Biochemical Mechanism of Human Immunodeficiency Virus Type 1 Reverse Transcriptase Resistance to Stavudine

Abstract

We have found a close correlation between viral stavudine (d4T) resistance and resistance to d4T-triphosphate at the human immunodeficiency virus type 1 reverse transcriptase (RT) level. RT from site-directed mutants with 69S-XX codon insertions and/or conventional zidovudine resistance mutations seems to be involved in an ATP-dependent resistance mechanism analogous to pyrophosphorolysis, whereas the mechanism for RT with the Q151M or V75T mutation appears to be independent of added ATP for reducing binding to d4T-triphosphate.

Although the use of nucleoside analog reverse transcriptase (RT) inhibitors (NRTIs) (in combination with nonnucleoside RT inhibitors and/or protease inhibitors) has proved highly successful in human immunodeficiency virus type 1 (HIV-1) therapy, this has not eliminated the selection of drug-resistant isolates (13, 22, 25). The mutational profile of these resistant viruses usually reflects which drugs have been used. For instance, the mutation M184V in RT results in specific and high-level resistance to lamivudine (24), and the changes at codons 41, 67, 70, 210, 215, and 219 contribute to high-level zidovudine (AZT) resistance (4, 8, 14). However, patient virus samples that are stavudine (d4T) resistant are also cross resistant to other nucleoside analogs rather than having d4T-specific resistance, and the magnitude of d4T resistance is usually low (6, 25). The biochemical mechanism of HIV-1 RT resistance to NRTIs is due in many cases to decreased binding of mutated RT to the respective NRTI-triphosphates (NRTI-TPs) (9). However, this does not appear to be the situation for AZT resistance, as no significant differences in discrimination between AZT-TP and dTTP in the polymerase reaction are seen when studying RTs with conventional AZT resistance the mutation (9, 11, 16). Recently, a new biochemical mechanism of AZT resistance has been reported where the RTs containing mutations D67N, K70R, T215F, or K219Q are able to remove AZT-monophosphate (AZT-MP) from blocked primers more efficiently than wild-type RT, thereby enabling resumption of DNA synthesis. This has been demonstrated to occur by either pyrophosphate-dependent pyrophosphorolysis (1) or by an ATP-mediated mechanism similar to pyrophosphorolysis, in which ATP at a physiological concentration (3.2 mM) acts as an acceptor to form, together with AZT-MP, a dinucleoside polyphosphate product (19). We have recently reported a close correlation between resistance to AZT-TP at the enzyme level and viral AZT resistance (16a). When we studied 11 mutants with either conventional AZT resistance mutations or multidrug resistance mutations (T69S-XX insertions or Q151M), we found this biochemical resistance mechanism to be mainly ATP dependent, with the exception of the Q151M mutant. Without added ATP, all the mutants (except the Q151M mutant) behaved similarly to wild-type RT. Mutations that appeared to be dominant in the ATP-dependent reaction were those in the β2-β3 loop in the RT “fingers” domain, i.e., D67N, K70R, and insertions T69S-SG and T69S-AG. In contrast, RT with the Q151M mutation seems to be involved in decreased binding to AZT-TP independently of added ATP.

As there has been little reported on the biochemical mechanism of resistance to d4T, our present study was designed to investigate such mechanisms by using the same RT assay system that we previously used to measure AZT resistance. We have studied a large group of mutants constructed by site-directed mutagenesis that we knew or suspected would confer resistance to d4T. Specific mutations were also selected for studying the potential involvement of the RT fingers domain and the likelihood of cross-resistance to AZT.

