Synthesis, Molecular Docking and Molecular Dynamics Simulation of 2- Thioxothiazolidin-4-One Derivatives against Gp41.

INTRODUCTION: Gp41 and its conserved hydrophobic groove on the N-terminal heptad repeat region are attractive targets in the design of HIV-1 entry inhibitors. Linearly extended molecules have shown potent anti-HIV-1 activity for their effective interactions with the gp41 binding pocket. Rhodanine ring attached to substituted pyrrole or furan rings has been proved a preferred moiety to be inserted inside the molecular structure of the gp41 inhibitors. OBJECTIVES: Based on the previous findings we are going to describe some rhodanine derivatives in which a substituted imidazole ring is introduced in place of the pyrrole or furan rings. The compounds' flexibility is increased by inserting methylene groups inside the main scaffold. METHODS: Molecular docking and molecular dynamics simulations approaches were exploited to investigate the chemical interactions and the stability of the designed ligands-gp41 complex. All compounds were synthesized and their chemical structures were elucidated by 1HNMR, 13CNMR, FTIR and Mass spectroscopy. Biological activities of the compounds against HIV-1 and HIV-2 and their cellular toxicities against the T-lymphocyte (MT-4) cell line were determined. RESULTS: All the designed compounds showed proper and stable chemical interactions with gp41 according to the in silico studies. The results of the biological tests proved none of the compounds active against HIV-1 replication in cell cultures. CONCLUSION: Since all the studied compounds were potently toxic for the host cell; it was therefore not possible to assess their anti-HIV activities.

Probing Vulnerability of the gp41 C-Terminal Heptad Repeat as Target for Miniprotein HIV Inhibitors.

One of the therapeutic strategies in HIV neutralization is blocking membrane fusion. In this process, tight interaction between the N-terminal and C-terminal heptad-repeat (NHR and CHR) regions of gp41 is essential to promote membranes apposition and merging. We have previously developed single-chain proteins (named covNHR) that accurately mimic the complete gp41 NHR region in its trimeric conformation. They tightly bind CHR-derived peptides and show a potent and broad HIV inhibitory activity in vitro. However, the extremely high binding affinity (sub-picomolar) is not in consonance with their inhibitory activity (nanomolar), likely due to partial or temporal accessibility of their target in the virus. Here, we have designed and characterized two single-chain covNHR miniproteins each encompassing one of the two halves of the NHR region and containing two of the four sub-pockets of the NHR crevice. The two miniproteins fold as trimeric helical bundles as expected but while the C-terminal covNHR (covNHR-C) miniprotein is highly stable, the N-terminal counterpart (covNHR-N) shows only marginal stability that could be improved by engineering an internal disulfide bond. Both miniproteins bind their respective complementary CHR peptides with moderate (micromolar) affinity. Moreover, the covNHR-N miniproteins can access their target in the context of trimeric native envelope proteins and show significant inhibitory activity for several HIV pseudoviruses. In contrast, covNHR-C cannot bind its target sequence and neither inhibits HIV, indicating a higher vulnerability of C-terminal part of CHR. These results may guide the development of novel HIV inhibitors targeting the gp41 CHR region.

Conserved Residue Asn-145 in the C-Terminal Heptad Repeat Region of HIV-1 gp41 is Critical for Viral Fusion and Regulates the Antiviral Activity of Fusion Inhibitors.

