeras. (A) Schematic representation of the subtype B luciferase-expressing HIV-1 vector used for antiretroviral drug testing, pHL(B-WT) (white boxes). All of the HIV-1 genes, except for nef and env ...

For ease of reference throughout the paper, vectors will be characterized according to the subtypes of the POL, CN, and RH domains, in respective order. For example, pHL(B-WT), which harbors the subtype B POL, CN, and RH domains, is B-B-B, pHL(AE-WT) is AE-AE-AE, and pHL(AET1) is AET1-AET1-AET1. Chimeric vectors will be referenced similarly: for example, a wild-type subtype B RT containing a subtype B POL domain, a subtype AE CN subdomain, and a subtype B RH domain will be designated B-AE-B.

We have previously shown that increases in AZT resistance associated with CN subdomain mutations occur in the presence of TAMs (4, 5, 19). To first determine if any NRTI resistance was associated with the CN subdomain of AET1 in the context of its own TAMs-containing POL domain, the POL plus CN subdomain from pHL(AET1) was subcloned into pHL(B-WT) to create AET1-AET1-B, which was then assayed for AZT resistance (Fig. (Fig.2B).2B). The resulting increase in AZT resistance was extremely high, >10,000-fold (50% inhibitory concentration [IC50] of >500 μM) compared to the B-B-B control (defined as onefold AZT resistance; IC50 of 0.05 ± 0.003 μM). AET1-AE-B, which contained the AET1 POL domain and the wild-type AE CN subdomain (Fig. (Fig.2B),2B), still exhibited a >10,000-fold increase in AZT resistance (IC50 of >500 μM). This was to be expected as it was previously reported that this specific 11-amino-acid insert in the AET1 POL domain in combination with TAMs resulted in a >400-fold increase in AZT resistance (IC50 of >10 μM) (28). To determine whether CN subdomain mutations contributed to increased AZT resistance before AET1 acquired the 11-amino-acid insertion, we analyzed AET1 CN subdomain mutations in the presence of a subtype B with a POL domain that contained TAMs D67N/K70R/T215Y/K219Q (BTAMs-B-B). Our previous studies have shown that this combination of TAMs in subtype B increases AZT resistance approximately 10- to 14-fold compared to wild-type subtype B (4, 5, 19, 20). This BTAMs POL domain was subsequently used to determine the levels of AZT resistance associated with the AET1 CN subdomain.

As shown in Fig. Fig.2C,2C, the addition to vector B-B-B of the AET1 CN subdomain plus the RH domain (B-AET1-AET1) (1.4-fold AZT resistance of the control; IC50 of 0.07 ± 0.01 μM), CN subdomain alone (B-AET1-B) (1.6-fold; IC50 of 0.085 ± 0.03 μM), or RH domain alone (B-B-AET1) (1.1-fold; IC50 of 0.054 ± 0.002 μM) did not increase the level of AZT resistance significantly above the B-B-B control (1-fold; IC50 of 0.05 ± 0.003 μM). However, in the presence of TAMs (Fig. (Fig.2D),2D), the addition of the AET1 CN subdomain (BTAMs-AET1-B) as well as CN subdomain plus RH domain (BTAMs-AET1-AET1) showed significant increases in AZT resistance (83-fold with IC50 of 4.2 ± 0.12 μM and 96-fold with an IC50 of 4.8 ± 0.41 μM, respectively) compared to the BTAMs-B-B (13-fold; IC50 = 0.67 ± 0.05 μM). Since the AET1 RH domain alone in BTAMs-B-AET1 (13-fold; IC50 of 0.63 ± 0.04 μM;) did not increase resistance above the BTAMs-B-B, the increases in AZT resistance were attributed mainly to the AET1 CN subdomain.

The wild-type AE CN subdomain increases AZT resistance in the presence of TAMs.