Site-directed mutagenesis and sequence analysis have been described elsewhere (12, 16a). Nucleotide changes were introduced into the RT-coding region of a wild-type HXB2-D EcoRI-NdeI restriction enzyme fragment cloned into the expression vector pKK233-2 (15), with transformation into Escherichia coli strain XL1-Blue. Using the HIV-1 drug susceptibility assay, phenotypic susceptibilities of these viable recombinant viruses to d4T and AZT were determined as described previously (5). Briefly, the proviral molecular clone pHIVΔRTBstEII (7) was cotransfected with the RT-PCR product into MT-4 cells (3, 5). Levels of fold resistance were calculated by dividing the mean 50% inhibitory concentration (IC₅₀) of drug for a recombinant virus from a mutant clone by the mean IC₅₀ for recombinant wild-type virus (HXB2). Expression and purification of RT have been described elsewhere (16a, 23). The final RT product, in a heterodimeric form (p66/p51), had a purity of >90% by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified RTs were assayed for the effect of d4T-TP with the same RT assay procedure as previously described for our study of AZT-TP inhibition (16a). Separate kit components were obtained from Cavidi Tech, Uppsala, Sweden, and the principle, performance, and composition of this non-radioactive microtiter plate RT assay have been described previously (2, 21). In brief, the prototype used in this study consisted of microtiter plates coated with poly(A), RT reaction buffer with oligo(dT)₂₂ set to 40 pM (64 pg/well) and MgCl₂ set to 10 mM as final concentrations, and bromodeoxyuridine triphosphate (BrdUTP) was used as deoxynucleoside triphosphate (dNTP). The RT assay also involved the addition of 3.2 mM ATP (Pharmacia Biotech). The RT reaction was started by addition of purified RT (mutant or wild-type enzyme) at 20 to 60 pM, 1 to 4 mU/well. The K_m values were determined using six different concentrations of BrdUTP from 30 nM to 16 μM. Within this substrate range the enzyme reaction was linear for the RT assay step (3 h) and, thereby, steady-state kinetics were assumed. The K_i values were determined for d4T-TP using three different concentrations (ranging from 1.5 to 50 nM) of the nucleotide, adjusted optimally for the expected K_i value of each mutant. Competitive inhibition can be assumed since the V_max values did not change with these different d4T-TP concentrations (data not shown). The K_m and K_i values were obtained by fitting the data to the Michaelis-Menten competitive inhibition equation using the Grafit 4.10 program (Erithacus Software).

Table 1 shows the results of HIV-1 drug susceptibility assays (virus assay) as fold increase in IC₅₀ with d4T and AZT. In a previous study, the V75T mutation was shown to confer a 7-fold increase in d4T resistance (10), but in our study only a 2.7-fold increase in resistance to d4T was seen. This discrepancy might be related to the fact that different virus drug susceptibility assay systems were used. Similar low levels of d4T resistance were also found with the multidrug-resistant Q15IM mutant. However, this mutant was only tested alone and not together with its cluster of associated mutations. As suspected, the RTs with codon 69 insertions, in an AZT resistance background, showed the highest level of resistance to d4T along with a broad spectrum of multinucleoside resistance (data not shown). The highest level (17.1-fold increase in IC₅₀) of d4T resistance was seen with the M41L, T69S-AG, L210W, R211K, L214F, T215Y mutant (designated 41/69S-AG/210/211/214/215).

TABLE 1.

Resistance of RT mutants and wild type, measured with the d4T-TP or AZT-TP inhibition assay with or without 3.2 mM ATP and by HIV-1 drug susceptibility assay^a

Mutation(s)	D4T									AZT
	RT assay without ATP				RT assay with 3.2 mM ATP				Virus assay (fold resist.)	RT assay without ATP (fold resist.)	RT assay with 3.2 mM ATP (fold resist.)	Virus assay (fold resist.)
	Ki (nM)	Km (nM)	Ki/Km	Fold resist.	Ki (nM)	Km (nM)	Ki/Km	Fold resist.
HXB2	1.0	140	0.0071	1.0 (0.1)	1.6	160	0.01	1.0 (0.1)	1.0	1.0 (0.1)	1.0 (0.1)	1.0
M41L, D67N, K70R, T215Y	0.4	110	0.0036	0.5 (0.2)	3.0	130	0.023	2.3 (0.2)	4.1 (1.3)	0.7 (0.1)	10.3 (1.2)	24.1 (7.7)
M41L, D67N, K70R, M184V, T215Y	0.7	110	0.0064	0.9 (0.3)	5.1	150	0.034	3.4 (0.4)	3.7 (1.0)	0.9 (0.5)	4.3 (0.5)	6.4 (1.5)
M41L, D67N, K70R, M184V, L210W,	1.5	220	0.0068	1.0 (0.3)	6.9	250	0.028	2.8 (0.1)	4.0 (1.3)	0.7 (0.1)	5.5 (1.9)	12.0 (2.6)
R211K, L214F, T215Y, T69S-AG	1.9	130	0.015	2.1 (0.4)	6.2	120	0.051	5.1 (0.2)	6.9 (2.7)	1.3 (0.2)	10.5 (2.1)	7.0 (2.0)
M41L, T69S-AG, L210W, R211K, L214F, T215Y	1.3	130	0.01	1.4 (0.3)	27.0	230	0.117	11.7 (1.4)	17.1 (0.6)	0.9 (0.0)	15.8 (4.0)	>36
T69S-SG	3.7	240	0.015	2.1 (0.2)	6.8	230	0.03	3.0 (0.6)	4.4 (1.1)	1.4 (0.4)	6.7 (1.7)	8.1 (3.1)
M41L, T69S-SG, L210W, R211K, L214F, T215Y	0.9	100	0.009	1.3 (0.3)	15.2	190	0.08	8.0 (0.5)	8.3 (1.8)	ND	ND	>36
V75T	2.6	90	0.029	4.1 (0.6)	6.6	160	0.041	4.1 (0.6)	2.7 (0.4)	2.8 (0.3)	2.3 (0.2)	1.4 (0.3)
Q151M	3.0	120	0.025	3.5 (0.9)	7.1	180	0.039	3.9 (0.2)	3.0 (0.8)	4.0 (0.2)	3.8 (0.4)	2.1 (0.4)

