Introduction
Antiviral agents preventing HIV-1 entry offer promising new prospects for therapy because they are active against viruses resistant to protease and reverse transcriptase (RT) inhibitors [1,2]. T-20 (Enfuvirtide, Fuzeon) is the first HIV-1 entry inhibitor approved by the Food and Drugs Administration for use in humans. Addition of T-20 to hightly active antiretroviral therapy (HAART) resulted in a significant reduction of viral load and immunological benefit [3,4]. T-20 is a 36-amino acid peptide based on the HR2 region of the HIV-1 type B gp41 envelope glycoprotein [5,6]. HIV-1 entry into target cells involves the formation of a trimer of antiparallel dimers of HR1 and HR2. This six-helix bundle induces a hairpin structure bringing the viral and cellular membranes into close proximity for fusion [7,8]. Fusion inhibitors block transition from the prehairpin intermediate to the fusion-active six-helix hairpin structure by competitive binding of HR1 and preventing normal intramolecular interactions between HR1 and HR2. Resistance to T-20 is associated with changes in the HR1 region, particularly in or near the GIV motif [9-11].
T-1249 is a second-generation fusion inhibitor with greater antiviral potency than T-20 [12,13]. It corresponds to an overlapping sequence within HR2 but targets a distinct region within HR1 and remains active against most T-20-resistant HIV-1 isolates [12]. The non-overlapping residues of T-20 and T-1249 encompass three highly conserved hydrophobic residues predicted to project into the deep hydrophobic grooves of the HR1 trimer which are highly important for HR2 binding [14]. Notably, T-1249 decreases the viral load in patients harboring T-20-resistant viruses [15].
Although T-1249 seems to be more effective than T-20, its clinical development is currently on hold due to formulation problems. Nonetheless, modified more stable and potent forms of T-1249 or related peptides that require fewer injections and are more appropriate for chronic administration might become the next generation of HIV-1 fusion inhibitors. Therefore, further evaluation of HIV-1 resistance to both T-20 and T-1249 remains of great interest. Several studies have investigated the variability of the HR1 region in untreated HIV-1-infected patients to evaluate the frequency of primary resistance to fusion inhibitors [16-20]. The results demonstrated that the HR1 region is usually highly conserved. However, some variations near the GIV motif and in the hydrophobic pocket were identified, particularly in non-B HIV-1 strains [16-20]. We examined whether these naturally occurring sequence variations affect HIV-1 infectivity and susceptibility to inhibition by T-20 and T-1249.
Methods
Generation of HIV-1 gp41 mutants
Splice-overlap extension PCR was used to introduce point mutations in the gp41 HR1 region of the HIV-1 NL4-3 molecular clone (Fig. 1a). PCR fragments were gel purified and cloned into NL4-3 by using the NheI and BamHI restriction sites flanking the HR1 region. Sequence analysis confirmed the presence of the mutations and the absence of undesired changes.
Cells, virus stocks, and infectivity assays
Virus stocks were generated by transient transfection of 293T cells and p24 antigen levels were quantified as described [21]. TZM-bl cells were kindly provided by Drs. Kappes and Wu through the NIH AIDS Reagent Program and were kept in Dulbecco's Modified Eagle's medium supplemented with 10% foetal calf serum. TZM-bl cells express large amounts of CD4, CCR5 and CXCR4 and contain the β-galactosidase gene under the control of the HIV-1 promoter [10,22]. Cells were seeded in flat-bottomed 96-well dishes and cultured overnight. Infections were performed in the absence of inhibitor (control) or in the presence of 20, 100 and 500 nM T-20 or 1, 10 and 100 nM T-1249, respectively, in triplicate with virus containing 1 ng of p24. Infectivity was measured in a luminometer at 2 days post-infection using the β-Gal screen Kit from TOPIX (Bedford, Massachusetts, USA) as recommended by the manufacturer. Virus infectivity was calculated by dividing the β-Gal activity (relative light units per second; RLU/s) produced at each concentration of inhibitor by the RLU measured in the absence of inhibitor. The mean 50% inhibitory concentrations (IC50) were calculated as described previously [22] and compared by using Student's t test to determine whether the observed differences were statistically significant.
Results
We generated mutants of HIV-1 NL4-3 containing changes of L33V, L34M, S35F, Q39R, L54M, Q56K and Q56R in the gp41 HR1 region (Fig. 1a). These variations have been observed in some treatment-naive patients infected with diverse HIV-1 group M subtypes [16-20] but it remained elusive whether they affect HIV-1 sensitivity to fusion inhibitors. We also constructed HIV-1 variants containing combined mutations to assess whether they might affect drug sensitivity more severely than individual changes. To generate a control HIV-1 variant insensitive to fusion inhibitors we introduced the L33S mutation into gp41. This mutation confers resistance to C-34 [23], which is highly related to T-1249 and also targets the hydrophobic cavity.
Infection of TZM-bl indicator cells demonstrated that most variations in the HR1 region do not impair viral infectivity (Fig. 1b). Exceptions were S35F and Q56R which reduced infectivity of HIV-1 NL4-3 about fivefold. The disruptive effect of Q56R was unexpected because an arginine is present at this position in the majority of HIV-1 group O gp41 sequences (Table 1). However, the Q56R substitution did not impair HIV-1 infectivity in the presence of an additional L54M change (Fig. 1b). Thus, its effect on gp41 function is context-dependent and it might not impair the fitness of group O strains.