As a control, viruses containing the wild-type AE CN and RH domains were also tested for AZT resistance. Surprisingly, in the context of a subtype B POL domain containing TAMs, the addition of the wild-type AE CN subdomain (BTAMs-AE-B), as well as a CN subdomain plus RH domain (BTAMs-AE-AE), also exhibited high levels of AZT resistance(99-fold with an IC50 of 4.9 ± 1.05 μM and 53-fold with an IC50 of 2.6 ± 0.19 μM, respectively) (Fig. (Fig.2E).2E). Again, the increase in AZT resistance was localized to the CN subdomain since addition of the wild-type AE RH domain (BTAMs-B-AE) (9-fold; IC50 = 0.46 ± 0.07 μM) did not further increase AZT resistance above the BTAMs-B-B control (13-fold) (Fig. (Fig.2E).2E). In this case, the combination of the wild-type AE CN and RH domains (BTAMs-AE-AE) reduced AZT resistance twofold compared to the AE CN subdomain (BTAMs-AE-B) alone (53-fold versus 99-fold), suggesting that some interactions between the CN and RH domains may influence AZT resistance. Nevertheless, the level of AZT resistance associated with BTAMs-AE-AE (53-fold) was still fourfold above the level of the BTAMs-B-B control (13-fold). These results demonstrated that if TAMs are present, the wild-type AE CN subdomain exhibits a higher level of AZT resistance than the subtype B CN subdomain. In the absence of TAMs, the AE CN subdomain (in B-AE-B) exhibited a twofold increase in AZT resistance (0.10 ± 0.01 μM) above the B-B-B control (onefold) (Fig. (Fig.2E2E).

The observed increases in resistance associated with the wild-type AE and AET1 CN subdomains were observed with only AZT; other NRTIs such as d4T and 3TC or non-NRTIs efavirenz and nevirapine did not show a significant enhancement of resistance (data not shown).

Wild-type AE POL domain does not exhibit higher resistance to AZT than the subtype B wild-type POL domain.

Since the CN subdomain from wild-type AE exhibited a higher level of AZT resistance than that from subtype B, the wild-type AE POL domain was also tested for its effect on AZT resistance. As shown in Fig. Fig.2F,2F, the wild-type AE POL domain alone (AE-B-B) did not confer increased AZT resistance compared to the B-B-B control (onefold; IC50 of 0.05 ± 0.01 μM). Similarly, further addition of the wild-type AE CN subdomain (AE-AE-B) (2.1-fold; IC50 of 0.10 ± 0.02 μM) or the CN subdomain plus RH domain (AE-AE-AE) (1.2-fold; IC50 of 0.06 ± 0.02 μM) to the wild-type AE POL domain did not significantly increase AZT resistance compared to the B-B-B control (P > 0.05). Thus, the wild-type AE POL domain did not exhibit higher resistance to AZT than the subtype B POL domain.

AE CN subdomain increases AZT resistance in the context of both AE and B POL domains containing TAMs.

To verify that the increase in resistance associated with the wild-type AE CN subdomain was not a consequence of forming a chimeric protein with subtype B POL domains containing TAMs, the same TAMs were introduced into pHL(AE-WT) to create pHL(AE-TAMs) (AETAMs-AE-AE). As shown in Fig. Fig.2G,2G, introducing these TAMs into the AE POL domain of the B-B-B control (AETAMs-B-B) produced a similar level of AZT resistance (10-fold; IC50 of 0.51 ± 0.07 μM) as the same combination with the BTAMs (BTAMs-B-B) (13-fold) (Fig. (Fig.2D).2D). The addition of the wild-type AE CN subdomain plus the RH domain (AETAMs-AE-AE) (64-fold; IC50 of 3.2 ± 0.39 μM) or of the CN subdomain alone (AETAMs-AE-B) (120-fold; IC50 of 6.0 ± 0.84 μM) still enhanced AZT resistance significantly above the level of AETAMs-B-B, indicating that the wild-type AE CN subdomain increased AZT resistance independently of the POL domain subtype. Furthermore, the combination of the AET1 CN subdomain with the AETAMs POL (AETAMs-AET1-B) also conferred a high level of AZT resistance (92-fold; IC50 of 4.6 ± 0.66 μM). The addition of the wild-type AE RH domain to the AETAMs POL domain (AETAMs-B-AE) (6-fold; IC50 of 0.28 ± 0.02 μM) did not further enhance AZT resistance compared to the AETAMs-B-B control (10-fold), again supporting the role of the wild-type AE CN subdomain in enhancing AZT resistance.

