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The Journal of Immunology, 2006, 176: 6900-6905.
Copyright © 2006 by The American Association of Immunologists

HIV-1 Adapts to a Retrocyclin with Cationic Amino Acid Substitutions That Reduce Fusion Efficiency of gp411

Amy L. Cole*, Otto O. Yang{ddagger}, Andrew D. Warren*, Alan J. Waring{ddagger}, Robert I. Lehrer{ddagger} and Alexander M. Cole2,*,{dagger}

* Department of Molecular Biology and Microbiology and {dagger} Biomolecular Science Center, Burnett College of Biomedical Sciences, University of Central Florida, Orlando, FL 32816; and {ddagger} Department of Medicine, David Geffen School of Medicine, University of California, Los Angeles, CA 90095


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Retrocyclin (RC)-101 is a cationic {theta}-defensin that inhibits HIV-1 entry. Passaging HIV-1BAL under selective pressure by this cyclic minidefensin resulted in only a 5- to 10-fold decrease in viral susceptibility to RC-101. Emergent viral isolates had three amino acid substitutions in their envelope glycoprotein. One was in a CD4-binding region of gp120, and the others were in the heptad repeat (HR) domains of gp41 (HR1 and HR2). Each mutation replaced an electroneutral or electronegative residue with one that was positively charged. These mutations were evaluated either alone or in combination in a single-round viral entry assay. Although the mutation in gp120 did not affect viral entry, the mutation in HR1 of gp41 conferred relative resistance to RC-101. Interestingly, the envelope with the HR2 mutation was less efficient and became codependent on the presence of RC-101 for entry. The adaptive response of HIV-1 to this cationic host defense peptide resembles the responses of bacteria that modulate their surface or membrane charge to evade analogous host defense peptides. These findings also suggest that interactions between {theta}-defensins and gp41 may contribute to the ability of these cyclic minidefensins to prevent HIV-1 entry into target cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Host defense peptides contribute to innate immunity by protecting the host from bacterial, fungal, and viral invaders. The defensins are cationic host defense peptides that contain six cysteine residues in one of three motifs ({alpha}, beta, and {theta}). All defensins have a largely beta sheet structure that is stabilized by three intramolecular disulfide bonds (1, 2, 3). All human {alpha}-defensin genes, most {theta}-defensin pseudogenes, and several beta-defensin genes are clustered on chromosome 8. The {alpha}-defensins are expressed by neutrophils, certain lymphocytes, and intestinal Paneth cells. beta-Defensins are expressed by epithelia throughout the body and are prominent in the skin, urogenital tract, and respiratory tract. {theta}-Defensins are circular octadecapeptides found only in certain nonhuman primates. They were originally identified in leukocytes and bone marrow of rhesus macaques (4, 5, 6). Human {theta}-defensin genes have a mutation that introduces a premature stop codon into the signal peptides. Although human {theta}-defensin pseudogenes are transcribed, the mRNA cannot be translated effectively (7, 8).

Our group used the sequence of this expressed human pseudogene to deduce and synthesize retrocyclin (RC)3-100, a {theta}-defensin with antimicrobial and lectin-like properties (7, 9). RC-100 inhibited infection of human primary CD4+ lymphocytes by the laboratory-adapted IIIB and JR-CSF strains of HIV-1 as well as by a number of primary clinical HIV-1 isolates, with IC50 values of 1–5 µg/ml for most strains (7, 10). RC-101, which differs from RC-100 by a single Arg->Lys substitution, was twice as active as RC-100 against 17 primary HIV-1 isolates (11). RC-101 was also nonhemolytic for human RBC and noncytotoxic for several human cell lines, even at concentrations of 200–500 µg/ml. Because RC-101 is active against HIV-1 in the presence of vaginal fluid (7, 12), it has potential use as a topical microbicide to prevent sexually transmitted HIV-1 infection.

