- Email: [email protected]
Identification of the potential regions of Epap-1 that interacts with V3 loop of HIV-1 gp120
Biochimica et Biophysica Acta 1834 (2013) 780–790
Contents lists available at SciVerse ScienceDirect
Biochimica et Biophysica Acta journal homepage: www.elsevier.com/locate/bbapap
Identification of the potential regions of Epap-1 that interacts with V3 loop of HIV-1 gp120 C. Bhaskar a, Palakolanu S. Reddy a, K. Sarath Chandra a, Sudeep Sabde a, b, Debashis Mitra a, b, Anand K. Kondapi a,⁎ a b
Department of Biotechnology, School of Life Sciences, University of Hyderabad, PO Central University, Hyderabad 500 046, Andhra Pradesh, India National Centre for Cell Science, Pune University Campus, Ganeshkhind, Pune - 411007 India
a r t i c l e
i n f o
Article history: Received 18 August 2012 Received in revised form 14 December 2012 Accepted 16 January 2013 Available online 27 January 2013 Keywords: Epap-1 HIV-1 Pregnancy Peptide gp120-binding
a b s t r a c t Early pregnancy associated protein-1 (Epap-1), a 90 kDa glycoprotein present in first trimester placental tissue, inhibits HIV-1 entry through interaction with HIV-1 gp120 at V3 and C5 regions. In the present study, we have identified the specific 32 mer region of Epap-1 that can interact with V3 loop. This was achieved by docking between Epap-1 molecular model and gp120 and studying the interaction of peptides with gp120 in vitro. Out of four peptides analyzed, two peptides (P-2 and P-3) showed significant interaction with V3 domain (N = 8; N = 7) of gp120. In the studies conducted using soluble gp120 and virus, peptide P-2 has shown conserved interaction at V3 loop regions recognized by 257D and F425 antibodies and higher anti-viral activity. Also, P-2 inhibited cell fusion mediated dye transfer between gp120 expressing HL2/3 and CD4 expressing Sup T1 cells suggesting its inhibition of viral entry, which is further confirmed by its action on HIV infection mediated by Tat activated beta gal expression in TZM-bl cells. Further optimization of P-2 peptide showed that the anti-viral activity and gp120 interaction residues lie in the N-terminal region of the peptide. These results together suggest that P-2 inhibits viral entry through specific interaction at V3 loop region. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Human immunodeficiency virus (HIV) is a lentivirus (a member of the retrovirus family) that causes acquired immunodeficiency syndrome (AIDS), a condition in humans in which the immune system fails, finally leading to life-threatening opportunistic infections. Current treatment for HIV infection consists of highly active antiretroviral therapy (HAART) which combines at least two classes of antiretroviral agents. Even though the HAART regimens are effective in many patients various off-target effects [1] were reported which include long term toxicities [2], drug–drug interactions [3] and emergence of drug resistant strains leading to their decreased potency [4–7]. As a consequence, the identification of new drugs that inhibit viral replication is a pressing need for the treatment of HIV patients. One stage of the HIV life cycle that presents targets for therapeutic intervention is the entry of virus into host cells. Drugs that block HIV entry are collectively known as entry inhibitors, which comprise a complex group of drugs with multiple mechanisms of action. Neutralization of viral surface antigen gp120–gp41 is a natural defense phenomena occurring in infected condition [8]. Immune system uses its arms in targeting envelope through neutralizing antibodies [9–14], cytotoxic T cell responses [15,16] and innate responses [17–22]. Early pregnancy associated protein-1 (Epap-1) is a 90 kDa protein expressed during the first trimester of pregnancy [23] that inhibits ⁎ Corresponding author. Tel.: +91 40 3134571; fax: +91 40 23010145/120. E-mail address: [email protected] (A.K. Kondapi). 1570-9639/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.bbapap.2013.01.017
the viral entry into the host cell by binding to multiple regions of gp120 spanning V3 and C5 regions [24]. Since the recombinant form expressed in Escherichia coli exhibits gp120 binding and anti-HIV-1 activity, the unmodified Epap-1 is adequate for biological activity. Further, the protease digest of Epap-1 retained anti-HIV-1 activity (data not shown) which suggests that the peptide components of Epap-1 are competent for anti-HIV-1 activity. Thus, the present study involved the use of molecular modeling techniques to simulate binding of Epap-1 and gp120 for the identification of specific regions in Epap-1 involved in gp120 interaction. The results presented in the manuscript show neutralization of HIV-1 infection by Epap-1 derived peptide, P-2 with significant binding to gp120 at V3 loop domain. 2. Materials 2.1. Reagents The reagents were 30-azido-30-deoxythymidine (AZT), T-20 (Virchow Biotech), Calcein AM and Calcein Blue AM (Molecular Probes, USA), RPMI 1640, DMEM and FBS (Invitrogen, Carlsbad, CA), and RITC. Peptides were synthesized at Vimta Labs, Hyderabad. 2.2. Cell lines The following reagents were obtained through the AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH, USA:
C. Bhaskar et al. / Biochimica et Biophysica Acta 1834 (2013) 780–790
HL2/3 cells (Dr. B. Felbar and G. Pavlakis) expressing IIIB HIV-1 Env, CD4+ SupT1 cells (Dr. J. Hoxie), colo205, cos7 cells, TZM-bl cells and SK-N-SH cells.
781
GROMACS (unpublished results). This model was validated by L-ALIGN and EMBOSSES. 3.2. Molecular docking
2.3. Antibodies The various epitope specific gp120 monoclonal antibodies were V2 specific: 697-30D, V3 epitope: 257-DIV (cf 257D) and C5 region: 670-30D (Dr. S. Zolla-Pazner); V3 loop: V3-21, SVEINCTRPNNNTRKSI, 298–315 (Dr. J. Laman); V3 loop: F425 B 4a.1 (cf F425) and gp41: F240 (Dr. M. Posner and Dr. L. Cavacini); CD4 Mab: SIM4 (Dr. J.E.K. Hildreth); C3 reactive: B32-FFY, 382–384; C1 reactive: B2 — FNMW, 94–97; C2: B13-TQLLLN, 257–262; and V4 domain: B15 (Dr. G.W. Lewis).
Molecular docking was carried out between Epap-1 and gp120 trimers [25] (obtained from PDB) using HEX 5.6 software [26,27]. Based on the docking conformation, potential gp120 interacting peptides of Epap-1 were selected for analysis of their interaction with gp120. 3.3. Analysis of cytotoxicity of peptides
The following two HIV-1 strains were procured from NIH–AIDS Research and reference reagent program, USA: HIV-193IN101 is of biotype-NSI (R5) (Dr. R. Bollinger), isolated from a seropositive individual in India. HIV-1MN and HIV-1III B are of biotype-SI (X4) (Dr. R. Gallo). HIV-1CEM-50 was obtained from Dr. Robin Mukhopadhyaya, Cancer Research Institute Tata Memorial centre, Mumbai, India. HIV-193RW024 (The UNAIDS Network for HIV Isolation and Characterization, and the DAIDS, NIAID). HIV-1NL-43.
Reduction of 3-(4,5-dimethylthiozol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, Sigma) is chosen as a cell viability measurement. Cell lines (0.2×106) in RPMI 1640, 10% FBS were seeded in 96 well plates. Increasing concentrations of peptides (50, 100, 150 μg/ml) were added to the cells and incubated at 37 °C for 14 h in a CO2 incubator with 5% CO2. The media was replaced with a fresh growth medium along with 20 μl of 3-(4,5-dimethylthiozol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, Sigma). After incubation for 4 h in a humidified atmosphere, the media was removed and 200 μl of 0.1 N acidic isopropyl alcohol was added to the wells to dissolve the MTT-formazan crystals. The absorbance was recorded at 570 nm immediately after the development of a purple color. Each experiment was conducted in triplicate and the data was represented as an average with standard deviation.