Entry of HIV-1 into target cells is mediated by its envelope (Env) glycoprotein composed of the receptor binding subunit gp120 and the fusion protein gp41. Refolding of the gp41 N- and C-terminal heptad repeats (NHR and CHR) into a six-helix bundle (6-HB) conformation drives the viral and cellular membranes in close apposition and generates huge amounts of energy to overcome the kinetic barrier leading to membrane fusion. In this study, we focused on characterizing the structural and functional properties of a single Asn-145 residue, which locates at the middle CHR site of gp41 and is extremely conserved among all the HIV-1, HIV-2, and simian immunodeficiency virus (SIV) isolates. By mutational analysis, we found that Asn-145 plays critical roles for Env-mediated cell-cell fusion and HIV-1 entry. As determined by circular dichroism (CD) spectroscopy and isothermal titration calorimetry (ITC), the substitution of Asn-145 with alanine (N145A) severely impaired the interactions between the NHR and CHR helices. Asn-145 was also verified to be important for the antiviral activity of CHR-derived peptide fusion inhibitors and served as a turn-point for the inhibitory potency. Intriguingly, Asn-145 could regulate the functionality of the M-T hook structure at the N-terminus of the inhibitors and displayed comparable activities with the C-terminal IDL anchor. Crystallographic studies further demonstrated the importance of Asn-145-mediated interhelical and intrahelical interactions in the 6-HB structure. Combined, the present results have provided valuable information for the structure-function relationship of HIV-1 gp41 and the structure-activity relationship of gp41-dependent fusion inhibitors.

Investigation of the inhibition effect of arachidonic acid on the core structure of the HIV-1 gp41.

The gp41 transmembrane domain of the envelope glycoprotein of the human immunodeficiency virus (HIV) modulates the conformation of the viral envelope spike. During the HIV fusion process, C-terminal heptad repeat (CHR, C34) wrap antiparallel to the N-terminal heptad repeat (NHR, N36) helices to form a stable six-helix bundle (6-HB) core structure, which brings the viral and cell membranes into close proximity for fusion. Therefore, inhibiting the formation of 6-HB is considered to be a key activity of an effective HIV-1 fusion inhibitor. The level of arachidonic acid (AA) is increased in HIV infected patients. Our study provides a new insight into the functional role of AA during the formation of HIV-1 gp41 6-HB. Native polyacrylamide gel electrophoresis (N-PAGE), enzyme-linked-immunosorbent serologic assay (ELISA) and circular dichroism (CD) spectroscopy were used to investigate the inhibition of AA for the formation of 6-HB. Molecular docking technique was adopted to explore the underlying mechanism. HIV-1 JR-FL (R5 strain) Envelope was adopted to determine the inhibition effect of AA. AA is shown to interfere with the formation of α-helical complexes of N36 and C34 by N-PAGE, ELISA and CD spectroscopy. The isotherm titration microcalorimetry (ITC) results indicate there is a single class of binding site on N36. ΔH and ΔS are -12.43 kJ mol-1 and 70.07 J mol-1 K-1, respectively, indicating hydrophobic interaction and electrostatic forces are the main acting forces. The molecular docking results manifest that AA interacts with the hydrophobic residues (Trp-571, Leu-568, Val-570 and Leu-576) and ionic interactions occur between Arg-579 and the -COOH of AA. The inhibitory activity of AA on HIV-1 JR-FL is quantified by 50% effective concentration (EC50) and 90% effective concentration (EC90), which are 31.42 ± 1.08 and 133.47 ± 18.10 µg mL-1, respectively. All the results indicate that AA is able to inhibit the formation of 6-HB but cannot disrupt the preformed 6-HB. Therefore, AA is a potential inhibitor for the viral fusion/entry.

Alpha-1-antitrypsin interacts with gp41 to block HIV-1 entry into CD4+ T lymphocytes.