The microbicidal properties of defensins have been attributed primarily to their cationic nature, which enables them to bind and subsequently disrupt negatively charged microbial membranes (reviewed in Refs. 2 and 3). Bacteria can acquire resistance to cationic antimicrobial peptides via genetic or regulatory responses that decrease the negative charge of their membrane phospholipids, teichoic acids, or regions of their LPS (13, 14, 15, 16). Enveloped viruses, such as HIV-1, HSV-1, and HSV-2, are believed to be susceptible to RCs, because their net positive charge and lectin-like behavior permit binding to anionic or carbohydrate-containing viral or host cell membrane domains, including sites implicated in membrane fusion and viral uptake (7, 9, 17, 18). This binding is thought to antagonize HIV-1 entry into target cells, but the precise interactions and mechanism of viral inhibition by RCs are poorly understood.

Inhibition of HIV-1 target cell entry has been the target of peptides and small molecules developed for therapeutic purposes. These bind envelope gp41 or the CXCR4 or CCR5. Some initially act at low (nanomolar) concentrations, only to have highly resistant variants emerge within weeks of their administration (19, 20, 21, 22); thus, resistance may be a substantial barrier to their clinical use.

The acquisition of resistance to RCs by HIV-1 and the mechanisms that may underlie this effect are not described. In the current studies we measured the activity of RC-101 against HIV-1 over the course of 100 days of selection in human cells. We examined the evolution of env under selective pressure by RC-101 at a dose previously determined to reduce viral replication by ~70%. Phenotypic resistance after selection under these conditions resulted in only a modest increase in the IC50 and was associated with a distinct evolution of mutations in env. From a practical standpoint, these relatively small changes in susceptibility do not negate developing RC-101 as a topical microbicide, because topical microbicides are usually formulated at concentrations of 1–10 mg/ml. From a theoretical standpoint, the findings provide an insight into how HIV-1 may adapt to the presence of cationic antimicrobial peptides in its environment.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Peptide synthesis and purification

The 18-aa RC analogs were prepared in our laboratory as previously described (4, 6, 7) with sequences as follows: RC-101, GICRC ICGKG ICRCI CGR; RC-106, GICYC ICGRG ICRCI CGR; RC-100, GICRC ICGRG ICRCI CGR; RC-100b, GICRC ICGRR ICRCI CGR; RC-119, GICKC ICGKG ICKCI CGR; and RC-valine, GVCRC ICGRG VCRCI CRR. After each purification step, peptides were subjected to MALDI-TOF mass spectrometry to assess homogeneity (typically ~95%) and to confirm that the observed masses agreed well with the theoretical masses. Peptide concentrations were determined by quantitative amino acid analysis.

Maintenance of cells and virus

The human cell lines TZM-bl (from Dr. J. C. Kappes (University of Alabama, Birmingham, AL), Dr. X. Wu (University of Alabama, Birmingham, AL), and Tranzyme (Research Triangle Park, NC)), PM1 (Dr. M. Reitz, Institute of Human Virology, Baltimore, MD), and HOS-CD4-CCR5 and HOS-CD4-fusin (Dr. N. Landau, Salk Institute for Biological Studies, La Jolla, CA) were acquired from the AIDS Research and Reference Reagent Program, Division of AIDS, National Institute of Arthritis and Infectious Diseases, National Institutes of Health. PM1 and TZM-bl cells express CD4, CCR5, and CXCR4 and thus are infectable by both R5 (macrophage-tropic) and X4 (T cell-tropic) strains of HIV-1. TZM-bl cells were grown in DMEM (4.5 g/L glucose) containing penicillin, streptomycin, and 10% (v/v) FBS. PM1 promyelocytic cells were maintained in RPMI 1640 supplemented with penicillin, streptomycin, 10 mM HEPES, and 20% FBS (R20). HOS-CD4-CCR5 and HOS-CD4-fusin cells, which permit R5 or X4 HIV-1 entry, respectively, were grown in DMEM supplemented with penicillin, streptomycin, 10% FBS, 1 µg/ml puromycin, and mycophenolic acid selection medium according to the manufacturers’ instructions. HIV-1 BaL, an R5 strain, was acquired from the National Institutes of Health AIDS Research and Reference Reagent Program (Dr. J. Levy, University of California, San Francisco, CA), and stocks were prepared by infecting 3 x 106 PM1 cells with the contents of the obtained vial for 3 h, washing the cells to remove excess virus, then growing the cells at 0.5–0.75 x 106/ml. Cell supernatants from 5 days after infection were clarified by centrifugation, followed by filtration through a 0.45-µm pore size nylon syringe filter, and stored at –80°C in aliquots for use as virus stocks. These HIV-1 BaL viral stocks were quantified using an HIV-1 p24 ELISA (PerkinElmer).