3. Methods
3.4. Anti-viral activity in SupT1 cells
3.1. Epap-1 model
One million SupT1 cells with 100% viability were seeded with RPMI 1640, 0.1% FBS in 12-well plates. Increasing concentrations of peptides (50, 100, 150 μg/ml) were added to the cells and they were infected with HIV-1IIIB at a final concentration of virus equivalent to 2 ng of
2.4. Viruses
Structural modeling of Epap-1 was done using the threading technique on WURST server and the energy minimization was done by
Fig. 1. Molecular structure of Epap-1 and Epap-1 — gp120 docked conformation: Epap-1 was modeled and docked with gp120 trimeric complex for the identification of potential interacting regions.
782
C. Bhaskar et al. / Biochimica et Biophysica Acta 1834 (2013) 780–790
p24 per ml. The infected cells were incubated at 37 °C and 5% CO2 incubator for 2 h. After 2 h, the cells were pelleted at 350 ×g for 10 min, the supernatant was discarded and the cells were washed with RPMI 1640 containing 10% FBS. The cells were resuspended in the same medium and incubated for 96 h. The supernatants were collected after 96 h and analyzed using p24 antigen capture assay kit (ABL kit).The infection in the absence of Epap-1 was considered to be 0% inhibition. T20 is taken as positive control. The result given is an average of three independent experiments. Percent inhibition is computed by percent ratio of virus replicated in the presence of test sample to that in the absence of test sample. 3.5. Conjugation of peptides with RITC and binding to gp120 expressing HL2/3 cells Rhodamine isothiocyanate (30 μl, 1 mg/ml PBS) was added to 50 μg of peptide and kept for overnight incubation at 4 °C. The conjugated peptide was dialysed for 1 h against PBS. The RITC conjugated peptides were incubated with HL2/3 cells for 1 h. The cells were washed with PBS and checked for peptide binding using confocal microscope. CD4 expressing SupT1 cells were used as control.
3.6. Dye transfer assay to examine cell fusion 3.6.1. Calcein AM labeling (ex/em 496/517) HL2/3 cells expressing HIV-1IIIB gp120 on surface were incubated with 0.5 μM of Calcein AM for 1 h at 37 °C. After washing, the cells were incubated in fresh medium for 30 min at 37 °C. The cells were again washed and resuspended in complete medium at 1 million cells/ml.
3.6.2. Calcein Blue loading (ex/em 354/469) SupT1 cells were loaded with 20 μM of Calcein Blue for 1 h at 37 °C. After washing, the cells were incubated in fresh medium for 30 min at 37 °C. The cells were again washed and resuspended in complete medium at 1 million cells/ml. Fluorescently labeled gp120–41 expressing (HL2/3) cells and CD4+, CXCR4+ (SupT1) cells were co-cultured at 1:1 ratio for 2 h at 37 °C. Cell fusion was monitored using a dye redistribution assay. The fusion inhibition was checked in the presence of active peptides (100 μg/ml). The cell fusion in the absence of peptides was considered to be control cell fusion.
Fig. 2. Structure and molecular interactions of peptides: The above boxes show the structure and the Ramachandran plot in the upper panel of all the five peptides whereas the lower panel indicates the number of interactions showed by each peptide with gp120. Here peptides P-1 and P-4 exhibited only one interaction whereas peptides P-2, P-3 and P-2.1 showed 8, 7 and 10 interactions respectively.
C. Bhaskar et al. / Biochimica et Biophysica Acta 1834 (2013) 780–790
3.7. Reporter based assay in TZM-bl cells Action of peptides on virus infection is further confirmed by using a reporter cell line, TZM-bl which upon HIV infection leads to Tat induced LTR-associated β-gal expression in them. In the control HIV infection, the cells express β-gal which reduces the substrate, X-gal when added. The reduced X-gal fluoresces in the presence of azo dye (fast red violet). 1 million TZM-bl cells with 100% viability were seeded with DMEM, 0.1% FBS in 12-well plates. The concentration of peptides and T-20 used was 100 μg/ml and 1 μg/ml respectively. The cells were incubated at 37 °C in a CO2 (5%) incubator for 48 h. X-gal with azo dye was added to count the number of fluorescent cells. No fluorescence was observed in the presence of T20 which is used as positive control. 3.8. Peptide interaction with gp160 in the presence of receptor Mouse monoclonal anti-human gp160 antibodies spanning different regions of HIV-1 gp160 were added into wells of 96-well RIA plate at 10 ng per well in PBS and the plates were incubated overnight. The following day the wells were blocked with 3% BSA for 2 h at 37 °C. Binary complexes containing gp160-peptides were formed by incubation of 10 ng of gp160 in PBS with increasing concentrations of peptides (100–400 ng/ml) at 37 °C for 1 h. These complexes were captured with gp160 monoclonal antibody pre-coated wells and incubated for 1 h at 37 °C. The unbound complexes were removed by washing thrice with wash buffer. Captured binary complexes were probed for the peptides using 10 ng of affinity purified rabbit polyclonal anti-human Epap-1 antibody by incubating for 1 h at 37 °C. After washing the wells with wash buffer the bound rabbit polyclonal was probed with 1:2000 dilution of goat-anti-rabbit IgG-peroxidase antibody by incubating at
783
37 °C for 30 min. Finally the wells were washed and developed with TMB substrate system. The reaction was stopped after 30 min with 1 N HCl and the plates were read at 450 nm. Each experiment was done in triplicates for which average and standard deviations were calculated. The same experiment was repeated in the presence of 10 ng of receptor. 3.9. Peptide interaction with virus surface gp120 HIV-1 virus (10 ng) was incubated in the presence of different concentrations of peptides (100–400 ng/ml). Bound complex of HIV-1 and peptide was captured by mouse monoclonal gp160 antibodies spanning different regions of gp160 as mentioned in the previous protocol. The captured complex was estimated for HIV-1 bound in terms of p24 released with 1% of Triton X-100 using p24 antigen capture assay kit of Advanced Bioscience Laboratories. 4. Results 4.1. Molecular analysis of Epap-1 and gp120 interaction Epap-1 amino acid sequence was derived from the cDNA sequence based on degeneracy code [24]. Since the homology of Epap-1 was b35% with the existing sequence data, the structure was derived by fold recognition using threading on WURST server [28]. The structure was optimized by the energy minimization using GROMACS software [29]. Optimized structure (Fig. 1A) was further validated with L ALIGN [30] and EMBOSSES [31] which has given 78.4% and 78.8% homology. Molecular docking was carried out between Epap-1 (Fig. 1A) and gp120 trimers (obtained from PDB, Fig. 1B) and based on the
Fig. 3. Cytotoxic profile of peptides: All the 4 panels represent the cytotoxic profile of the five peptides in different cell lines viz., SupT1, colo205, SK-N-SH and cos7 cell lines. Even at 150 μg/ml concentration only up to 20% toxicity was exhibited by the peptides (n = 3,*p b 0.05, **p b 0.001).
784
C. Bhaskar et al. / Biochimica et Biophysica Acta 1834 (2013) 780–790
Fig. 4. Binding affinity of peptides: First panel shows the confocal images of binding affinity of all the peptides to HL2/3 cells. Here all the peptides bound to HL2/3 cells with the high efficiency. Whereas none of the peptides bound to SupT1 (second panel) cells that possess CD4 on its surface and the scrambled peptide bound to neither of the cells.
docking conformation (Fig. 1C), four potential regions of Epap-1 were selected for analysis of their interaction with gp120 (indicated in parenthesis): P-1. H2N-CNPSLVPPSILISFAATRTKRMAYTPFTSNIC-COOH (114–147) (GLU246 of gp120) P-2. H2N-RSTCALTAATAKAYATRSLEHRVVYRILHDC-COOH (383–412) (VAL270-GLN352 of gp120) P-3. H2N-VYNLDCLFRSICLHPWWWWMGVYNLVRDFITLH-COOH (614–646) (GLU268-LYS357 of gp120) P-4. H2N-SIMCLVLVISSKNKTPGPLTIVQVPAVATCFML-COOH (685– 718) (GLN267 of gp120) Scr-P-2. H2N-RKLTADVRACYELRITVAASTRHYCSHALAT-COOH (scrambled peptide as control). Fig. 5. Antiviral activity of peptides: Panel shows the antiviral activity of peptides in SupT1 cells. Out of all peptides P-2 has shown 80% antiviral activity and P-3 has shown ~40% activity at 100 μg/ml concentration. Peptides P-1, P-4 and Scr-P-2 were inactive (n=3, *pb 0.05, **pb 0.001).