BACKGROUND: Study of a clinic case reveals that alpha-1-antitrypsin (AAT) deficiency is related to CD4+ T cell count decline and AIDS progression, suggesting that AAT might be an endogenous inhibitor of HIV/AIDS. Previous study shows that AAT inhibits HIV-1 replication in infected host cells and the C-terminus fragment of AAT, VIRIP, interferes with HIV-1 infection. However, it is still unclear whether and how intact AAT inhibits HIV-1 infection. It is also unknown what the mechanism of AAT is and which critical step(s) are involved. RESULTS: In the present study, the C-terminus of AAT (C) was synthesized. C terminus-truncated AAT (ΔAAT) was also prepared by digesting AAT with metalloproteinase. Primary CD4+ T cells were then co-cultured with HIV-1 with the presence or absence of AAT/C/ΔAAT to detect cis-infection of HIV-1. The interaction between AAT/C/ΔAAT and gp120/gp41 was also measured. Meanwhile, HIV-1 reverse transcriptase activity and viral DNA integration were also detected in these lymphocytes. The results demonstrated that AAT and C, not ΔAAT, inhibited HIV-1 entry by directly interacting with gp41. Meanwhile, AAT, C and ΔAAT could not directly interfere with the steps of viral RNA reverse transcription and viral DNA integration. CONCLUSION: AAT inhibits HIV-1 entry by directly interacting with gp41 through its C-terminus and thereby inhibits HIV-1 infection.

Design, synthesis and activity evaluation of novel peptide fusion inhibitors targeting HIV-1 gp41.

Human immunodeficiency virus type 1 (HIV-1), the pathogen of acquired immunodeficiency syndrome (AIDS), causes about 2 million people to death every year. Fusion inhibitors targeted the envelope protein (gp41) represent a novel and alternative approach for anti-AIDS therapy, which terminates the HIV-1 life cycle at an early stage. Using CP621-652 as a template, a series of peptides were designed, synthesized and evaluated in vitro assays. An interesting phenomenon was found that the substitution of hydrophobic residues at solvent accessible sites could increase the anti-HIV activity when the C-terminal sequence was extended with an enough numbers of amino acids. After the active peptides was synthesized and evaluated, peptide 8 showed the best anti-HIV-1 IIIB whole cell activity (MAGI IC50=53.02 nM). Further study indicated that peptide 8 bound with the gp41 NHR helix, and then blocked the conformation of 6-helix, thus inhibited virus-cell membrane fusion. The results would be helpful for the design of peptide fusion inhibitors against HIV-1 infection.

Discovery of small molecule fusion inhibitors targeting HIV-1 gp41.

Gp41 is regarded as an attractive target for development of HIV-1 entry inhibitors since it mediates the fusion process of HIV- 1 entry into the target cell through the six-helix bundle (6-HB) formation between its N-heptad repeat (NHR) and C-heptad repeat (CHR). Any chemical entity that disrupts the six-helix bundle formation may inhibit the fusion process, thereby blocking HIV-1 entry into the target cells. A brief review of discovering small molecule inhibitors targeting gp41 is presented here, including the development of assay methods, current known small molecule inhibitors and their binding mode studies. Lessons learned and challenges remained in view of blocking protein-protein interaction between NHR and CHR are also discussed.

Development of resistance to VIR-353 with cross-resistance to the natural HIV-1 entry virus inhibitory peptide (VIRIP).

OBJECTIVE: Virus-inhibitory peptide (VIRIP) has been identified as a component of human hemofiltrate that blocks HIV-1 gp41-dependent fusion by interacting with the fusion peptide. A VIRIP analogue (VIR-576) has been shown to be effective in a phase I/II clinical trial. We have evaluated the activity and mechanism of HIV-1 resistance to VIRIP and its analogue, VIR-353. METHODS: Anti-HIV activity and passage of HIV-1 strains in cell culture were used to generate and identify mutations that confer resistance to VIRIP and VIR-353. Recombinant viruses harboring the most relevant mutations were generated and characterized. RESULTS: VIRIP and VIR-353 showed anti-HIV-1 activity with EC(50) of 28 and 0.3 µmol/l, respectively, and were active against virus resistant to BMS-155, AMD3100, T20, TAK-779 or nevirapine. Time of addition experiments showed that VIR-353 targets a time/site of action corresponding to gp41-dependent fusion. VIR-353-resistant virus was generated after 450 days in cell culture, suggesting a high genetic barrier for resistance. The VIR-353-resistant virus was cross-resistant to VIRIP but remained sensitive to T20, AMD3100 or zidovudine. Recombination of gp41 into a wild-type backbone partially recovered the resistant phenotype, but both gp120 and gp41 from the resistant virus were necessary to restore resistance to VIRIP or VIR-353. Site-directed mutagenesis confirmed the role of specific mutations and identified a combination of three mutations (A433T/V489I/V570I) as the most relevant to VIRIP resistance. CONCLUSION: VIRIP may interact with a region of gp41 that is essential for fusion but not the fusion peptide. Our results highlight interactions between gp41 and gp120 that may be required during the fusion process.