To confirm that the increase in resistance associated with the wild-type AE CN subdomain was not dependent upon the specific TAMs present in POL, AZT sensitivity was determined in the presence of a combination of mutations associated with the TAM1 pathway, specifically M41L/L210W/T215Y (7, 16, 42). This combination of TAMs was originally present in the POL domain of the AET1 virus from the treatment-experienced patient (Fig. (Fig.1).1). As shown in Fig. Fig.2H,2H, introducing M41L/L210W/T215Y to the AE POL domain (AETAMsLWY) in the B-B-B control (AETAMsLWY-B-B) led to an 18-fold increase in AZT resistance (IC50 of 0.89 ± 0.15 μM), similar to the level of AZT resistance observed with these same mutations in the subtype B POL domain (17-fold) (19). Addition of the wild-type AE CN subdomain plus RH domain to AETAMsLWY POL (AETAMsLWY-AE-AE) increased the level of AZT resistance 51-fold (2.5 ± 0.45 μM), similar to the level of AZT resistance observed with AETAMs-AE-AE (64-fold) (Fig. (Fig.2G).2G). Therefore, the increase in AZT resistance observed with the wild-type AE CN subdomain was independent of the combination of TAMs in the POL domain.

The T400 amino acid in the wild-type AE CN subdomain is associated with increased AZT resistance.

There are 13 amino acid positions that differ between the CN subdomains of wild-type AE and B; the AET1 contains two additional amino acid differences compared to subtype B (Fig. (Fig.1A1A and and3A).3A). As described earlier, the wild-type AE CN subdomain (in BTAMs-AE-B) and AET1 CN subdomain (in BTAMs-AET1-B) exhibited similar levels of AZT resistance (99-fold and 83-fold compared to B-B-B, respectively), indicating that the substitutions P313Q, R395K, and E404D in the AET1 CN subdomain did not substantially affect AZT resistance. To determine which amino acids in the wild-type AE CN subdomain were responsible for the increase in AZT resistance, additional mutational analysis was performed. Mutant M1, containing subtype B amino acid substitutions T312E/V329I/R395K/E404D exhibited a similar 107-fold (IC50 of 5.3 ± 0.30 μM) increase in AZT resistance versus the wild-type AE CN subdomain (99-fold); further addition of substitutions V326I/D335G/K357M/R358K/S359G/R366K/V371A to mutant M1 to create mutant M2 did not significantly decrease AZT resistance (96-fold; IC50 of 4.8 ± 0.61 μM). Mutant M3, which retained only the substitutions A371V/A400T/T403M, also had a high level of AZT resistance (90-fold; IC50 of 4.5 ± 0.70 μM). Similarly, mutant M4, which retained only the A400T/T403M substitutions, exhibited a high (90-fold; IC50 of 4.5 ± 0.80 μM) increase in AZT resistance. Single mutants were then created and tested for AZT resistance. Mutant M5, which contained only amino acid substitution T403M, exhibited a 25-fold (IC50 of 1.2 ± 0.22 μM) increase in AZT resistance; however, mutant M6, which contained only amino acid substitution A400T, exhibited a 50-fold (IC50 of 2.5 ± 0.38 μM) increase in AZT resistance. The A400T CN subdomain substitution itself could therefore account for at least 50% of the AZT resistance associated with the AE CN subdomain. Additional support for the role of T400 in AZT resistance was obtained when the subtype B-specific T400A substitution was introduced into AETAMs-AE-AE (Fig. (Fig.3B).3B). The T400A substitution in AETAMs-AE-AE (AETAMs-AET400A-AE) reduced AZT resistance from 64-fold to 6-fold (IC50 of 0.28 ± 0.03 μM). This level of AZT resistance was similar to that of the vector in which the entire AE CN subdomain was replaced with the B CN subdomain in the background of AE TAMs (AETAMs-B-AE) (sixfold; IC50 = 0.28 ± 0.02 μM;) (Fig. (Fig.3B).3B). Overall, the mutational analysis indicated that the combination of T400 and M403 led to the same level of AZT resistance as the entire AE CN subdomain, with T400 playing a larger role in contributing to the resistance.