These peptides possess molecular interactions with gp120 in terms of 1, 8, 7, and 1 numbers of interactions for P-1, P-2, P-3 and P-4 respectively (Fig. 2). These were synthesized commercially and analyzed for their affinity to gp120 and anti-HIV activity.
C. Bhaskar et al. / Biochimica et Biophysica Acta 1834 (2013) 780–790
4.2. Action of peptides on viability of various cell lines Peptides were analyzed for their cytotoxicity against four cell lines, viz. SupT1, colo205, SK-N-SH, and cos7 by using MTT as per protocol mentioned. The results showed that all five peptides were not cytotoxic (b20%) in these cell lines at 150 μg/ml concentration (Fig. 3). 4.3. Interaction of peptides with HL2/3 cells To assess the affinity of peptides toward gp120, they were conjugated with RITC. HL2/3 cells which express gp120 on their surface and SupT1 cells that express CD4 and CXCR-4 were incubated with RITC conjugated peptides (50 μg/ml) separately for 1 h at 37 °C in CO2 incubator. The results in Fig. 4 showed the peptides bound significantly to HL2/3 and did not bind to SupT1 cells whereas the scrambled peptide exhibited interaction to neither of the cells. 4.4. Anti-HIV-1 activity of peptides CD4 positive, SupT1 cells (0.5 million) were challenged with HIV-1IIIB (2 ng/ml) in the presence of 50–150 μg/ml of peptides respectively. The results presented in Fig. 5 show that peptides, P-2 and P-3 significantly inhibit the replication of HIV-1, while peptides P-1, P-4 and Scr-P-2 were inactive. Since P-2 was potent among four peptides with the IC-50 value of 59 μg (data not shown), it was taken as the lead molecule to determine the mode of action. 4.5. Interaction of P-2 with soluble gp160 To understand the molecular interaction of P-2 with gp160, the gp160 was incubated with increasing concentrations of P-2 (100– 400 ng) and the bound complexes were captured onto various anti-gp160 monoclonal antibody coated wells. The binary complexes were detected with Epap-1 polyclonal antibody. The results shown in Fig. 6 (panel A) suggest that P-2 interacts specifically with V3 domain and V3 loop of gp160 indicated by its inhibition of gp160 binding to F425 and 257D antibodies. Further, the interaction of P-2 with V3
785
domain not only remains unaltered but also increases in the presence of CD4 (Fig. 6, panel B). A separate complex of gp160-CD4 in the absence of P-2 was incubated with SIM.4 and V3 region antibodies and developed with anti-gp120 antibody which confirmed the activity of soluble CD4 employed (supplementary data, panel A). 4.6. Interaction of P-2 with virus surface gp120 Since the gp120 epitopes exposed in soluble gp160 may be different from that of the epitopes exposed on the viral surface, studying the interaction of P-2 with virus surface gp120 would be functionally relevant. The virus was captured using monoclonal antibodies spanning gp120 and gp41 regions in the presence of P-2. The exposure of various epitopes of gp120 in the presence of P-2 was monitored in terms of the amount of the virus bound to the corresponding antibody. The results show that P-2 binds to V3 domain of the virus surface gp120 (Fig. 7A). The same result was repeated when the virus was pre-incubated with P-2 and CD4 before the addition of CD4 (Fig. 7B) and P-2 (Fig. 7C) correspondingly. A separate complex of virus-CD4 in the absence of P-2 was incubated with SIM.4 and V3 region antibodies and detected for p24 levels which confirmed the activity of soluble CD4 employed (supplementary data, panel B). Further molecular analysis of P-2 peptide with gp120 revealed that all the major possible interactions lie in the N-terminal region. The docking was performed by taking the first 15 residues of P-2 peptide with gp120. The first 15 residues of P-2 which was named as P-2.1 (H2N-RSTCALTAATAKAYA-COOH) showed 10 interactions (Fig. 2). To experimentally confirm these interactions, this peptide was synthesized commercially and analyzed for their anti-HIV activity and molecular action on gp120 mediated cell fusion. 4.7. Action of peptides on reporter based TZM-bl cells Anti-HIV-1 activity of peptides was confirmed by reporter based assay. TZM-bl cells were challenged with HIV-1 in the presence and absence of peptides. Expression of β-gal occurred during HIV infection in the control TZM-bl cells and the reduction of X-gal took place. The presence of T20 (1 μg/ml) and peptides (100 μg/ml)
Fig. 6. Peptide binding with soluble gp160: Panel (A) shows the interaction of peptide P-2 with V3 region which is proven by the masking of V3 region of soluble gp160 by it, thereby preventing the interaction of anti-V3 antibodies viz., F425 and 257-D respectively. The same interaction is enhanced in the presence of CD4 which is reported in panel (B) (n = 3, *p b 0.05, **p b 0.001).
786 C. Bhaskar et al. / Biochimica et Biophysica Acta 1834 (2013) 780–790 Fig. 7. Peptide interaction with virus surface gp120: Panel (A) shows the interaction of peptide P-2 with V3 region which is shown by the masking of V3 region of virus surface gp120 and preventing the interaction of anti-V3 antibodies viz., F425 and 257-D respectively. The same interaction is enhanced when the assay is performed with gp120–P2 complex prior to the addition of CD4 (panel B) and also in another combination of gp120–CD4 complex prior to the addition of P2 (panel C) (n = 3,*p b 0.05, **pb 0.001).
C. Bhaskar et al. / Biochimica et Biophysica Acta 1834 (2013) 780–790
787
Fig. 8. Inhibition of viral entry by peptides in reporter cell line, TZM-bl: Panel (A) indicates the confocal image of the TZM-bl cells in which lane 1 shows the expression of β-gal upon HIV-1 infection in control cells. Lanes below indicate the prevention of infection by T-20 (1 μg/ml-positive control) and peptides, P-2 and P-2.1 (100 μg/ml) which was reflected in terms of reduction in β-gal expression. The same assay was repeated with FACS to count the number of cells fluoresced which were shown in panel (B). Scrambled peptide which is used as negative control did not stop the infection.
inhibited the viral infection and the subsequent expression of β-gal (Fig. 8, panel A). The results of FACS analysis presented in Fig. 8 (panel B) confirm that peptides possess affinity to gp120 and inhibit HIV-1 infection. Here the scrambled peptide could not prevent the HIV infection confirming the specificity of peptides.
4.8. Action of peptides on gp120 mediated cell fusion HL2/3 cells and SupT1 cells were incubated in the presence and absence of peptides (100 μg/ml) for 2 h. After extensive washing with PBS the cells were analyzed using confocal microscopy. The
788
C. Bhaskar et al. / Biochimica et Biophysica Acta 1834 (2013) 780–790
results show that peptides P-2 and P-2.1 indeed bound to HL2/3 cells and inhibited the fusion of SupT1 cells and HL2/3 cells, whereas the scrambled peptide failed to prevent the fusion (Fig. 9).
4.9. Action of peptides on multiple strains of HIV-1 The peptides, P-2 and P-2.1 exhibited neutralization activity against viral strains namely CEM 50, 93RW024, MN and NL-43 of HIV-1. At 50 μg/ml concentration, the peptides showed 40–80% antiviral activity against the HIV-1 isolates analyzed (Fig. 10).