Measurement of anti-HIV-1 activity of RC-101 and RC analogs

Activity of RC-101 against HIV-1 BaL was measured using TZM-bl reporter cells to determine infection by measuring long terminal repeat-driven luciferase expression (23). Cells (5 x 103/well; 96-well plates) were treated with 100 µl of growth medium containing HIV-1 BaL (2 ng of p24/ml) in the presence of vehicle (0.01% acetic acid) or RC-101 or RC-106 (inactive control) at 1.25–10 µg peptide/ml for 24 h. Supernatants were then removed, and cells were lysed with 100 µl of 1x Glo Lysis Buffer (Promega). Luciferase activity was measured using Bright Glo luciferase assay buffer (Promega). Protection from infection was measured as the percentage of reduction in luciferase activity (relative light units) compared with the HIV-1 BaL-infected control (vehicle only, no peptide). To confirm that the observed reductions in luciferase activity were not due to nonspecific effects of RC-101 on the cells, MTT assays (measuring cellular metabolic activity) were performed on identically treated TZM-bl cells.

Serial passaging of HIV-1 with RC-101 to generate HIV-1 escape mutants

PM1 cells (1 x 105/0.1 ml) were infected with 1) HIV-1 BaL (0.2 ng of p24/105 cells) and 0.01% acetic acid (vehicle), 2) HIV-1 BaL and RC-106, or 3) HIV-1 BaL and RC-101 in a total of 100 µl of R20 medium for 3 h with periodic mixing of the cell suspension. Cells were then washed with 2 ml of R20 medium, pelleted, resuspended in 0.5 ml of R20 medium containing vehicle or peptide, and left to incubate (37°C; 5% CO2). On day 3 after infection, the supernatants were removed, and the cells were resuspended in 1 ml of fresh R20 medium containing vehicle or peptide. On day 5, cell supernatants were collected, filtered, and stored in aliquots at –80°C for 1) quantitation by HIV-1 p24 ELISA, 2) viral RNA extraction (QIAamp viral RNA kit; Qiagen) for subsequent cloning and sequence analysis, and 3) subsequent rounds of continued passaging under the same conditions. Cells were initially infected with HIV-1 BaL in the presence of 2 µg/ml peptide, an effective, but suboptimal (70 ± 10% inhibition in the TZM-bl cell assay), dose of RC-101. The peptide concentration was increased when the preceding concentration lost inhibitory activity (<60% inhibition of viral propagation, as measured by HIV-1 p24 ELISA) against a particular passage of HIV-1 BaL.

