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
). 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 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
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 (more ...)
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. ) the POL, CN, and RH domains from wild-type CRF01_AE [Fig. , pHL(AE-WT)] or treatment-experienced CRF01_AE [Fig. , 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 (more ...)
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
). 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
-B, which was then assayed for AZT resistance (Fig. ). 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. ), 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
). This BTAMs POL domain was subsequently used to determine the levels of AZT resistance associated with the AET1
As shown in Fig. , 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. ), 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. ). 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. ). 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. ).
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. , 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. , 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. ). 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
). This combination of TAMs was originally present in the POL domain of the AET1
virus from the treatment-experienced patient (Fig. ). As shown in Fig. , 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
-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. ). 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. and ). 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. ). 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. ). 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. (more ...) 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. ) and a DNA template (Fig. ) 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 (more ...)
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. ) and the DNA template (Fig. ). Quantification of the proportions of total primer that were fully extended to the 42-mer product is shown on Fig. ; 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. ) and 2.3-fold more efficient on the DNA template (Fig. ). 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. ). 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. ) and on a DNA template by 5-fold (0.2-fold) (Fig. ) 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. ). Furthermore, the T400A substitution (AETAMs-AE400A-AE) decreased the efficiency of AZTMP excision 1.7-fold on an RNA template (Fig. , 0.6-fold) and 1.3-fold on a DNA template (Fig. , 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. ). As in the results shown in Fig. , 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. ). 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. ). 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. and ). 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. ). 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. , 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. ) 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. ). As shown in Fig. , 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
). 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 (more ...)
We next examined the role of the wild-type AE CN subdomain and the A400T substitution in subtype B on RNase H activity (Fig. ). 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. ), 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. ). 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. ). 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.