5. Discussion Even though Epap-1 is a human protein, its immunological properties were unexplored. The stability of this 90 kDa protein and its bioavailability are the major concerns if it was promoted as a lead molecule for therapeutic application. In previous studies, Epap-1 was shown to interact with V3 and C5 regions of gp120 and the identification of specific region in Epap-1 interacting with these regions will decipher the mode of action of naturally occurring Epap-1. Since the crystal structure of Epap-1 was unavailable, we have modeled its structure and docked it with gp120 to obtain the exact region that will help in virus neutralization. Molecular analysis of
Fig. 9. Inhibition of cell fusion by peptides: Top 2 panels showed the interaction of CD4+ SupT1 cells (blue) and gp120+ HL2/3 cells (yellow) with each other in the absence of an inhibitor and presence of scrambled peptide which results in the successful fusion of cells (arrows in red shows the fusion of cells). Whereas the presence of peptides (100 μg/ml) prevented the interaction between both the cells, indicated by the inhibition of dye transfer (lower panels).
C. Bhaskar et al. / Biochimica et Biophysica Acta 1834 (2013) 780–790
789
One of the major limitations in the present study is the inability in predicting the C5 binding region of the Epap-1 with the existing molecular model. The other is the higher IC-50 value of the peptides. The identification of the C5 binding domain in the Epap-1 may result in getting potent peptides which can act in a concerted manner with the existing peptides that can enhance the possibility of reducing the IC-50 value to the nanomolar level. The important and immediate focus is to reduce the IC-50 value of the peptide by measuring the binding strength between the peptides and gp120. Further studies are under way to assess the activity after creating mutations in the potent peptides by scanning mutagenesis approach. 6. Conclusion
Fig. 10. Antiviral action of peptides on different strains of HIV: Here the peptides show antiviral action on the different viral strains namely CEM 50, 93RW024, MN and NL-43 of HIV-1. At 50 μg/ml of final concentration the peptides showed 40–80% antiviral action on all the strains (n = 3,*p b 0.05, **p b 0.001).
The present study identified Epap-1 derived peptide with multiple interactions in V3 domain of gp120 which can block the viral entry and thereby leading to the neutralization of virus infection. Supplementary data to this article can be found online at http:// dx.doi.org/10.1016/j.bbapap.2013.01.017. Role of the funding source
peptides in the presence of gp160 helped in the identification of a 32 mer peptide with significant activity. Further, molecular analysis yielded 15 mer peptide with significant HIV-1 neutralizing activity. Epap-1 derived 32 mer peptides principally interact with gp120 in a region surrounding the V3 loop involving Glu 268 to Lys 357, while V3 region spans from aa 296 to 331 depending upon the viral isolate. Most importantly residues Alanine and Argenine of Epap-1 are involved in interaction with gp120. These results can be confirmed though our observation of the region in gp120 is blocked by the binding of peptides to the epitopes recognized by 257D and F425 monoclonal antibodies. It was shown that F425 is a broadly neutralizing antibody that binds to the tip of V3 and the presence of Ile309, Arg315 and Phe317 is important for high-affinity binding [32]. On the other hand, mAb 257D binds strongly to two adjacent peptides, RKRIHI and KRIHIG, showing that it recognizes the smallest reactive peptide KRIHI which is located to the left of the conserved tip of the V3 loop. The flanking N- and C-terminal Arginine and Glycine residues of V3 play a role in the binding to this mAb [33]. The V3 loop region is of high importance since it is involved in many aspects of HIV-1 viral infectivity. The V3 loop was termed as the “principal neutralizing determinant” [34] because peptides containing the V3 region can induce neutralizing antibodies [35–37] and is arguably a potential target for HIV vaccine design [38]. The possibility of generating an effective vaccine based on intact gp120 or peptides containing the V3 loop region was decreased because even though the constructs induced a strong immune response toward V3, the antibodies generated happened to neutralize only laboratory-adapted isolates (TCLA) [39,40] and are specific for only a small number of strains [41]. This paradox is due to the differential exposure of V3 on various strains, and at different stages of the viral entry thereby decreasing the accessibility of the V3 region to antibody. Primary viral isolates, which utilize the CCR5 coreceptor are extremely resistant to neutralization by the majority of antibodies, including directed against the V3 loop. This may be due to the inaccessibility of V3 loop in primary isolates, either due to carbohydrate masking or because of interactions with the gp120 complex. Recent studies have shown that V3 may be shielded by the V1/V2 region [42,43], thus supporting the results from earlier investigations into the functional properties of V1/V2 [44,45]. Even if V3 region is not exposed on the surface, an interaction with adjoining residues of V3 loop such as V2 region may initiate nucleation of peptide interaction followed by alignment at V3 domain. So the identification of the potential regions in Epap-1, especially peptides may provide insight into a mechanism that will assist in addressing the above difficulties to develop a therapy with broadly neutralizing effect.
Funding source has no role in design, execution and interpretation of the results incorporated in the manuscript. Acknowledgements We acknowledge the financial assistance under DBT sponsored research project. CB was ICMR SRF and currently UoH-DBT-CREBB postdoctoral fellow. Infrastructure developed through UGC funded CAS and XI plan used for the work. We acknowledge contributors of antibodies and viral strains from NIH–AIDS reagent and reference program. Antibodies: C5 region: 670-30D (Dr. S. Zolla-Pazner); V3 loop: V3-21, 298–315 (Dr. J. Laman); V3 loop: F425 B 4a.1 (cf F425) and gp41: F240 (Dr. M. Posner and Dr. L. Cavacini); CD4 Mab: SIM4 (Dr. J.E.K. Hildreth); C3 reactive: B32-FFY, 382–384 (Dr. G.W. Lewis). Viruses: HIV-193IN101 (Dr. R. Bollinger), HIV-1MN is of biotype-SI (X4) (Dr. R. Gallo), HIV-1CEM-50 (Dr. Robin Mukhopadhyaya) HIV-1 93RW024, and HIV-1 94UG103 (The UNAIDS Network for HIV Isolation and Characterization, and the DAIDS, NIAID). References [1] G.M. Lucas, R.E. Chaisson, R.D. Moore, Highly active antiretroviral therapy in a large urban clinic: risk factors for virologic failure and adverse drug reactions, Ann. Intern. Med. 131 (1999) 81–87. [2] A. Carr, D.A. Cooper, Adverse effects of antiretroviral therapy, Lancet 356 (2000) 1423–1430. [3] S.C. Piscitelli, C. Flexner, J.R. Minor, M.A. Polis, H. Masur, Drug interactions in patients infected with human immunodeficiency virus, Clin. Infect. Dis. 23 (1996) 685–693. [4] D. Boden, A. Hurley, L. Zhang, Y. Cao, Y. Guo, E. Jones, J. Tsay, J. Ip, C. Farthing, K. Limoli, N. Parkin, M. Markowitz, HIV-1 drug resistance in newly infected individuals, JAMA 282 (1999) 1135–1141. [5] D. Finzi, J. Blankson, J.D. Siliciano, J.B. Margolick, K. Chadwick, T. Pierson, Latent infection of CD4+ T cells provides a mechanism for lifelong persistence of HIV-1, even in patients on effective combination therapy, Nat. Med. 5 (1999) 512–517. [6] S.A. Wegner, S.K. Brodine, J.R. Mascola, S.A. Tasker, R.A. Shaffer, M.J. Starkey, A. Barile, G.J. Martin, N. Aronson, W.W. Emmons, K. Stephan, S. Bloor, J. Vingerhoets, K. Hertogs, B. Larder, Prevalence of genotypic and phenotypic resistance to anti-retroviral drugs in a cohort of therapy-naive HIV-1 infected US military personnel, AIDS 14 (2000) 1009–1015. [7] S. Yerly, L. Kaiser, E. Race, J.P. Bru, F. Clavel, L. Perrin, Transmission of antiretroviral-drug-resistant HIV-1 variants, Lancet 354 (1999) 729–733. [8] Q.J. Sattentau, J.P. Moore, Human immunodeficiency virus type 1 neutralization is determined by epitope exposure on the gp120 oligomer, J. Exp. Med. 182 (1995) 185–196. [9] E. Emini, P. Nara, W. Schlief, J. Lewis, J. Davide, D. Lee, J. Kessler, S. Conley, M. Matsushita, S. Putney, R. Gerety, J. Eichberg, Antibody-mediated in vitro neutralization of human immunodeficiency virus type 1 abolishes infectivity for chimpanzees, J. Virol. 64 (1990) 3674–3678.