Sequence and structural analysis of env

HIV-1 BaL env from each treatment group was cloned and sequenced after infection days 25, 50, 75, 100, and 125 (rounds 5, 10, 15, 20, and 25). Extracted HIV-1 BaL viral RNA was reverse transcribed (iScript cDNA synthesis kit; Bio-Rad) and amplified using five sets of PCR primer pairs (PP), PP1-PP5, which generated overlapping PCR products (600–800 bp each) that collectively spanned the entire length of env. The primer sequences were: PP1: sense, 5'-AGAAGACAGTGGCAATG-3'; antisense, 5'-CAGCAGTTGAGTTGATAC-3'; PP2: sense, 5'-AACACCTCAGTCATT ACAC-3'; antisense, 5'-TACATTGCTCTTCCTACTTC-3'; PP3: sense, 5'-GCTGAATGA ATCTGTAG-3'; antisense, 5'-GGTGCTACTCCTAATG-3'; PP4: sense, 5'-GGCTGCTATTAACAAGAGATGG-3'; antisense, 5'-GTGGGTCTGAAACGATAATGG-3'; and PP5: sense, 5'-TACACAAGCATAATATACAG-3'; antisense, 5'-GCCATACGACTATACTAC-3'. High-fidelity PCR (Invitrogen Life Technologies) was conducted as follows: denaturation of template and activation of Taq at 95°C for 2 min; amplification for 35 cycles of 95°C for 30 s; annealing at 56.6°C (pp1, pp2, and pp4), 55.2°C (pp3), or 51.7°C (pp5) for 45 s; and extension at 68°C for 45 s. PCR products were checked for purity by agarose gel electrophoresis, then gel purified or ligated directly into pCR4-TOPO (Invitrogen Life Technologies) and cloned according to the manufacturer’s instructions. Plasmid clones were sequenced from T3 and T7 (i.e., both directions) using a Beckman Coulter automated sequencer (UCF BMSC Genomics Core Laboratory). Sequences were aligned and analyzed using ClustalW 1.8 with boxshade (<http://searchlauncher.bcm.tmc.edu/multialign/multialign.html>). Note that the overlapping sequences of the cloned PCR products accounted for 39.3% (1009 of 2568) of all nucleotides in BaL env. Alignment of these overlapping regions showed 99.7% sequence homology between PCR clones. Protein sequences were generated from DNA sequences using the Translate algorithm of The Sequence Manipulation Suite (<http://bioinformatics.org/sms/index.html>) and were aligned using ClustalW. A consensus sequence for BaL env on day 0 (i.e., the stock virus) to be used for comparison with viral clones from subsequent rounds and treatment groups was generated by analyzing three or four clones of each PCR product. The resulting stock consensus sequence was 98.98% identical with the published BaL env sequence (GenBank accession no. M68893).

Genetic analysis for selective pressure on env

Estimation of synonymous and nonsynonymous substitution rates (dS:dN ratio) was performed according to the method of Nei and Gojobori (24), using DnaSP (<http://ub.es/dnasp>). In the absence of selective pressure, synonymous substitutions will equal nonsynonymous (structure altering) mutations; thus, a dS:dN >1 indicates negative selection, whereas a dS:dN ≤1 suggests the presence of a positive selective force (antiviral drug, immune function, etc.).