HIV-1 gp41 fusion intermediate: a target for HIV therapeutics.

Human immunodeficiency virus (HIV)-1 infection is initiated by the binding of gp120 envelope glyco-protein to its cell receptor (CD4) and a coreceptor (CXCR4 or CCR5), followed by a series of conformational changes in the gp41 transmembrane subunit. These changes include insertion of fusion peptide into the target cell membrane and association of C-heptad repeat (CHR) peptide with the N-heptad repeat (NHR) trimer, a pre-hairpin fusion intermediate. A stable six-helix bundle core is then formed, bringing the viral envelope and target cell membrane into close proximity for fusion. Peptides derived from the CHR region, such as T20 and C34, inhibit HIV-1 fusion by interacting with the gp41 fusion intermediate. A number of anti-HIV-1 peptides and small molecule compounds targeting the gp41 NHR-trimer have been identified. By combining HIV fusion/entry inhibitors targeting different sites in the gp41 fusion intermediate, a potent synergistic effect takes place, resulting in a potential new therapeutic strategy for the HIV infection/AIDS. Here, we present an overview of the current development of anti-HIV drugs, particularly those targeting the gp41 fusion intermediate.

An albumin-conjugated peptide exhibits potent anti-HIV activity and long in vivo half-life.

The clinical application of conventional peptide drugs often is limited by their short in vivo half-life and potential immunogenicity. Frequent injection presents challenges to the treatment of chronic diseases, such as HIV infection. We chemically modified a peptide HIV fusion inhibitor with 3-maleimidopropionic acid (MPA), which allows rapid and irreversible conjugation with serum albumin at a 1:1 molar ratio. FB006M, with an MPA modification at the 13th amino acid, rapidly formed conjugate with albumin upon intravenous injection, and it exhibited a remarkably extended in vivo half-life. The albumin conjugate of FB006M displayed potent inhibitory activity against a number of laboratory and clinical isolates of HIV-1 in vitro and in vivo. No immunogenicity or antibody formation was detected after repeated dosing. The clinical application of FB006M may decrease the cost of treatment and improve treatment compliance and patient quality of life.

[Inhibition of HIV-1 mediated cell-cell fusion by saponin fraction from Psidium guajava leaf].

OBJECTIVE: To investigate the effects of the total saponin of Psidium guajava leaf (TSGL) on HIV-1 envelop proteins (env) mediated virus entry into target cells. METHODS: The TSGL was purified and concentrated using SA-1 macropore resin. The effect of TSGL on HIV-1 entry into target cells was tested using a cell-cell fusion assay by mixing CHO-WT and MT-2 cells. The cytotoxicity of TSGL was measured by MTT assay. The activity of TSGL on blocking the HIV-1 gp41 six helical bundle (6-HB) formation was analyzed by ELISA and Native-PAGE (N-PAGE). RESULTS: The TSGL could inhibit HIV env mediated cell-cell fusion with an IC50 of (7.33 +/- 0.40) microg/mL, and displayed little cytotoxicity at that concentration. ELISA assay showed that the TSGL could prevent gp41 6-HB formation with inhibitory activity of 95.93% at 25 microg/mL. N-PAGE study confirmed the inhibitory effect of TSGL on gp41 6-HB formation. CONCLUSIONS: The TSGL can inhibit HIV entry target cells by interfering the envelop subunit gp41 form the critical 6-HB structure.