790
C. Bhaskar et al. / Biochimica et Biophysica Acta 1834 (2013) 780–790
[10] D. Ho, et al., Conformational epitope on gp120 important in CD4 binding and human immunodeficiency virus type 1 neutralization identified by a human monoclonal antibody, J. Virol. 65 (1991) 489–493. [11] M. Posner, et al., An IgG human monoclonal antibody which reacts with HIV-1 gp120, inhibits virus binding to cells, and neutralizes infection, J. Immunol. 146 (1991) 4325–4332. [12] M. Thali, et al., Discontinuous, conserved neutralization epitopes overlapping the CD4 binding region of the HIV-1 gp120 envelope glycoprotein, J. Virol. 66 (1992) 5635–5641. [13] M. Thali, et al., Characterization of conserved human immunodeficiency virus type 1 gp120 neutralization epitopes exposed upon gp120-CD4 binding, J. Virol. 67 (1993) 3978–3988. [14] A. Trkola, et al., Human monoclonal antibody 2G12 defines a distinctive neutralization epitope on the gp120 glycoprotein of human immunodeficiency virus type 1, J. Virol. 70 (1996) 1100–1108. [15] L. Musey, Y. Hu, L. Eckert, M. Christensen, T. Karchmer, M.J. McElrath, HIV-1 induces cytotoxic T lymphocytes in the cervix of infected women, J. Exp. Med. 185 (2) (1997) 293–303. [16] B. Rosok, P. Voltersvik, B.M. Larsson, J. Albert, J.E. Brinchmann, B. Asjö, CD8+ T cells from HIV type 1-seronegative individuals suppress virus replication in acutely infected cells, AIDS Res. Hum. Retroviruses 13 (1) (1997) 79–85. [17] C. Hayano, Accumulation of CD16+ cells with secretion of Ksp37 in decidua at the end of pregnancy, Am. J. Reprod. Immunol. 48 (1) (2002) 57–62. [18] J.S. Hunt, M.G. Petroff, T.G. Burnett, Uterine leukocytes: key players in pregnancy, Semin. Cell Dev. Biol. 11 (2) (2000) 127–137. [19] K. Ohtani, Y. Suzuki, S. Eda, T. Kawai, T. Kase, H. Keshi, Y. Sakai, A. Fukuoh, T. Sakamoto, H. Itabe, T. Suzutani, M. Ogasawara, I. Yoshida, N. Wakamiya, The membrane-type collectin CL-P1 is a scavenger receptor on vascular endothelial cells, J. Biol. Chem. 276 (2001) 44222–44228. [20] B.K. Patterson, H. Behbahani, W.J. Kabat, Y. Sullivan, M.R. O'Gorman, A. Landay, Z. Flener, N. Khan, R. Yogev, J. Andersson, Leukemia inhibitory factor inhibits HIV-1 replication and is upregulated in placentae from non transmitting women, J. Clin. Invest. 107 (2001) 287–294. [21] P. Sedlmyr, A. Blaschitz, R. Wintersteiger, M. Semlitsch, A. Hammer, C.R. MacKenzie, W. Walcher, O. Reich, O. Takikawa, G. Dohr, Localization of indoleamine 2,3-dioxygenase in human female reproductive organs and the placenta, Mol. Hum. Reprod. 8 (2002) 385–391. [22] P. Tiensiwakul, Stromal cell-derived factor (SDF) 1-3′A polymorphism may play a role in resistance to HIV-1 infection in seronegative high-risk Thais, Intervirology 47 (2) (2004) 87–92. [23] A.K. Kondapi, M.A. Hafiz, T. Sivaram, Anti-HIV activity of a glycoprotein from first trimester placental tissue, Antivir. Res. 54 (2002) 47–57. [24] K.P. Roda Rani, Dheeraj Pelluru, Anand K. Kondapi, A conserved molecular action of native and recombinant Epap-1 in inhibition of HIV-1 gp120 mediated viral entry, Arch. Biochem. Biophys. 456 (1) (2006) 79–92. [25] J. Liu, A. Bartesaghi, M.J. Borgnia, G. Sapiro, S. Subramaniam, Molecular architecture of native HIV-1 gp120 trimers, Nature 455 (7209) (2008) 109–113. [26] D.W. Ritchie, Recent progress and future directions in protein–protein docking, Curr. Protein Pept. Sci. 9 (1) (2008) 1–15. [27] G. Macindoe, L. Mavridis, V. Venkatraman, M.D. Devignes, D.W. Ritchie, HexServer: an FFT-based protein docking server powered by graphics processors, Nucleic Acids Res. 38 (2010) W445–W449. [28] A.E. Torda, J.B. Procter, T. Huber, Wurst: a protein threading server with a structural scoring function, sequence profiles and optimized substitution matrices, Nucleic Acids Res. 32 (2004) W532–W535, (Web Server issue). [29] D. Van Der Spoel, E. Lindahl, B. Hess, G. Groenhof, A.E. Mark, H.J. Berendsen, GROMACS: fast, flexible, and free, J. Comput. Chem. 26 (16) (2005) 1701–1718. [30] X. Huang, W. Miller, A time-efficient linear-space local similarity algorithm, Adv. Appl. Math. 12 (1991) 337–357.
[31] P. Rice, I. Longden, A. Bleasby, EMBOSS: the European Molecular Biology Open Software Suite, Trends Genet. 16 (6) (2000) 276–277. [32] R. Pantophlet, R.O. Aguilar-Sino, T. Wrin, L.A. Cavacini, D.R. Burton, Analysis of the neutralization breadth of the anti-V3 antibody F425-B4e8 and reassessment of its epitope fine specificity by scanning mutagenesis, Virology 364 (2007) 441–453. [33] J.Y. Xu, M.K. Gorny, T. Palker, S. Karwowska, S. Zolla-Pazner, Epitope mapping of two immunodominant domains of gp41, the transmembrane protein of human immunodeficiency virus type 1, using ten human monoclonal antibodies, J. Virol. 65 (9) (1991) 4832–4838. [34] K. Javaherian, A.J. Langlois, C. McDanal, K.L. Ross, L.I. Eckler, C.L. Jellis, A.T. Profy, J.R. Rusche, D.P. Bolognesi, S.D. Putney, T.J. Matthews, Principal neutralizing domain of the human immunodeficiency virus type 1 envelope protein, Proc. Natl. Acad. Sci. U. S. A. 86 (1989) 6768–6772. [35] J. Goudsmit, C. Debouck, R.H. Meloen, L. Smit, M. Bakker, D.M. Asher, A.V. Wolff, C.J. Gibbs Jr., D.C. Gajdusek, Human immunodeficiency virus type 1 neutralization epitope with conserved architecture elicits early type-specific antibodies in experimentally infected chimpanzees, Proc. Natl. Acad. Sci. U. S. A. 85 (1988) 4478–4482. [36] T.J. Palker, M.E. Clark, A.J. Langlois, T.J. Matthews, K.J. Weinhold, R.R. Randall, D.P. Bolognesi, B.F. Haynes, Type-specific neutralization of the human immunodeficiency virus with antibodies to env-encoded synthetic peptides, Proc. Natl. Acad. Sci. U. S. A. 85 (1988) 1932–1936. [37] J.R. Rusche, K. Javaherian, C. McDanal, J. Petro, D.L. Lynn, R. Grimaila, A. Langlois, R.C. Gallo, L.O. Arthur, P.J. Fischinger, D.P. Bolognesi, S.D. Putney, T.J. Matthews, Antibodies that inhibit fusion of human immunodeficiency virus-infected cells bind a 24-amino acid sequence of the viral envelope, gp120, Proc. Natl. Acad. Sci. U. S. A. 85 (1988) 3198–3202. [38] S. Zolla-Pazner, Improving on nature: focusing the immune response on the V3 loop, Hum. Antibodies 14 (3–4) (2005) 69–72. [39] J.R. Mascola, J. Louwagie, F.E. McCutchan, C.L. Fischer, P.A. Hegerich, K.F. Wagner, A.K. Fowler, J.G. McNeil, D.S. Burke, Two antigenically distinct subtypes of human immunodeficiency virus type 1: viral genotype predicts neutralization serotype, J. Infect. Dis. 169 (1994) 48–54. [40] T.J. Matthews, Dilemma of neutralization resistance of HIV-1 field isolates and vaccine development, AIDS Res. Hum. Retroviruses 10 (1994) 631–632. [41] T.J. Palker, M.E. Clark, A.J. Langlois, T.J. Matthews, K.J. Weinhold, R.R. Randall, D.P. Bolognesi, B.F. Haynes, Type-specific neutralization of the human immunodeficiency virus with antibodies to env-encoded synthetic peptides, Proc. Natl. Acad. Sci. U. S. A. 85 (6) (1988) 1932–1936. [42] C.P. Krachmarov, W.J. Honnen, S.C. Kayman, M.K. Gorny, S. Zolla-Pazner, A. Pinter, Factors determining the breadth and potency of neutralization by V3-specific human monoclonal antibodies derived from subjects infected with clade A or clade B strains of human immunodeficiency virus type 1, J. Virol. 80 (14) (2006) 7127–7135. [43] A. Pinter, W.J. Honnen, Y. He, M.K. Gorny, S. Zolla-Pazner, S.C. Kayman, The V1/V2 domain of gp120 is a global regulator of the sensitivity of primary human immunodeficiency virus type 1 isolates to neutralization by antibodies commonly induced upon infection, J. Virol. 78 (10) (2004) 5205–5215. [44] J. Cao, N. Sullivan, E. Desjardin, C. Parolin, J. Robinson, R. Wyatt, J. Sodroski, Replication and neutralization of human immunodeficiency virus type 1 lacking the V1 and V2 variable loops of the gp120 envelope glycoprotein, J. Virol. 71 (12) (1997) 9808–9812. [45] R. Wyatt, N. Sullivan, M. Thali, H. Repke, D. Ho, J. Robinson, M. Posner, J. Sodroski, Functional and immunologic characterization of human immunodeficiency virus type 1 envelope glycoproteins containing deletions of the major variable regions, J. Virol. 67 (8) (1993) 4557–4565; J. Virol. 76 (18) (Sep 2002) 9035–9045.