FIG. 3.
Subtype AE CN subdomain mutations associated with AZT resistance. (A) Schematic representation of the RT from the subtype B containing TAMs. The top numbers depict the position of the CN subdomain amino acids that differ between subtype B and CRF01_AE. ...

Wild-type AE CN subdomain enhances ATP-mediated excision and extension of AZTMP-terminated primers on both RNA and DNA templates.

To determine if the increase in AZT resistance associated with the wild-type AE CN subdomain was due to enhanced nucleotide excision, ATP-mediated AZTMP excision and extension assays were performed over time on both an RNA (Fig. (Fig.4A)4A) and a DNA template (Fig. (Fig.4B)4B) using virion-derived RTs. Briefly, a radiolabeled 18-mer DNA primer was annealed to a 42-nucleotide DNA or RNA template and blocked by incorporation of AZTMP at the 3′ end. Excision of the AZTMP, followed by extension of the primer in the presence of AZTTP, was carried out by RT that was obtained from viral lysates.

FIG. 4.
Subtype AE enhanced ATP-mediated AZTMP excision on RNA and DNA templates. Representative autoradiograms of the kinetics of ATP-mediated nucleotide excision and extension from virion-derived RTs determined using a 32P-labeled (star) 19-mer DNA primer (thick ...

The results showed that AETAMs-AE-AE RT carried out ATP-mediated AZTMP excision and extension more efficiently than BTAMs-B-B RT on both the RNA (Fig. (Fig.4A)4A) and the DNA template (Fig. (Fig.4B).4B). Quantification of the proportions of total primer that were fully extended to the 42-mer product is shown on Fig. 4C and D; the percentage of the primer that was fully extended to the 42-mer by the BTAMs-B-B RT (25% for DNA template; 20% for RNA template) was set to onefold. For reference, the structures of the vectors and their AZT resistance levels are shown. Relative to the BTAMs-B-B RT, the AETAMs-AE-AE RT was 1.9-fold more efficient on the RNA template (Fig. (Fig.4C)4C) and 2.3-fold more efficient on the DNA template (Fig. (Fig.4D).4D). As expected, B-B-B and AE-AE-AE RT, which lack TAMs, both exhibited little to no AZTMP excision and extension in this assay; less than 5% of the primer was fully extended (data not shown).

To determine which AE RT domain(s) enhanced AZTMP excision, vectors containing chimeric B/AE RTs were tested for their ability to carry out AZTMP excision (Fig. 4E and F). The percentage of the labeled primer that was converted to the 42-mer in the presence of AETAMs-AE-AE RT (38% on the RNA template and 57% on the DNA template) was set to onefold. Replacing the AE CN subdomain with the subtype B CN subdomain alone (AETAMs-B-AE) or in conjunction with the subtype B RH domain (AETAMs-B-B) reduced AZTMP excision on an RNA template by 2.5-fold (0.4-fold) (Fig. (Fig.4E)4E) and on a DNA template by 5-fold (0.2-fold) (Fig. (Fig.4F)4F) compared to the AETAMs-AE-AE RT control. Again, the reduced efficiency of excision correlated with a reduction in AZT resistance from 64-fold to 6-fold and 10-fold, respectively. A comparison of BTAMs-B-B with the vector in which the B TAMs POL domain was replaced with the AE TAMs POL domain (AETAMs-B-B) indicated that these chimeras were less efficient at AZTMP excision on both RNA and DNA templates (Fig. 4E and F). Furthermore, the T400A substitution (AETAMs-AE400A-AE) decreased the efficiency of AZTMP excision 1.7-fold on an RNA template (Fig. (Fig.4E,4E, 0.6-fold) and 1.3-fold on a DNA template (Fig. (Fig.4F,4F, 0.8-fold), which resulted in lower AZT resistance (6-fold) than with the AETAMs-AE-AE control.