Next, we analysed the HR1 variants for sensitivity to T-20 and T-1249. Several changes altered HIV-1 susceptibility to inhibition by T-20 (Fig. 1c). In contrast, all mutant viruses, except the C-34 resistant L33S variant [23], were efficiently blocked by T-1249 (Table 1). For T-20, the mean IC50 obtained for wild type NL4-3 was 96.7 nM. This value is higher than those documented in the literature [9-11]. However, since T-20 binds directly to the HR1 region to inhibit fusion the IC50 correlates directly with the virus inoculum (data not shown). Thus, these variations might result from variable experimental conditions, particularly utilization of different doses of virus for infection. Concordant with previous studies [12,13], T-1249 was more effective than T-20 in inhibiting HIV-1 infection (Table 1).
Among the variations analyzed, L33V reduced HIV-1 sensitivity to T-20 most severely (fivefold; P = 0.0007; Table 1). Nonetheless, the effect was less dramatic than that of the L33S mutation (Fig. 1c). Furthermore, only L33S but not L33V affected sensitivity of HIV-1 to T-1249 inhibition. The remaining individual changes reduced viral susceptibility to T-20 inhibition only about twofold (L34M, L54M), had no significant effect (Q56K), or even slightly enhanced sensitivity (S35F, Q39R, Q56R). In contrast, combined mutations of L54M/Q56K and L34M/L54M/Q56R reduced the susceptibility of HIV-1 to inhibition by T-20 about four- to fivefold (Fig. 1c, Table 1). Altogether, the effects of the naturally occurring sequence variations on the susceptibility of HIV-1 to T-20 were subtle but highly reproducible. In contrast, none of these gp41 HR1 changes had marked effects on T-1249-mediated inhibition (Fig. 1c).
Discussion
We analysed the effect of sequence variations in the gp41 HR1 region previously observed in treatment-naive patients infected with different subtypes of HIV-1 group M [16-20] on sensitivity to entry inhibitors. Our data show that some of these mutations reduce the sensitivity of HIV-1 to inhibition by T-20. Extending a previous study on C-34 resistance [23], we also demonstrate that the L33S change confers cross-resistance to T-20 and T-1249 (Table 1). It will be of interest to further clarify whether the L33S change mediates resistance to most or all HR2-derived peptidic inhibitors. Concordant with the finding that position 33 in gp41 is critical for inhibition, the naturally occurring L33V change also conferred resistance to T-20, although it did not allow to overcome T-1249 inhibition. The impact of these variations on the clinical response in patients treated with fusion inhibitors needs further evaluation. Notably, some HIV-1-infected individuals on HAART show increasing CD4 cell counts in the presence of virus variants resistant against protease or RT inhibitors and ongoing viral replication [24]. Some natural variations in the HR1 region reducing the sensitivity of HIV-1 to fusion inhibitors might also affect viral fitness and still allow immune recovery.
T-20 was generated based on the HIV-1Lai subtype B gp41 sequence [6] raising the question whether some subtypes might be less susceptible to inhibition by fusion inhibitors. For example, the L33V change, which conferred about fivefold resistance to T-20 is frequently observed in subtype D isolates (Table 1). Such a reduction in drug sensitivity might affect the efficiency of viral suppression and hence the response to treatment. Accordingly, some subtype D strains might be partially resistant to T-20. It needs to be considered, however, that the effect of these HR1 variations on drug sensitivity might depend on the specific envelope backbone. For example, substitution of L54M slightly reduced susceptibility to T-20 inhibition (Table 1) and has been detected in HIV-1-infected individuals who did not respond to T-20 treatment [25,26]. This change is present in the majority of subtype C strains. However, a recent study proposed that subtype C might be even more susceptible to T-20 than subtype B [27]. Both need to be tested under the same experimental conditions to evaluate possible differences in susceptibility to fusion inhibitors.
Notably, changes in the hydrophobic pocket altered the sensitivity of HIV-1 to T-20 although this inhibitor does not bind directly to this region (Fig. 1a). L54 is at a 'g' position in the coiled coil structure of HR1 and might affect the interaction with the antiparallel HR2 helix [7,8]. Similarly, changes at Q56 in the hydrophobic pocket could impact the interaction of the HR1 and HR2 domains and the formation of the fusogenic six-helix bundle [14]. Structural changes in the trimeric gp41 complex induced by the L54M and Q56K changes might alter the accessibility of the HR1 region to T-20, thereby reducing sensitivity to inhibition.
Our data were obtained using the CXCR4-tropic HIV-1 NL4-3 clone. The coreceptor tropism might impact the susceptibility of HIV-1 to fusion inhibitors. Therefore, we also introduced the changes in the gp41 HR1 region in a CCR5-tropic derivative of NL4-3 containing the 005pf135 V3 loop [28]. Some polymorphisms in the gp41 HR1 region also affected the sensitivity of the CCR5-tropic HIV-1 variants to T-20. However, as previously suggested [22], the CCR5-tropic derivatives were less sensitive to inhibition by T-20 and T-1249 than their CXCR4-tropic counterparts. Thus, both sequence variations in the gp41 HR1 region and the coreceptor tropism can modulate the sensitivity of HIV-1 to fusion inhibitors.
In conclusion, our study shows that some natural variations detected in the HIV-1 gp41 region of treatment-naive patients affect the sensitivity to inhibition by T-20. However, the mean IC50 increased only up to fivefold and most HIV-1 variants analysed remained fully susceptible to T-20 inhibition. Considering previous studies demonstrating that the HR1 region is usually highly conserved [16-20] our results further confirm that primary resistance to T-20 and T-1249 is rare although the impact of some subtype-specific variations on the susceptibility of HIV-1 to fusion inhibitors needs further evaluation.
Acknowledgements
We thank Thomas Mertens for support Nicola Bailer and Daniela Krnavek for excellent technical assistance and Ingrid Bennett for critical reading of the manuscript.
Sponsorship: Supported by grants from the Deutsche Forschungsgemeinschaft (DFG) and the Landesstiftung Baden-Württemberg.
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