Contents lists available at SciVerse ScienceDirect
Biochimica et Biophysica Acta journal homepage: www.elsevier.com/locate/bbapap
Identification of the potential regions of Epap-1 that interacts with V3 loop of HIV-1 gp120 C. Bhaskar a, Palakolanu S. Reddy a, K. Sarath Chandra a, Sudeep Sabde a, b, Debashis Mitra a, b, Anand K. Kondapi a,⁎ a b
Department of Biotechnology, School of Life Sciences, University of Hyderabad, PO Central University, Hyderabad 500 046, Andhra Pradesh, India National Centre for Cell Science, Pune University Campus, Ganeshkhind, Pune - 411007 India
a r t i c l e
i n f o
Article history: Received 18 August 2012 Received in revised form 14 December 2012 Accepted 16 January 2013 Available online 27 January 2013 Keywords: Epap-1 HIV-1 Pregnancy Peptide gp120-binding
a b s t r a c t Early pregnancy associated protein-1 (Epap-1), a 90 kDa glycoprotein present in first trimester placental tissue, inhibits HIV-1 entry through interaction with HIV-1 gp120 at V3 and C5 regions. In the present study, we have identified the specific 32 mer region of Epap-1 that can interact with V3 loop. This was achieved by docking between Epap-1 molecular model and gp120 and studying the interaction of peptides with gp120 in vitro. Out of four peptides analyzed, two peptides (P-2 and P-3) showed significant interaction with V3 domain (N = 8; N = 7) of gp120. In the studies conducted using soluble gp120 and virus, peptide P-2 has shown conserved interaction at V3 loop regions recognized by 257D and F425 antibodies and higher anti-viral activity. Also, P-2 inhibited cell fusion mediated dye transfer between gp120 expressing HL2/3 and CD4 expressing Sup T1 cells suggesting its inhibition of viral entry, which is further confirmed by its action on HIV infection mediated by Tat activated beta gal expression in TZM-bl cells. Further optimization of P-2 peptide showed that the anti-viral activity and gp120 interaction residues lie in the N-terminal region of the peptide. These results together suggest that P-2 inhibits viral entry through specific interaction at V3 loop region. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Human immunodeficiency virus (HIV) is a lentivirus (a member of the retrovirus family) that causes acquired immunodeficiency syndrome (AIDS), a condition in humans in which the immune system fails, finally leading to life-threatening opportunistic infections. Current treatment for HIV infection consists of highly active antiretroviral therapy (HAART) which combines at least two classes of antiretroviral agents. Even though the HAART regimens are effective in many patients various off-target effects [1] were reported which include long term toxicities [2], drug–drug interactions [3] and emergence of drug resistant strains leading to their decreased potency [4–7]. As a consequence, the identification of new drugs that inhibit viral replication is a pressing need for the treatment of HIV patients. One stage of the HIV life cycle that presents targets for therapeutic intervention is the entry of virus into host cells. Drugs that block HIV entry are collectively known as entry inhibitors, which comprise a complex group of drugs with multiple mechanisms of action. Neutralization of viral surface antigen gp120–gp41 is a natural defense phenomena occurring in infected condition [8]. Immune system uses its arms in targeting envelope through neutralizing antibodies [9–14], cytotoxic T cell responses [15,16] and innate responses [17–22]. Early pregnancy associated protein-1 (Epap-1) is a 90 kDa protein expressed during the first trimester of pregnancy [23] that inhibits ⁎ Corresponding author. Tel.: +91 40 3134571; fax: +91 40 23010145/120. E-mail address: [email protected] (A.K. Kondapi). 1570-9639/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.bbapap.2013.01.017
the viral entry into the host cell by binding to multiple regions of gp120 spanning V3 and C5 regions [24]. Since the recombinant form expressed in Escherichia coli exhibits gp120 binding and anti-HIV-1 activity, the unmodified Epap-1 is adequate for biological activity. Further, the protease digest of Epap-1 retained anti-HIV-1 activity (data not shown) which suggests that the peptide components of Epap-1 are competent for anti-HIV-1 activity. Thus, the present study involved the use of molecular modeling techniques to simulate binding of Epap-1 and gp120 for the identification of specific regions in Epap-1 involved in gp120 interaction. The results presented in the manuscript show neutralization of HIV-1 infection by Epap-1 derived peptide, P-2 with significant binding to gp120 at V3 loop domain. 2. Materials 2.1. Reagents The reagents were 30-azido-30-deoxythymidine (AZT), T-20 (Virchow Biotech), Calcein AM and Calcein Blue AM (Molecular Probes, USA), RPMI 1640, DMEM and FBS (Invitrogen, Carlsbad, CA), and RITC. Peptides were synthesized at Vimta Labs, Hyderabad. 2.2. Cell lines The following reagents were obtained through the AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH, USA:
C. Bhaskar et al. / Biochimica et Biophysica Acta 1834 (2013) 780–790
HL2/3 cells (Dr. B. Felbar and G. Pavlakis) expressing IIIB HIV-1 Env, CD4+ SupT1 cells (Dr. J. Hoxie), colo205, cos7 cells, TZM-bl cells and SK-N-SH cells.
781
GROMACS (unpublished results). This model was validated by L-ALIGN and EMBOSSES. 3.2. Molecular docking
2.3. Antibodies The various epitope specific gp120 monoclonal antibodies were V2 specific: 697-30D, V3 epitope: 257-DIV (cf 257D) and C5 region: 670-30D (Dr. S. Zolla-Pazner); V3 loop: V3-21, SVEINCTRPNNNTRKSI, 298–315 (Dr. J. Laman); V3 loop: F425 B 4a.1 (cf F425) and gp41: F240 (Dr. M. Posner and Dr. L. Cavacini); CD4 Mab: SIM4 (Dr. J.E.K. Hildreth); C3 reactive: B32-FFY, 382–384; C1 reactive: B2 — FNMW, 94–97; C2: B13-TQLLLN, 257–262; and V4 domain: B15 (Dr. G.W. Lewis).