HIV-1 plasmid constructs and viral entry assay

The expression vectors pNL-LucR–E– and JR.FL env were gifts from Dr. N. R. Landau (The Salk Institute for Biological Studies, La Jolla, CA). JR.FL is an R5 strain of HIV-1. The JR.FL env nucleotide sequence is 95% identical with BaL, and both strains are inhibited by RC-101 in human cell HIV-1 entry assays. The three mutations observed in RC-101-exposed HIV-1 BaL were created either individually or in combination in JR.FL env using the QuikChange Multi Site-Directed Mutagenesis Kit (Stratagene) and were verified by sequencing (University of Central Florida Biomolecular Science Center Genomics Core Laboratory, Orlando, FL). In all, four JR.FL mutant plasmids were created: JR.FL M1 (GAA (Glu)->AAA (Lys) at aa 426), JR.FL M1+2 (M1 combined with CAG (Gln)->CGG (Arg) at aa 574), JR.FL M1+2+3 (M1 combined with both M2 and AAT (Asn)->AAA (Lys) at aa 634), and JR.FL M2+3. Then, HIV-1 single-cycle (replication incompetent) luciferase reporter viruses were produced by cotransfecting 293T cells (AIDS Research and Reference Reagent Program) with 10 µg each of pNL-LucR–E– and one of the JR.FL env clones (25). Virus-containing, clarified supernatants were collected after 48 h and quantified by p24 Ag ELISA. HOS-CD4-CCR5 cells (4 x 103/well; 96-well plate) were infected with 20 ng p24/well virus in the presence or the absence of RC-101 (1.25–10 µg/ml), and luciferase activity was measured 2 days later.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
We previously demonstrated RC-101 activity against multiple R5 and X4 strains of HIV-1 (7, 11, 17). In this study we tested RC-101 activity against HIV-1 BaL, an R5 strain, because R5 strains of HIV-1 are overwhelmingly the predominant phenotype of transmitted virus (26). Initially, we used HIV-1 entry indicator cells to examine the effective concentration range of RC-101 for activity against HIV-1 BaL (Fig. 1). Subconfluent TZM-bl cells were infected with HIV-1 BaL (0.2 ng p24/5000 cells) containing 1.25–10 µg/ml RC-101 or RC-106 (inactive, negative control peptide) or vehicle (0.01% acetic acid, no peptide) for 24 h, then luciferase activity was measured. RC-101 potently inhibited HIV-1 BaL infection at 2.5–10 µg/ml in this assay. As expected, RC-106 was not inhibitory in this concentration range. When administered to TZM-bl cells at concentrations up to 20 µg/ml, neither peptide had an appreciable effect on basal luciferase levels or MTT activity (data not shown), demonstrating that the observed decrease in luciferase values reflected specific antiviral activity of the RC.


Figure 1
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  		FIGURE 1. RC-101 inhibits HIV-1 infection in a dose-dependent manner. Anti-HIV-1 activity of RC-101 and RC-106 (inactive peptide) against HIV-1 BaL (R5) was measured using a luciferase expression system. Activity is expressed as the percentage of inhibition of infection compared with control (vehicle-only) cells (n = 4–6). Error bars represent the SEM.

 
We next measured RC-101 activity over the course of repeated HIV-1 BaL infections. PM1 cells were infected with BaL combined with vehicle, RC-106, or RC-101, initially using a peptide concentration of 2 µg/ml (~1 µM). For each infectious round, virus collected on the fifth day after infection from each treatment group was used for quantitation of HIV-1 by p24 ELISA, viral RNA extraction for cloning and sequencing, or the next round of infection of the PM1 cells. Peptide concentration was increased when viral inhibition was determined to be <60% as measured by p24 ELISA. RC-101 effectiveness, displayed as the percentage of inhibition of infection compared with RC-106, was measured after each round of infection and is shown in Fig. 2. It should be noted that although the initial peptide concentration (2 µg/ml; ~1 µM) was suboptimal for HIV-1 BaL infection of TZM-bl cells (Fig. 1), it potently inhibited infection of PM1 cells for 15 days (three infectious rounds). When administered at 5 µg/ml, a concentration >50-fold lower than that tolerated by human cells, RC-101 inhibited infection by at least 88% for an additional nine rounds of infection. These data suggest that RC-101 induces minimal HIV-1 resistance compared with other candidate entry inhibitors, such as CCR5 antagonists, that have been shown to induce highly resistant (10,000- to 20,000-fold) HIV-1 isolates in similarly designed serial passage experiments (20, 22, 27).


Figure 2
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  		FIGURE 2. RC-101 effectively inhibits HIV-1 BaL infection through 18 serial passages. PM1 cells were initially infected with HIV-1 BaL combined with vehicle, RC-106 (inactive peptide), or RC-101 (2 µg/ml). Propagated virus, collected 5 days after infection, was quantified by p24 ELISA to monitor the effectiveness of RC-101 and was also used to perform subsequent rounds of infection. Data are expressed as the percentage of inhibition of infection compared with RC-106. The RC-101 concentration was increased when the preceding concentration demonstrated reduced inhibition of the passaged BaL.