Asymmetric deactivation of HIV-1 gp41 following fusion inhibitor binding.

Both equilibrium and nonequilibrium factors influence the efficacy of pharmaceutical agents that target intermediate states of biochemical reactions. We explored the intermediate state inhibition of gp41, part of the HIV-1 envelope glycoprotein complex (Env) that promotes viral entry through membrane fusion. This process involves a series of gp41 conformational changes coordinated by Env interactions with cellular CD4 and a chemokine receptor. In a kinetic window between CD4 binding and membrane fusion, the N- and C-terminal regions of the gp41 ectodomain become transiently susceptible to inhibitors that disrupt Env structural transitions. In this study, we sought to identify kinetic parameters that influence the antiviral potency of two such gp41 inhibitors, C37 and 5-Helix. Employing a series of C37 and 5-Helix variants, we investigated the physical properties of gp41 inhibition, including the ability of inhibitor-bound gp41 to recover its fusion activity once inhibitor was removed from solution. Our results indicated that antiviral activity critically depended upon irreversible deactivation of inhibitor-bound gp41. For C37, which targets the N-terminal region of the gp41 ectodomain, deactivation was a slow process that depended on chemokine receptor binding to Env. For 5-Helix, which targets the C-terminal region of the gp41 ectodomain, deactivation occurred rapidly following inhibitor binding and was independent of chemokine receptor levels. Due to this kinetic disparity, C37 inhibition was largely reversible, while 5-Helix inhibition was functionally irreversible. The fundamental difference in deactivation mechanism points to an unappreciated asymmetry in gp41 following inhibitor binding and impacts the development of improved fusion inhibitors and HIV-1 vaccines. The results also demonstrate how the activities of intermediate state inhibitors critically depend upon the final disposition of inhibitor-bound states.

[Isolation and characterization of the anti-HIV active component from Eucommia ulmoides].

OBJECTIVE: To investigate the anti-HIV effects of Eucommia ulmoides Oliver, so as to provide experimental basis for searching a new efficacious drug for treatment of AIDS. METHODS: Using phytochemistry to isolate compounds from Eucommia ulmoides Oliver, the inhibitory activity of Samples on the HIV gp41 six-helix bundle formation was determined by a modified sandwich ELISA and PAGE. RESULTS: The Samples from Eucommia ulmoides Oliver had potent inhibitory activity on the HIV gp41 six-helix bundle formation. CONCLUSION: Eucommia uloides Oliver can inhibit HIV by targeting HIV gp41.

The application of perfluorooctanoate to investigate trimerization of the human immunodeficiency virus-1 gp41 ectodomain by electrophoresis.

The transmembrane glycoprotein gp41 of human immunodeficiency virus has been proposed to form trimer-of-hairpin during virus-cell membrane fusion. To investigate its oligomerization propensity under soluble and membrane-mimic conditions, sodium salt of perfluorooctanoate (PFO) was applied. A recombinant gp41 ectodomain devoid of disulfide linkage was overexpressed in Escherichia coli and characterized by MS and circular dichroism spectropolarimetry in PFO solution in comparison to SDS. The helical content of this ectodomain in PFO is higher than that in SDS. Notably, PFO employed in PAGE clearly conduced to the formation of trimer under the optimized condition as visualized in the gel. In addition, the proteins expressed from the two mutants in the heptad repeat (HR) domains of gp41, I62P, and N126K, were also examined by the PFO-PAGE analysis for functional ramification of molecular organization. Remarkably, the I62P mutation completely abolished the gp41 trimer formation, whereas the N126K mutation resulted in a more stable trimer. The data suggested that PFO-PAGE analysis is appropriate for evaluating the effect of mutations on the trimerization of gp41 and other fusion proteins which may be implicated in the alteration of their fusogenicity.

Increased polymorphism in the HR-1 gp41 env gene encoding the enfuvirtide (T-20) target in HIV-1 variants harboring multiple antiretroviral drug resistance mutations in the pol gene.