^aFold resistance data for AZT and AZT-TP are from reference 16a. The RT d4T-TP and AZT-TP assay fold resistance (fold resist.) values were calculated as the increase of the K_i (d4T-TP or AZT-TP)/K_m (BrdUTP) ratio compared to the wild-type (HXB2) corresponding ratio. The virus d4T and AZT assay fold resistance values are based on IC₅₀ relative to wild-type HXB2 virus control. The mean IC₅₀s of d4T and AZT for the wild-type control virus were 1.2 and 0.012 μM, respectively. All values are the averages from at least two separate experiments. Standard errors are indicated in parentheses. ND, not determined. 

We knew from our previous study that the addition of ATP was the most effective for demonstrating AZT resistance at the enzyme level in our assay system (Table 1) (16a). Therefore, we included ATP at physiological concentration to study d4T resistance in the RT assay. Kinetic data for purified wild-type RT and nine mutants are shown in Table 1. The level of resistance was designated as the K_i/K_m ratio of the mutant divided by that of the wild type. The 41/69S-AG/210/211/214/215 mutant displayed the highest level of resistance (11.7-fold) when ATP was added to the RT assay, but in the absence of ATP only a 1.4-fold increase was observed. As shown in Table 1, in the absence of ATP all codon 69 insertion mutants behaved more as wild type (1.3- to 2.1-fold resistance), whereas a better correlation with the virus inhibition assay data was seen when ATP was included in the RT assays. An ATP-mediated effect was also observed for the mutants with specific AZT resistance mutations. In contrast, the codon 75 and 151 mutants gave a similar fourfold level of d4T resistance, both with and without added ATP, which also matched the virus inhibition data.

The ATP-dependent d4T resistance effect was most apparent for the codon 69 insertion mutants in an AZT resistance background. The effect of these 69-insertion mutations seems to overlap with the mechanism of resistance to AZT-TP. It is of interest that the corresponding ATP-dependent d4T resistance effect was also observed for mutants with AZT-specific resistance mutations at codons 41, 67, 70, and 215. However, the magnitude of resistance observed for d4T was much lower than that for AZT. A recent study has reported that the ATP-dependent removal of blocked d4T-MP is elevated to the same level as the removal of AZT-MP for RT containing the combination of D67N, K70R, T215F, and K219Q (20). On the other hand, the lower level of resistance to d4T is explained by the fact that the removal of d4T-MP is more sensitive to inhibition by levels of dNTP complementary to the next nucleotide position on the template (20). Nevertheless, the degree of cross-resistance to AZT is consistent with previous reports of a resistance pathway for thymidine analogue genotypic resistance (17, 18). The Q151M and V75T mutations seem to be associated with decreased binding to d4T-TP independently of ATP. The same trend was also seen in the RT AZT-TP assay, which suggests that these mutations discriminate binding to nucleoside analogs from natural dNTP substrate and not by the enhanced ATP-dependent mechanism analogous to pyrophosphorolysis.

It is anticipated that studies of this nature to elucidate biochemical mechanisms of NRTI resistance will lead to a better understanding of the interactions between the RT and inhibitors and provide valuable information for the future design of novel RT inhibitors.