Cell-penetrating peptides (CPP), which are short peptides that are capable of crossing the plasma membrane of a living cell, are under development as delivery vehicles for therapeutic agents that cannot themselves enter the cell. One well-studied CPP is the 10-amino acid peptide derived from the human immunodeficiency virus type 1 (HIV-1) Tat protein. In experiments to test the hypothesis that multiple cationic amino acids within Tat peptide confer antiviral activity against HIV-1, introduction of Tat peptide resulted in concentration-dependent inhibition of HIV-1 IIIB infection. Using Tat peptide variants containing arginine substitutions for two nonionic residues and two lysine residues, HIV-1 inhibition experiments demonstrated a direct relationship between cationic charge and antiviral potency. These studies of Tat peptide as an antiviral agent raise new questions about the role of Tat in HIV-1 replication and provide a starting point for the development of CPPs as novel HIV-1 inhibitors.
Cell penetrating peptides (CPP) are short peptides that can efficiently cross the plasma membrane, which is otherwise a formidable barrier to many extracellular molecules [
Of the numerous peptides shown to have cell penetrating properties, a 10-amino acid (aa) peptide derived from the human immunodeficiency virus type 1 (HIV-1) Tat protein has been well studied as an effective CPP and an attractive drug delivery agent [
Sequences of peptides examined. Peptide sequences are shown relative to the primary amino acid sequence of the Tat peptide. Position numbers are derived from the full-length Tat protein amino acid sequence (HIV-1 strain SF2) [
Peptide | Sequence | Charge | |||||||||
---|---|---|---|---|---|---|---|---|---|---|---|
Tat peptide | G | R | K | K | R | R | Q | R | R | R | +8 |
TPvar1 | R | — | — | — | — | — | — | — | — | — | +9 |
TPvar2 | — | — | — | — | — | — | R | — | — | — | +9 |
TPvar3 | R | — | — | — | — | — | R | — | — | — | +10 |
R-10 | R | — | R | R | — | — | R | — | — | — | +10 |
aa position | 48 | 49 | 50 | 51 | 52 | 53 | 54 | 55 | 56 | 57 |
CPPs such as the Tat peptide, the 16-aa penetratin peptide derived from the Drosophila melanogaster Antennapedia homeodomain protein, and nona-arginine contain numerous cationic arginine (R) and lysine (K) residues [
The present studies were conducted to test the hypothesis that Tat peptide, because of the numerous cationic amino acids contained within its primary sequence, can effectively inhibit HIV-1 infection. In vitro experiments involving Tat peptide and an HIV-1-susceptible indicator cell line demonstrated concentration-dependent inhibition of the X4 HIV-1 strain IIIB, which uses CXCR4 as a coreceptor. Additional experiments involving variants of Tat peptide with increased cationic charge suggested a direct relationship between charge magnitude and antiviral potency. These results provide further insights into a potential role for Tat as an HIV-1 inhibitor and suggest a novel anti-HIV-1 activity attributed to the family of CPPs.
Tat peptide (Table
P4-R5 MAGI indicator cells (NIH AIDS Research and Reference Reagent Program number 3580) were maintained in Dulbecco’s modified eagle’s media (DMEM) supplemented with 10% fetal bovine serum (FBS), 0.05% sodium bicarbonate, antibiotics (penicillin, streptomycin, and kanamycin at 40
Peptide effectiveness was determined in an HIV-1 infection inhibition assay using P4-R5 MAGI indicator cells. P4-R5 MAGI cells were plated at a concentration of 1.5 × 104 cells/well in a flat-bottom 96-well plate (BD Biosciences, Bedford, MA). The cells were then infected with HIV-1 strain IIIB (Advanced Biotechnologies, Inc., Columbia, MD; 107.8 TCID50/mL) at multiplicities of infection (MOI) of 0.6, 0.05, or 0.03 in the presence or absence of peptide or dextran sulfate (DS) (Sigma, St. Louis, MO). Following a 2 h incubation at 37°C, the cells were washed with PBS, provided with 200
P4-R5 MAGI cells were plated at a concentration of 1.5 × 104 cells/well in a flat-bottom 96-well plate
Mean values and standard deviations were calculated from two independent assays in which each concentration was examined in quadruplicate. Calculations of EC50 (concentrations that resulted in 50% reductions in infection relative to mock-treated, HIV-1-infected cells) were calculated using the Forecast function of Microsoft Excel.
Initial experiments were performed to test the hypothesis that Tat peptide, by virtue of its cationic charge, was capable of inhibiting HIV-1 infection. HIV-1-susceptible P4-R5 MAGI indicator cells were exposed to HIV-1 strain IIIB (0.6 MOI) for 2 h while in the presence of half log concentrations of Tat peptide up to 1 mg/mL. The presence of Tat peptide inhibited HIV-1 infection in a concentration-dependent manner (Figure
Viral titer does not affect the antiviral activity of Tat peptide. EC50 values were calculated from the results of antiviral assays (as described in Section
Virus concentration during infection (103 infectious virions/mL) | MOI | EC50 |
---|---|---|
88 | 0.6 | 0.094 mg/mL |
8.8 | 0.05 | 0.14 mg/mL |
4.4 | 0.03 | 0.10 mg/mL |
Tat peptide inhibits infection by HIV-1 strain IIIB. P4-R5 MAGI cells were exposed to half log concentrations of Tat peptide (TP) or dextran sulfate (DS) in the presence of HIV-1 strain IIIB for 2 h. Reductions in HIV-1 infection (%) were calculated relative to mock-exposed HIV-1 infected cells. The graph represents data from two independent assays in which infections at each concentration were repeated in quadruplicate. Error bars represent standard deviations.