Molecular docking was carried out between Epap-1 and gp120 trimers [25] (obtained from PDB) using HEX 5.6 software [26,27]. Based on the docking conformation, potential gp120 interacting peptides of Epap-1 were selected for analysis of their interaction with gp120. 3.3. Analysis of cytotoxicity of peptides
The following two HIV-1 strains were procured from NIH–AIDS Research and reference reagent program, USA: HIV-193IN101 is of biotype-NSI (R5) (Dr. R. Bollinger), isolated from a seropositive individual in India. HIV-1MN and HIV-1III B are of biotype-SI (X4) (Dr. R. Gallo). HIV-1CEM-50 was obtained from Dr. Robin Mukhopadhyaya, Cancer Research Institute Tata Memorial centre, Mumbai, India. HIV-193RW024 (The UNAIDS Network for HIV Isolation and Characterization, and the DAIDS, NIAID). HIV-1NL-43.
Reduction of 3-(4,5-dimethylthiozol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, Sigma) is chosen as a cell viability measurement. Cell lines (0.2×106) in RPMI 1640, 10% FBS were seeded in 96 well plates. Increasing concentrations of peptides (50, 100, 150 μg/ml) were added to the cells and incubated at 37 °C for 14 h in a CO2 incubator with 5% CO2. The media was replaced with a fresh growth medium along with 20 μl of 3-(4,5-dimethylthiozol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, Sigma). After incubation for 4 h in a humidified atmosphere, the media was removed and 200 μl of 0.1 N acidic isopropyl alcohol was added to the wells to dissolve the MTT-formazan crystals. The absorbance was recorded at 570 nm immediately after the development of a purple color. Each experiment was conducted in triplicate and the data was represented as an average with standard deviation.
3. Methods
3.4. Anti-viral activity in SupT1 cells
3.1. Epap-1 model
One million SupT1 cells with 100% viability were seeded with RPMI 1640, 0.1% FBS in 12-well plates. Increasing concentrations of peptides (50, 100, 150 μg/ml) were added to the cells and they were infected with HIV-1IIIB at a final concentration of virus equivalent to 2 ng of
2.4. Viruses
Structural modeling of Epap-1 was done using the threading technique on WURST server and the energy minimization was done by
Fig. 1. Molecular structure of Epap-1 and Epap-1 — gp120 docked conformation: Epap-1 was modeled and docked with gp120 trimeric complex for the identification of potential interacting regions.
782
C. Bhaskar et al. / Biochimica et Biophysica Acta 1834 (2013) 780–790
p24 per ml. The infected cells were incubated at 37 °C and 5% CO2 incubator for 2 h. After 2 h, the cells were pelleted at 350 ×g for 10 min, the supernatant was discarded and the cells were washed with RPMI 1640 containing 10% FBS. The cells were resuspended in the same medium and incubated for 96 h. The supernatants were collected after 96 h and analyzed using p24 antigen capture assay kit (ABL kit).The infection in the absence of Epap-1 was considered to be 0% inhibition. T20 is taken as positive control. The result given is an average of three independent experiments. Percent inhibition is computed by percent ratio of virus replicated in the presence of test sample to that in the absence of test sample. 3.5. Conjugation of peptides with RITC and binding to gp120 expressing HL2/3 cells Rhodamine isothiocyanate (30 μl, 1 mg/ml PBS) was added to 50 μg of peptide and kept for overnight incubation at 4 °C. The conjugated peptide was dialysed for 1 h against PBS. The RITC conjugated peptides were incubated with HL2/3 cells for 1 h. The cells were washed with PBS and checked for peptide binding using confocal microscope. CD4 expressing SupT1 cells were used as control.
3.6. Dye transfer assay to examine cell fusion 3.6.1. Calcein AM labeling (ex/em 496/517) HL2/3 cells expressing HIV-1IIIB gp120 on surface were incubated with 0.5 μM of Calcein AM for 1 h at 37 °C. After washing, the cells were incubated in fresh medium for 30 min at 37 °C. The cells were again washed and resuspended in complete medium at 1 million cells/ml.
3.6.2. Calcein Blue loading (ex/em 354/469) SupT1 cells were loaded with 20 μM of Calcein Blue for 1 h at 37 °C. After washing, the cells were incubated in fresh medium for 30 min at 37 °C. The cells were again washed and resuspended in complete medium at 1 million cells/ml. Fluorescently labeled gp120–41 expressing (HL2/3) cells and CD4+, CXCR4+ (SupT1) cells were co-cultured at 1:1 ratio for 2 h at 37 °C. Cell fusion was monitored using a dye redistribution assay. The fusion inhibition was checked in the presence of active peptides (100 μg/ml). The cell fusion in the absence of peptides was considered to be control cell fusion.
Fig. 2. Structure and molecular interactions of peptides: The above boxes show the structure and the Ramachandran plot in the upper panel of all the five peptides whereas the lower panel indicates the number of interactions showed by each peptide with gp120. Here peptides P-1 and P-4 exhibited only one interaction whereas peptides P-2, P-3 and P-2.1 showed 8, 7 and 10 interactions respectively.
C. Bhaskar et al. / Biochimica et Biophysica Acta 1834 (2013) 780–790
3.7. Reporter based assay in TZM-bl cells Action of peptides on virus infection is further confirmed by using a reporter cell line, TZM-bl which upon HIV infection leads to Tat induced LTR-associated β-gal expression in them. In the control HIV infection, the cells express β-gal which reduces the substrate, X-gal when added. The reduced X-gal fluoresces in the presence of azo dye (fast red violet). 1 million TZM-bl cells with 100% viability were seeded with DMEM, 0.1% FBS in 12-well plates. The concentration of peptides and T-20 used was 100 μg/ml and 1 μg/ml respectively. The cells were incubated at 37 °C in a CO2 (5%) incubator for 48 h. X-gal with azo dye was added to count the number of fluorescent cells. No fluorescence was observed in the presence of T20 which is used as positive control. 3.8. Peptide interaction with gp160 in the presence of receptor Mouse monoclonal anti-human gp160 antibodies spanning different regions of HIV-1 gp160 were added into wells of 96-well RIA plate at 10 ng per well in PBS and the plates were incubated overnight. The following day the wells were blocked with 3% BSA for 2 h at 37 °C. Binary complexes containing gp160-peptides were formed by incubation of 10 ng of gp160 in PBS with increasing concentrations of peptides (100–400 ng/ml) at 37 °C for 1 h. These complexes were captured with gp160 monoclonal antibody pre-coated wells and incubated for 1 h at 37 °C. The unbound complexes were removed by washing thrice with wash buffer. Captured binary complexes were probed for the peptides using 10 ng of affinity purified rabbit polyclonal anti-human Epap-1 antibody by incubating for 1 h at 37 °C. After washing the wells with wash buffer the bound rabbit polyclonal was probed with 1:2000 dilution of goat-anti-rabbit IgG-peroxidase antibody by incubating at
783
37 °C for 30 min. Finally the wells were washed and developed with TMB substrate system. The reaction was stopped after 30 min with 1 N HCl and the plates were read at 450 nm. Each experiment was done in triplicates for which average and standard deviations were calculated. The same experiment was repeated in the presence of 10 ng of receptor. 3.9. Peptide interaction with virus surface gp120 HIV-1 virus (10 ng) was incubated in the presence of different concentrations of peptides (100–400 ng/ml). Bound complex of HIV-1 and peptide was captured by mouse monoclonal gp160 antibodies spanning different regions of gp160 as mentioned in the previous protocol. The captured complex was estimated for HIV-1 bound in terms of p24 released with 1% of Triton X-100 using p24 antigen capture assay kit of Advanced Bioscience Laboratories. 4. Results 4.1. Molecular analysis of Epap-1 and gp120 interaction Epap-1 amino acid sequence was derived from the cDNA sequence based on degeneracy code [24]. Since the homology of Epap-1 was b35% with the existing sequence data, the structure was derived by fold recognition using threading on WURST server [28]. The structure was optimized by the energy minimization using GROMACS software [29]. Optimized structure (Fig. 1A) was further validated with L ALIGN [30] and EMBOSSES [31] which has given 78.4% and 78.8% homology. Molecular docking was carried out between Epap-1 (Fig. 1A) and gp120 trimers (obtained from PDB, Fig. 1B) and based on the
Fig. 3. Cytotoxic profile of peptides: All the 4 panels represent the cytotoxic profile of the five peptides in different cell lines viz., SupT1, colo205, SK-N-SH and cos7 cell lines. Even at 150 μg/ml concentration only up to 20% toxicity was exhibited by the peptides (n = 3,*p b 0.05, **p b 0.001).