BACKGROUND: Sequence variations in HR-1 gp41 env gene region encoding the target for T-20 have previously been reported among patients naive to inhibitory fusion. OBJECTIVE: To evaluate whether a previous therapeutic history of patients could have an impact on a differential evolution of the gp41 polymorphism. METHODS: We assessed the genetic polymorphism within the critical HR-1 gp41 env gene region in HIV-1 variants from 108 T-20-naive patients (Groups I-III) and 12 patients receiving T-20 as part of a salvage regimen (Group IV). T-20-naive patients included 50 patients exhibiting variants harboring resistance mutations to NRTIs, NNRTIs, and PIs (Group I), 24 patients with variants harboring resistance mutations for NRTIs and/or NNRTIs (Group II), and 34 antiretroviral drug-naive patients (Group III). RESULTS: In T-20-naive patients whose HIV harbored resistance mutations to NRTIs, NNRTIs, and/or PIs, the mean number of synonymous mutations (ds) per patient was decreased and the mean number of nonsynonymous (da) mutations per patient was increased, resulting in a significant decrease in the mean Sigmads/Sigmada ratio as compared with antiretroviral drug-naive patients (Group III; 4.1 vs. 11.6; P < 0.0001). The mean number of polymorphic mutations in HR-1 gp41 per patient was two-fold higher in patients exhibiting antiretroviral drug resistance mutations (Groups I and II) than in antiretroviral drug-naive patients (Group III; 0.41 vs. 0.20; P < 0.05). CONCLUSION: Our observations indicate that the HR-1 gp41 T-20 target is subjected to high genetic variability, including intrinsic polymorphism and selection of T-20 resistance mutations under T-20 intake, that is increased by the presence of resistance mutations to NRTIs, NNRTIs, and/or PIs. Our data provide a basis for a potential impact of previous antiretroviral drug history on the therapeutic efficacy of T-20.

Rapid emergence of enfuvirtide resistance in HIV-1-infected patients: results of a clonal analysis.

OBJECTIVES: To study the dynamics of enfuvirtide (T-20) resistance development in HIV-1-infected subjects. PATIENTS AND METHODS: Clonal analysis of gp41 sequences was performed on serial samples obtained from HIV-1-infected subjects with early virologic failure of T-20-based regimens. RESULTS: Enfuvirtide resistance mutations at codons 36 to 45 in the first heptad repeat of gp41 emerged within 2 weeks in most subjects and were associated with the return of plasma HIV-1 RNA level toward baseline by weeks 4 to 8. Mutations at codons 36 (G36E, G36D, or G36S) and 38 (V38A, V38G, or V38M) were the most commonly detected resistance mutations at week 2. Mutations at codons 40 (Q40H) and 43 (N43D) were more prevalent at week 4 than at week 2 and seemed to emerge more slowly than mutations at codons 36 and 38. CONCLUSIONS: The rapid emergence of mutations associated with T-20 resistance in the absence of a fully suppressive antiretroviral regimen demonstrates a low genetic barrier to resistance and underscores the importance of combining T-20 with other active drugs when constructing regimens for highly treatment-experienced patients.

Theaflavin derivatives in black tea and catechin derivatives in green tea inhibit HIV-1 entry by targeting gp41.

Theaflavin derivatives and catechin derivatives are the major polyphenols in black tea and green tea, respectively. Several tea polyphenols, especially those with galloyl moiety, can inhibit HIV-1 replication with multiple mechanisms of action. Here we showed that the theaflavin derivatives had more potent anti-HIV-1 activity than catechin derivatives. These tea polyphenols could inhibit HIV-1 entry into target cells by blocking HIV-1 envelope glycoprotein-mediated membrane fusion. The fusion inhibitory activity of the tea polyphenols was correlated with their ability to block the formation of the gp41 six-helix bundle, a fusion-active core conformation. Computer-aided molecular docking analyses indicate that these tea polyphenols, theaflavin-3,3'-digallate (TF3) as an example, may bind to the highly conserved hydrophobic pocket on the surface of the central trimeric coiled coil formed by the N-terminal heptad repeats of gp41. These results indicate that tea, especially black tea, may be used as a source of anti-HIV agents and theaflavin derivatives may be applied as lead compounds for developing HIV-1 entry inhibitors targeting gp41.