To confirm that any adverse effects of Tat peptide on reporter cell viability had not compromised the antiviral assays, MTT cytotoxicity assays were performed using conditions identical to those used in the antiviral assays. In these assays, 2 h exposures to Tat peptide at concentrations below 1 mg/mL had no effect on P4-R5 MAGI cell viability, as measured immediately after exposure or after extended postexposure maintenance (24 h or 48 h) in the absence of Tat peptide (Figure
Tat peptide has no effect on reporter cell viability. P4-R5 MAGI cells were exposed to half log concentrations of Tat peptide for 2 h, washed, and assessed immediately for changes in cell viability or after extended maintenance (24 h or 48 h after exposure) in the absence of Tat peptide. Percent changes in cell viability were calculated relative to mock-exposed cells. The graph represents data from two independent assays in which exposure to each concentration of peptide was repeated in quadruplicate. Error bars represent standard deviations.
Having demonstrated the anti-HIV-1 activity of the Tat peptide, additional experiments were performed to investigate the role of charge in determining antiviral efficacy. Of the 10 aa residues in Tat peptide, eight are cationic (six arginine and two lysine residues) and the remaining two are uncharged (G48, nonpolar and aliphatic; Q54, polar). To increase the net peptide charge, arginine residues were substituted for one or both of the noncationic residues in the native Tat peptide sequence (Table
Concurrent incubation of HIV-1 IIIB and each peptide with P4-R5 MAGI cells again resulted in concentration-dependent inhibition of HIV-1 infection (Figure
Increased peptide antiviral potency is associated with increased peptide cationic charge. P4-R5 MAGI cells were exposed to half log concentrations of Tat peptide (TP), three Tat peptide variants (TPvar1-3), decaarginine (R-10), or dextran sulfate (DS) in the presence of HIV-1 strain IIIB for 2 h. Peptide sequences are depicted in Table
Results presented in this paper demonstrate that Tat peptide, a CPP that is capable of delivering molecules intracellularly [
In a broader virologic context, observations reported herein provide new information about Tat protein and its contributions to HIV-1-associated pathogenesis. In previous studies, soluble full-length Tat protein, which is secreted from infected cells [
These studies also provide two starting points for the development of novel inhibitors of HIV-1. First, Tat peptide can serve as a prototype for the development of novel agents effective against HIV-1. Such agents may take the form of cationic peptides or small molecule inhibitors that mimic a peptide structure. Second, Tat peptide provides the basis for multifunctional therapeutic agents that combine the intrinsic and specific anti-HIV-1 activity of Tat peptide with its ability to deliver therapeutic agents that by themselves do not readily penetrate cells and tissues [
Our experiments also indicate that this intrinsic antiviral activity is not limited to the Tat-derived CPP alone. In the present studies, the R-10 peptide was also an effective HIV-1 inhibitor, despite changes in four out of ten amino acids with respect to the Tat peptide. Preliminary studies have also demonstrated anti-HIV-1 activity associated with the well-studied CPP nona-arginine (R-9) and a 20-aa peptide consisting solely of alternating arginine and glycine residues (data not shown). The finding that antiviral activity is not limited to Tat peptide suggests that a key characteristic common to these molecules (i.e., multiple cationic charges) confers activity against HIV-1.
The involvement of cationic charge in CPP antiviral activity is also supported by the results of experiments involving the Tat peptide variants. Those results indicated a direct relationship between charge and antiviral activity. Tat peptide (+8 charge) was the least active while R-10 (+10 charge) was the most active, and variants with intermediate levels of cationic charge had intermediate levels of antiviral activity. Despite the fact that R-10 and TPvar3 had the same charge, these two peptides differed in their effects on HIV-1 infection, likely due to the replacement of two lysine residues with two arginine residues. Lysine has a single positive charge associated with a terminal amino group while arginine has a single positive charge associated with a terminal guanidinium group. The charge in arginine is delocalized across the guanidinium group, supporting the formation of multiple hydrogen bonds [
Related studies have also indicated the importance of charge in cationic HIV-1 inhibitors and provided further evidence for a mechanism of CPP antiviral activity. We previously demonstrated that charge distribution plays a key role in the antiviral activity of biguanide-based molecules [
These results provide the basis for further basic science and translational studies. Expanded studies will be necessary to investigate the antiviral effect of Tat and Tat peptide on HIV-1 replication in natively HIV-1-susceptible immune cell populations and to better understand the contribution of the potential bias toward R5 virus replication to viral pathogenesis and disease progression. Related efforts will be directed toward the development of novel CPP-based antiviral agents that can serve as multifunctional HIV-1 inhibitors. These efforts will address CPP potency, stability, mechanism of action, and combined activity as these agents are advanced into preclinical investigations and clinical trials.
These studies were supported by faculty development funds provided by the Department of Microbiology and Immunology, Drexel University College of Medicine and the Institute for Molecular Medicine and Infectious Disease. The authors would like to thank Dr. Brian Wigdahl for lively discussions and insightful contributions relevant to this work, and the critical review of this paper prior to its submission.