 
We next sequenced the env gene of viral isolates collected on days 25, 50, 75, and 100 (rounds 5, 10, 15, and 20) from each treatment group to determine whether specific mutations mediated the observed decreased viral susceptibility to RC-101. Sequence analysis of virus collected after 25 days of RC-101 selection at 2 µg/ml (which had become resistant, as shown in Fig. 2) revealed two novel amino acid substitutions that were not present in the control culture viruses (Fig. 3A). The Glu(E)->Lys(K) substitution at gp160 residue 426 switches a negatively charged to a positively charged amino acid in a 6-aa region important for binding of the env gp120 domain to CD4 on host cells (28) (Fig. 3B, structure I). Interestingly, we detected no mutations in the highly mutable first three variable loop regions (V1–V3) of gp120 after exposure of HIV-1 BaL to RC-101, consistent with our previous finding that RCs do not inhibit binding of gp120 to CCR5 (7, 17). Another change was noted in the env gp41 domain, which contains the heptad repeat (HR)1 and HR2 motifs that mediate fusion of viral and target cell membranes by arranging in coils that pack tightly with each other to form a six-helix bundle to bring the viral and host cell membranes into apposition (19, 29). A Gln(Q)->Arg(R) substitution at gp160 residue 574, switching a hydrophilic residue to a positive one, occurred in the HR1 domain of gp41 (Fig. 3B, structures II–IV). Together, the introduction of these positively charged amino acids after HIV-1 exposure to a low dose of peptide supports the concept that RC-101 activity involves binding to the HIV-1 envelope glycoproteins at multiple neutral or negatively charged sites that are critical for infection of host cells.


Figure 3
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  		FIGURE 3. RC-101 induces mutations that increase the positive charge of membrane-active regions of gp120 and gp41. PM1 cells were treated as described in Fig. 2. A, Virus collected from each treatment group was cloned and sequenced after infectious rounds 5, 10, 15, and 20 and was aligned with the stock (round 0) env sequence. Arrows show the M1 and M2 mutations (Glu(E)->Lys(K) and Gln(Q)->Arg(R), respectively) detected in all the aforementioned infectious rounds in the RC-101-exposed virus. An additional mutation (M3; *, Asn(N)->Lys(K)) was detected in the RC-101 treatment group at rounds 15 and 20. Dots signify residues that were conserved across all treatment groups. Numbers refer to the amino acid number in gp160 for our stock (round 0) viral sequence. M1, M2, and M3 correspond to the mutations described in Fig. 4. B, The three amino acid substitutions were visualized using available three-dimensional models of gp120 complexed with CD4 (structure I, PDB accession code 1G9M) (28 ) and core gp41 (structures II-IV, PDB accession codes 1AIK, 1ENV, and ISZT, respectively) (29 ) and drawn using PyMOL (DeLano Scientific; <http://pymol.sourceforge.net>). Structure I: CD4 is orange, core gp120 is green, and residues of gp120 contacting CD4 are blue. The red residue (arrow) denotes the Glu (E)->Lys (K) amino acid substitution detected in the RC-101-exposed viral isolates. Structures II, III, and IV: The red residue (no arrow) corresponds to the Gln (Q)->Arg (R) substitution in HR1, and the red residue (arrow) corresponds to the Asn (N)->Lys (K) substitution in HR2.

 
Increasing the RC-101 concentration from 2 to 5 µg/ml restored peptide activity against HIV-1 BaL throughout infection days 25–65 (rounds 5–13; Fig. 2). Consistent with continued selective pressure against HIV-1, sequence analysis of day 50 (round 10) virus revealed maintenance of the two mutations observed on day 25 (round 5; Fig. 3A). However, by day 75 (round 15), RC-101 (5 µg/ml) reduced viral replication by only 30% (Fig. 2), and this reduced susceptibility coincided with the appearance of an additional cationic amino acid substitution (Asn(N)->Lys(K); Fig. 3A) at residue 634 in the HR2 domain of gp41 (Fig. 3B, structures II–IV, arrows). All three amino acid substitutions were present in treated virus isolated on day 100 (round 20), demonstrating their stability under selective pressure exerted by RC-101. Estimation of synonymous and nonsynonymous substitution rates (dS:dN ratio) comparing full-length env from RC-101- vs control RC-106-selected virus revealed a dS:dN of 0.88 on day 25 (round 5), 2.32 on day 50 (round 10), 0.94 on day 75 (round 15), and 2.34 on day 100 (round 20). Significantly, dS:dN ratios <1 corresponded to infectious rounds at which the cationic mutations were observed.