The role of the wild-type AE CN subdomain in enhancing excision was further confirmed by analyzing vectors in which the AE CN and RH domains were combined with the subtype B TAMs POL domain (Fig. 4G and H). As in the results shown in Fig. 4C and D, the percentage of the labeled primer that was converted to the 42-mer in the presence of the BTAMs-B-B control was set to an efficiency of onefold (20% on the RNA template and 25% on the DNA template). The addition of the wild-type AE CN subdomain alone (BTAMs-AE-B) or together with the AE RH domain (BTAMs-AE-AE) enhanced AZTMP excision on an RNA template 1.5- and 1.9-fold, respectively, compared to the BTAMs-B-B control (Fig. (Fig.4G).4G). Furthermore, the A400T substitution alone (BTAMs- BA400T-B) increased excision on an RNA template compared to the BTAMs-B-B control by 2.1-fold (Fig. (Fig.4G).4G). The enhancement in excision for these three chimeric RTs also resulted in an enhancement of AZT resistance 99-, 53-, and 50-fold, respectively, compared to the BTAMs-B-B control (13-fold) (Fig. (Fig.2E2E and and3A).3A). Therefore, the wild-type AE CN subdomain, and specifically the A400T mutation, were largely responsible for the increase in AZTMP excision on the RNA template.

AZTMP excision was also carried out on the DNA template for all of these chimeras (Fig. (Fig.4H).4H). Similar to the results obtained on the RNA template, addition of the wild-type AE CN subdomain, AE CN subdomain plus RH domain, or A400T increased AZTMP excision on the DNA template. The relative increase in AZTMP excision was similar on the RNA and DNA templates for BTAMs-AE-B (1.5 and 1.4-fold, respectively). The BTAMs-AE-AE and BTAMs-BA400T-B increased the AZTMP excision more efficiently on the DNA template than on the RNA template (compare 1.9- versus 3.2-fold increase for BTAMs-AE-AE, and 2.1- versus 3.4-fold increase for BTAMs-B400T-B). Overall, these results showed that in the context of TAMs, the wild-type AE CN subdomain and the threonine at position 400 enhance AZT resistance by increasing the efficiency of AZTMP excision on both RNA and DNA templates.

To verify that the increases in AZTMP excision-extension by RTs containing the AE CN subdomain or A400T mutation were not due to a higher efficiency of DNA polymerization, the rates of DNA polymerization in the absence of AZTTP on the same RNA and DNA templates used in the excision and extension assay were determined and normalized to the BTAMs-B-B (Fig. 4I and J, respectively). The parental BTAMs-B-B and AETAMs-AE-AE RTs had similar kinetics of polymerization on the RNA and DNA templates. On both templates, the chimeric RTs exhibited the same or lower rates of polymerization than the BTAMs-B-B or AETAMs-AE-AE controls, indicating that the observed increases in AZTMP excision and extension (Fig. 4I and J) were not due to increased polymerization activity. The addition of the A400T mutation to BTAMS-B-B had the lowest polymerization activity on both RNA and DNA templates.

AE RT has lower RNase H activity than subtype B RT.