Different from the HIV fusion inhibitor C34, the anti-HIV drug Fuzeon (T-20) inhibits HIV-1 entry by targeting multiple sites in gp41 and gp120.

Fuzeon (also known as T-20 or enfuvirtide), one of the C-peptides derived from the HIV-1 envelope glycoprotein transmembrane subunit gp41 C-terminal heptad repeat (CHR) region, is the first member of a new class of anti-HIV drugs known as HIV fusion inhibitors. It has been widely believed that T-20 shares the same mechanism of action with C34, another C-peptide. The C34 is known to compete with the CHR of gp41 to form a stable 6-helix bundle (6-HB) with the gp41 N-terminal heptad repeat (NHR) and prevent the formation of the fusogenic gp41 core between viral gp41 NHR and CHR, thereby inhibiting fusion between viral and target cell membranes. Here we present data to demonstrate that, contrary to this belief, T-20 cannot form stable 6-HB with N-peptides derived from the NHR region, nor can it inhibit the 6-HB formation of the fusogenic core. Instead, it may interact with N-peptides to form unstable or insoluble complexes. Our data suggest that T-20 has a different mechanism of action from C34. The interaction of T-20 with viral NHR region alone may not prevent the formation of the fusion active gp41 core. We also demonstrate that the T-20-mediated anti-HIV activity can be significantly abrogated by peptides derived from the membrane-spanning domain in gp41 and coreceptor binding site in gp120. These new findings imply that T-20 inhibits HIV-1 entry by targeting multiple sites in gp41 and gp120. Further elucidation of the mechanism of action of T-20 will provide new target(s) for development of novel HIV entry inhibitors.

Genotype and phenotype patterns of human immunodeficiency virus type 1 resistance to enfuvirtide during long-term treatment.

The human immunodeficiency virus type 1 (HIV-1) fusion inhibitor enfuvirtide has recently been introduced into clinical practice and has exhibited efficient anti-HIV-1 activity in combination with other antiretroviral agents. In the present study, we addressed the effect of long-term treatment with enfuvirtide on the intrahost evolution of HIV-1. The genotype and phenotype patterns and the relative replication capacity (rRC) of enfuvirtide-resistant HIV-1 mutants were evaluated in samples from 11 subjects (7 virological nonresponders and 4 responders) who received the compound for more than 1 year in combination with different regimens. Selection of one or more mutations clustering in a sequence (amino acids 36 to 45) of the gp41 N-terminal heptad repeat was observed in samples from the seven virological nonresponders but not in those from responders. In two subjects who discontinued enfuvirtide, reversion of the resistant genotype was detected within 3 months. Recombinant clones bearing mutated gp41 sequences displayed reduced susceptibilities to enfuvirtide, with the 50% inhibitory concentrations (IC(50)s) ranging from 0.6 to 12.8 microg/ml, whereas the IC(50) for isolates with baseline sequences was 0.013 +/- 0.010 microg/ml. Interestingly, long-term monitoring of resistant variants provided evidence that ongoing adaptation to the drug is paralleled by phenotypic changes. A limited drop in the rRC in the absence of drug was observed for clones from four of the seven nonresponders bearing mutations associated with resistance. Overall, the data indicate that the different genotype patterns associated with a detectable degree of HIV-1 resistance to enfuvirtide generated during long-term treatments are characterized by a substantially low genetic barrier, possible ongoing adaptation with increased degrees of resistance, and limited influence on the viral rRC.