We next created HIV-1 env molecular clones to confirm that any or all of the observed mutations would effectively alter HIV-1 susceptibility to RC-101. An available JR.FL env expression vector, an R5 pseudotype with 95% homology to BaL env, was subjected to site-directed mutagenesis to create clones containing various combinations of the three mutations (M1 = Glu->Lys mutation in gp120, M2 = Gln->Arg substitution in gp41 HR1, M3 = Asn->Lys in gp41 HR2). The stock (nonmutated) or mutant JR.FL env clones were then used to make single-cycle HIV-1 luciferase reporter viruses, and RC-101 activity against each viral clone was measured. Consistent with our previous finding that RCs do not fully inhibit binding of gp120 to CD4 or CCR5 (17) and the current data showing that the emergence of M1 together with M2 was required for viral escape from low dose RC-101 (Fig. 3), the stock JR.FL env and the JR.FL M1 env reporter viruses were equally inhibited by 2.5–10 µg/ml RC-101 (Fig. 4, A and B). JR.FL M1+2, analogous to the RC-101-resistant virus detected on day 25, was significantly less susceptible to RC-101, confirming that the M2 mutation is involved in resistance (Fig. 4, A and B). It should be noted that JR.FL M1+2+3, containing the mutations observed in RC-101-exposed virus collected on days 75 and 100, did not adequately infect HOS-CD4-CCR5 cells in the absence of RC-101 (Fig. 4C). The mutant clone JR.FL M2+3 was also unable to infect cells unless RC-101 was also present at 5–10 µg/ml (Fig. 4C), demonstrating that the amino acid substitutions observed in gp41 probably affected not only HIV-1 interaction with RC-101, but also the efficiency of viral target cell entry. Most interestingly, the same Asn(N)->Lys(K) substitution at residue 634 (M3 mutation) was observed in a drug-dependent variant of HIV-1 that emerged in a patient who did not respond to therapy with the fusion inhibitor T20 (19). Replication of this viral variant was dependent on the presence of T20, because the peptide probably prevented premature conformational changes in gp41. It is also noteworthy that the M3 mutation removes an N-linked glycosylation site of gp41, and that mutations to this site have previously been shown to reduce viral fitness (30).


Figure 4
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  		FIGURE 4. Specific mutations in gp41 affect RC-101 activity and the efficiency of viral target cell entry. HOS-CD4-CCR5 cells were infected with HIV-1 luciferase reporter viruses pseudotyped either by nonmutated JR.FL env (JR.FL stock) or one of four mutated JR.FL env clones, M1, M1+2, M1+2+3, or M2+3, corresponding to the mutations shown in Fig. 3 (M1 = Glu->Lys mutation in gp120; M2 = Gln->Arg substitution in gp41 HR1; M3 = Asn->Lys in gp41 HR2). Values are presented as relative luciferase units (RLU) in A and C or as the percentage of inhibition (mean ± SEM; n = 3) compared with virus alone in B and D. *, p < 0.05 vs JR.FL stock.

 
Because HIV-1 BaL gained 5-fold resistance to RC-101 by increasing the net positive charge of three separate envelope regions, we compared the activity of RC-101 with those of four different RC analogs against day 100 (round 20) virus (Fig. 5). The analogs, termed RC-100, RC-100b, RC-119, and RC-valine, were determined previously by our group to be 50–100% active against both R5 and X4 strains of HIV-1 at 0.5–2 µg/ml (7, 17) (our unpublished observations). Only RC-valine exhibited similar activity to RC-101, potently inhibiting infection at both 10 and 20 µg/ml. RC-valine was the only valine-containing peptide tested in these experiments, and its continued effectiveness suggests that compositional features other than charge may critically determine the anti-HIV-1 activity of RC analogs.