To further elucidate the mechanism by which the wild-type AE CN subdomain and the A400T substitution increased AZTMP excision, we determined the RNase H activities of subtype B and AE RTs as well as chimeric RTs. Viral lysates were normalized for the amounts of p24 CA protein by enzyme-linked immunosorbent assay, and virion-associated RTs from each vector were analyzed for their ability to carry out primary RNase H cleavage on an 18-mer RNA/DNA hybrid (Fig. (Fig.5A).5A). As shown in Fig. Fig.5B,5B, subtype B vectors with and without TAMs (BTAMs-B-B and B-B-B) exhibited higher levels of primary RNase H cleavage on an 18-mer RNA/DNA template (42% and 28%, respectively) than the similar AE vectors with and without TAMs (32% for AETAMs-AE-AE and 17% for AE-AE-AE). Thus, CRF01_AE exhibited significantly lower levels of RNase H cleavage than subtype B in either the presence or absence of TAMs (P < 0.05). Interestingly, in both subtype B and CRF01_AE, addition of TAMs enhanced RNase H cleavage in this assay, indicating that one or more of these TAMs in the POL domain can influence RNase H activity. This increase in RNase H activity is in agreement with our previous results, which show that TAMs in subtype B increase in vivo template switching compared to wild-type subtype B (4, 19). Reductions in RNase H activity decrease template switching while increases in RNase H activity enhance template switching.

FIG. 5.
Primary RNase H cleavages. (A) Representative autoradiogram of RNase H cleavage levels of virion-derived RTs on a 32P-labeled (star) 18-mer RNA (thin line)/DNA (thick line) hybrid. Vectors tested are shown pictorially above the lanes. The percentages ...

We next examined the role of the wild-type AE CN subdomain and the A400T substitution in subtype B on RNase H activity (Fig. (Fig.5C).5C). The wild-type AE CN subdomain in BTAMs-AE-B caused a significant decrease (P < 0.05) in RNase H cleavage (24%) compared to the BTAMs-B-B control (42%) (Fig. (Fig.5C),5C), as did the addition of the mutation A400T in BTAMs-BA400T-B (33%). These data suggest that the entire AE CN subdomain and the threonine substitution alone reduce RNase H cleavage, contributing to an enhancement in AZT resistance.

We also analyzed the effects on RNase H cleavage for the reverse chimeras in which the AE CN subdomain of AETAMS-AE-AE was replaced with either the B CN subdomain or the T400A substitution alone (Fig. (Fig.5D).5D). The subtype B CN subdomain in AETAMs-B-AE did not increase RNase H cleavage (33% versus 32% for the AETAMs-AE-AE control), as would be expected for the reverse vector BTAMs-AE-B (Fig. (Fig.4).4). However, replacing the T400 in AETAMS-AE-AE with alanine did slightly increase RNase H cleavage from 32% to 39% (P < 0.05).

Overall, these results suggest that AE RT exhibits lower RNase H activity than subtype B RT, which may contribute to its higher level of AZT resistance. The effect of the AE CN subdomain on RNase H activity appears to be dependent upon the context of the POL domain and RNase H sequences; the BTAMs-AE-B RT had decreased RNase H activity, but the reverse vector, AETAMS-B-AE, did not enhance RNase H activity. Thus, the AE CN subdomain in BTAMs-AE-B directly enhances the efficiency of AZTMP excision and may also increase AZT resistance by reducing RNase H activity; however, the AE CN subdomain in AETAMS-AE-AE does not reduce RNase H activity and appears to only directly enhance AZTMP excision.

Examination of the threonine at position 400 in CRF01_AE and the alanine at position 400 in subtype B indicated that the T400A substitution in CRF01_AE decreased AZTMP excision, whereas the A400T substitution in subtype B increased AZTMP excision. The result that these substitutions affected AZTMP excision on both RNA and DNA templates indicated that the threonine at position 400 primarily enhanced AZT resistance by directly increasing the efficiency of AZTMP excision. However, since the A400T substitution in subtype B reduced RNase H cleavage and since the T400A substitution in AE modestly increased RNase H cleavage, the threonine at position 400 may also contribute to AZTMP excision on the RNA template by influencing the balance between polymerase and RNase H activities.