Figure 5
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  		FIGURE 5. Activity of RC analogs against HIV-1 BaL escape mutant. Propagated virus from the round 19 µg/ml BaL plus 20 µg/ml RC-101 treatment group was incubated alone or in combination with 10 or 20 µg/ml RC-106 (inactive peptide), RC-101, RC-100, RC-100b, RC-119, or RC-valine. Supernatants containing replicated virus were collected on day 5 after infection, and virus was quantified by p24 ELISA. Values are presented as the percentage of inhibition (mean ± SEM) compared with RC-106 controls (n = 3). *, p < 0.05 vs RC-101.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Almost all endogenous antimicrobial peptides are cationic. It has also become apparent that bacterial stratagems to evade these host defense molecules frequently take the form of mutations or programmed responses that reduce the negative charges of molecules displayed on the microbial surface or in its membrane bilayer(s) (13, 14, 15, 16). HIV-1 was not highly successful in overcoming the effects of RC-101, increasing its resistance by only 5- to 10-fold. The viral envelope mutations mediating this effect were analogous to resistance stratagems used by bacteria in that they involved alterations of local charge in areas with critical function. For HIV-1, these critical sites were in HR1 and HR2 of gp41, and the mutations probably reduced the affinity with which {theta}-defensins bound to these areas.

It is noteworthy that HIV-1 is highly susceptible to RCs and other {theta}-defensins (e.g., rhesus{theta}-defensins), whereas HIV-2 and SIV are far less susceptible (R. J. Lehrer, unpublished observations). HIV-2 evolved from a simian retrovirus (SIV) whose natural host is an Old World monkey, Cercocebus torquatus atysan (i.e., the sooty mangaby), whereas HIV-1 evolved from a chimpanzee retrovirus, SIVcpz. Phylogenetic studies indicate that {theta}-defensins are expressed by Old World monkeys, but are lacking in humans and chimpanzees. Thus, it is tempting to speculate that HIV-2 could have evolved under selection pressure from {theta}-defensins, and HIV-1 may not have experienced similar selection, explaining the pattern of differential susceptibility to {theta}-defensins.

Modest resistance of HIV-1 against RC-101 developed stepwise over 100 days. Under similar conditions of selection by other antiviral compounds, HIV-1 has developed robust resistance, suggesting that defensins may be more difficult to evade. The rapid kinetics of HIV-1 replication (1–2 days) and the high error rate of reverse transcriptase (~10–4) ensure the continuous development of single-point mutations in this culture system. The slow development of resistance mutations suggests that such escape could incur significant replicative fitness costs that create a bottleneck. Whether this translates to less development of resistance in vivo (if RCs find clinical use) will remain to be determined definitively, but these findings are encouraging.


   Acknowledgments
 
We thank Martin Kline, Younghee Lee, Dharma Thapa, Hwee Ng, and Duy Pham for their expert technical assistance.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by National Institutes of Health Grants AI052017, AI065430, and AI060753 (to A.M.C.); AI056921 (to R.I.L.); and AI037945 (to A.J.W.). Back

2 Address correspondence and reprint requests to Dr. Alexander M. Cole, Molecular Biology and Microbiology, Biomolecular Science Center, University of Central Florida, 4000 Central Florida Boulevard Building 20, Room 236, Orlando, FL 32816. E-mail address: acole{at}mail.ucf.edu Back

3 Abbreviations used in this paper: RC, retrocyclin; dS:dN ratio, synonymous and nonsynonymous substitution rates; HR, heptad repeat; PP, primer pair. Back

Received for publication January 26, 2005. Accepted for publication March 9, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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