DISCUSSION

In these studies, we sought to determine whether mutations in the CN and RH domains contribute to AZT resistance in CRF01_AE. Interestingly, our results showed that CRF01_AE is inherently less susceptible to AZT than subtype B. Recent studies have begun to identify HIV-1 subtype-specific differences that influence drug susceptibility (3, 10, 11, 30). However, subtype-specific differences in the RT C-terminal CN and RH domains have not been examined, largely because the CN and RH domains are not routinely included in genotypic and phenotypic analyses. To our knowledge, these studies report for the first time that CRF01_AE in the context of TAMs is more resistant to AZT than subtype B and that the increased resistance is primarily due to the threonine at position 400 in the CN subdomain of CRF01_AE.

Santos et al. reported that a threonine at position 400 in subtype B HIV-1 is more frequent in NRTI-exposed patients (25, 26). Sequence database analysis of the CN and RH domains in subtype B (26) showed that a threonine at position 400 was present in 43% of naïve and 69% of NRTI-experienced subtype B patients, and selection of the threonine at this position in subtype B appeared to be linked to the acquisition of NRTI resistance mutations in the POL domain. Interestingly, analysis of 140 CRF01_AE genomes that were sequenced through the CN subdomain (Stanford University HIV Drug Resistance Database; http://hivdb.stanford.edu) showed that among both treatment-naïve (90%) and NRTI-experienced (96%) CRF01_AE patients, the threonine at position 400 is highly conserved. The frequency of the threonine at position 400 in other subtypes in the absence of treatment were as follows: subtype A, 78%; subtype C, 63%; subtype D, 65%; and subtype F, 81%. Unfortunately, the differences between treatment-naïve and treatment-experienced patients could not be determined because very few viral sequences that extend to the amino acid 400 position from treatment-experienced patients were available.

We examined for the first time the molecular mechanism by which the threonine at position 400 increases AZT resistance and observed that this substitution increases AZTMP excision on both RNA and DNA templates. These observations led us to conclude that, unlike most of the previously described CN subdomain mutations that enhance AZT resistance (5, 43), the threonine at position 400 in CRF01_AE increases AZT resistance by increasing the efficiency of AZTMP excision from the terminated primer. However, an alanine at position 400 in CRF01_AE also slightly increases RNase H activity, suggesting that reductions in RNase H activity may also contribute to the effect by influencing the balance between polymerase and RNase H activities.

To gain further insight into the mechanism by which the threonine at position 400 in the subtype B genome might increase AZTMP excision, we analyzed its location in the subtype B HIV-1 RT in complex with the template-primer (Fig. (Fig.6).6). The 400 residue in the P66 subunit is located far from the polymerase active site (~50 Å), the RNase H active site (~32 Å), the template (~20 Å), and the primer (~24 Å). Interestingly, the 400 residue in the p51 subunit is only 5 Å from K395 and 2.8 Å from E396 of the p51 subunit, which help form the RNase H primer grip (27). The RNase H primer grip has been shown to help position the template-primer at the RNase H active site (1, 9, 24, 27). Later studies by us (46) and others (1, 9) also showed that the RNase H primer grip can help position the template-primer at the polymerase active site. Based on its location, we propose that a threonine at position 400 in the p51 subunit of CRF01_AE may affect the positioning of the E396, and possibly the K395, with respect to the primer strand, in turn affecting the positioning of the template-primer at the polymerase active site and/or the RNase H active site. We hypothesize that altered positioning of the template-primer could result in more efficient excision and extension of the AZTMP from the blocked primer and/or reduced RNase H cleavage, resulting in enhanced resistance to AZT.

FIG. 6.
Molecular model of subtype B HIV-1 RT depicting the 400 residue in the p66 and p51 subunits of RT. The p51 subunit is magenta, while the fingers and palm, thumb, CN, and RH domains of p66 are pink, purple, cyan, and green, respectively. The RNA template ...

Although a threonine at position 400 in the subtype B CN subdomain clearly increased AZT resistance and AZTMP excision in these studies, our previous analyses of treatment-naïve and NRTI-experienced patients indicated that not all CN subdomains containing a threonine at position 400 increase AZT resistance (5). The CN subdomains from some subtype B treatment-experienced patients contained the A400T substitution, but these CN subdomains increased AZTMP excision only on an RNA template and not on the DNA template. This observation suggests that other substitutions in the CN subdomain modulate the effect of the threonine at position 400 on AZT resistance by affecting the positioning of the template-primer at the polymerase active site. Additional studies are needed to understand the interactions between the threonine at position 400 and other substitutions in the CN subdomain.

Commercial phenotypic assays for resistance to RTIs typically include only the POL domain subcloned into a subtype B CN subdomain and a subtype B RH domain; as a result, the potential contribution of CN and RH domain mutations to drug resistance for all subtypes, including CRF01_AE, has not been fully explored. Our results show that mixing the RT POL, CN, and RH domains from different subtypes, specifically the AE POL domain and the subtype B CN and RH domains, can lead to underestimation of the overall level of AZT resistance. These observations indicate the need to develop subtype-specific genotypic and phenotypic assays since they are more likely to reflect the level of drug resistance exhibited by the viruses in patients.

Circulation of subtype B and CRF01_AE viruses in Southeast Asia since the late 1980s has led to the emergence of AE/B recombinants. Identified recombinants were initially either mostly subtype B with the gp120 portion of the envelope from CRF01_AE (CRF01_AE/B) (37) or CRF01_AE with the gp120 from subtype B (99TH.MU2079 and CRF15_01B) (38, 40). However, as more and more AE/B recombinants are being isolated and sequenced, recombination breakpoints are being found not only in env but also in gag and the integrase and RT regions of pol (13, 14, 23, 31, 33-36, 41, 47). Figure Figure77 shows a compilation of some of the previously published AE/B recombinants from the literature depicting the recombination breakpoints in RT. As the figure shows, the AE/B recombinants can contain chimeric RT proteins with recombination breakpoints in the POL, CN, and RH domains. A brief survey of these AE/B recombinants suggests that some of the circulating recombinants may exhibit higher levels of AZT resistance than subtype B since they will contain the AE wild-type CN subdomain rather than the subtype B CN subdomain. Furthermore, under drug selection pressure, a recombinant between a subtype B POL domain containing TAMs and the wild-type AE CN subdomain may outcompete the parental subtype B containing the same TAMs.

FIG. 7.
CRF01_AE and subtype B/B′ recombinants. Schematic representation of RT from CRF01_AE and subtype B/B′ recombinants previously reported in the literature (see Materials and Methods). CRF01_AE and subtype B/B′ recombination breakpoints ...

In summary, the results of these studies show that the threonine at position 400 in the wild-type AE CN subdomain confers a higher level of AZT resistance in the presence of TAMs and that subtype-specific differences in the CN and RH domains may modulate AZT susceptibility. Thus, additional studies of the CN and RH domains in other subtypes may reveal important subtype-specific differences in their ability to acquire antiviral drug resistance.

Acknowledgments

We especially thank John Coffin, Wei-Shau Hu, and Steve Hughes for intellectual input and critical discussions of the manuscript. We also thank Andrea Galli for his expert advice on the phylogenetic analyses of AE/B recombinants.

This work was supported in part by the Intramural Research Program of the National Institutes of Health, National Cancer Institute, Center for Cancer Research, and the Intramural AIDS Targeted Antiviral Program.

The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. Government. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Footnotes

[down-pointing small open triangle]Published ahead of print on 24 June 2009.

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