784
C. Bhaskar et al. / Biochimica et Biophysica Acta 1834 (2013) 780–790
Fig. 4. Binding affinity of peptides: First panel shows the confocal images of binding affinity of all the peptides to HL2/3 cells. Here all the peptides bound to HL2/3 cells with the high efficiency. Whereas none of the peptides bound to SupT1 (second panel) cells that possess CD4 on its surface and the scrambled peptide bound to neither of the cells.
docking conformation (Fig. 1C), four potential regions of Epap-1 were selected for analysis of their interaction with gp120 (indicated in parenthesis): P-1. H2N-CNPSLVPPSILISFAATRTKRMAYTPFTSNIC-COOH (114–147) (GLU246 of gp120) P-2. H2N-RSTCALTAATAKAYATRSLEHRVVYRILHDC-COOH (383–412) (VAL270-GLN352 of gp120) P-3. H2N-VYNLDCLFRSICLHPWWWWMGVYNLVRDFITLH-COOH (614–646) (GLU268-LYS357 of gp120) P-4. H2N-SIMCLVLVISSKNKTPGPLTIVQVPAVATCFML-COOH (685– 718) (GLN267 of gp120) Scr-P-2. H2N-RKLTADVRACYELRITVAASTRHYCSHALAT-COOH (scrambled peptide as control). Fig. 5. Antiviral activity of peptides: Panel shows the antiviral activity of peptides in SupT1 cells. Out of all peptides P-2 has shown 80% antiviral activity and P-3 has shown ~40% activity at 100 μg/ml concentration. Peptides P-1, P-4 and Scr-P-2 were inactive (n=3, *pb 0.05, **pb 0.001).
These peptides possess molecular interactions with gp120 in terms of 1, 8, 7, and 1 numbers of interactions for P-1, P-2, P-3 and P-4 respectively (Fig. 2). These were synthesized commercially and analyzed for their affinity to gp120 and anti-HIV activity.
C. Bhaskar et al. / Biochimica et Biophysica Acta 1834 (2013) 780–790
4.2. Action of peptides on viability of various cell lines Peptides were analyzed for their cytotoxicity against four cell lines, viz. SupT1, colo205, SK-N-SH, and cos7 by using MTT as per protocol mentioned. The results showed that all five peptides were not cytotoxic (b20%) in these cell lines at 150 μg/ml concentration (Fig. 3). 4.3. Interaction of peptides with HL2/3 cells To assess the affinity of peptides toward gp120, they were conjugated with RITC. HL2/3 cells which express gp120 on their surface and SupT1 cells that express CD4 and CXCR-4 were incubated with RITC conjugated peptides (50 μg/ml) separately for 1 h at 37 °C in CO2 incubator. The results in Fig. 4 showed the peptides bound significantly to HL2/3 and did not bind to SupT1 cells whereas the scrambled peptide exhibited interaction to neither of the cells. 4.4. Anti-HIV-1 activity of peptides CD4 positive, SupT1 cells (0.5 million) were challenged with HIV-1IIIB (2 ng/ml) in the presence of 50–150 μg/ml of peptides respectively. The results presented in Fig. 5 show that peptides, P-2 and P-3 significantly inhibit the replication of HIV-1, while peptides P-1, P-4 and Scr-P-2 were inactive. Since P-2 was potent among four peptides with the IC-50 value of 59 μg (data not shown), it was taken as the lead molecule to determine the mode of action. 4.5. Interaction of P-2 with soluble gp160 To understand the molecular interaction of P-2 with gp160, the gp160 was incubated with increasing concentrations of P-2 (100– 400 ng) and the bound complexes were captured onto various anti-gp160 monoclonal antibody coated wells. The binary complexes were detected with Epap-1 polyclonal antibody. The results shown in Fig. 6 (panel A) suggest that P-2 interacts specifically with V3 domain and V3 loop of gp160 indicated by its inhibition of gp160 binding to F425 and 257D antibodies. Further, the interaction of P-2 with V3
785
domain not only remains unaltered but also increases in the presence of CD4 (Fig. 6, panel B). A separate complex of gp160-CD4 in the absence of P-2 was incubated with SIM.4 and V3 region antibodies and developed with anti-gp120 antibody which confirmed the activity of soluble CD4 employed (supplementary data, panel A). 4.6. Interaction of P-2 with virus surface gp120 Since the gp120 epitopes exposed in soluble gp160 may be different from that of the epitopes exposed on the viral surface, studying the interaction of P-2 with virus surface gp120 would be functionally relevant. The virus was captured using monoclonal antibodies spanning gp120 and gp41 regions in the presence of P-2. The exposure of various epitopes of gp120 in the presence of P-2 was monitored in terms of the amount of the virus bound to the corresponding antibody. The results show that P-2 binds to V3 domain of the virus surface gp120 (Fig. 7A). The same result was repeated when the virus was pre-incubated with P-2 and CD4 before the addition of CD4 (Fig. 7B) and P-2 (Fig. 7C) correspondingly. A separate complex of virus-CD4 in the absence of P-2 was incubated with SIM.4 and V3 region antibodies and detected for p24 levels which confirmed the activity of soluble CD4 employed (supplementary data, panel B). Further molecular analysis of P-2 peptide with gp120 revealed that all the major possible interactions lie in the N-terminal region. The docking was performed by taking the first 15 residues of P-2 peptide with gp120. The first 15 residues of P-2 which was named as P-2.1 (H2N-RSTCALTAATAKAYA-COOH) showed 10 interactions (Fig. 2). To experimentally confirm these interactions, this peptide was synthesized commercially and analyzed for their anti-HIV activity and molecular action on gp120 mediated cell fusion. 4.7. Action of peptides on reporter based TZM-bl cells Anti-HIV-1 activity of peptides was confirmed by reporter based assay. TZM-bl cells were challenged with HIV-1 in the presence and absence of peptides. Expression of β-gal occurred during HIV infection in the control TZM-bl cells and the reduction of X-gal took place. The presence of T20 (1 μg/ml) and peptides (100 μg/ml)
Fig. 6. Peptide binding with soluble gp160: Panel (A) shows the interaction of peptide P-2 with V3 region which is proven by the masking of V3 region of soluble gp160 by it, thereby preventing the interaction of anti-V3 antibodies viz., F425 and 257-D respectively. The same interaction is enhanced in the presence of CD4 which is reported in panel (B) (n = 3, *p b 0.05, **p b 0.001).
786 C. Bhaskar et al. / Biochimica et Biophysica Acta 1834 (2013) 780–790 Fig. 7. Peptide interaction with virus surface gp120: Panel (A) shows the interaction of peptide P-2 with V3 region which is shown by the masking of V3 region of virus surface gp120 and preventing the interaction of anti-V3 antibodies viz., F425 and 257-D respectively. The same interaction is enhanced when the assay is performed with gp120–P2 complex prior to the addition of CD4 (panel B) and also in another combination of gp120–CD4 complex prior to the addition of P2 (panel C) (n = 3,*p b 0.05, **pb 0.001).
C. Bhaskar et al. / Biochimica et Biophysica Acta 1834 (2013) 780–790
787
Fig. 8. Inhibition of viral entry by peptides in reporter cell line, TZM-bl: Panel (A) indicates the confocal image of the TZM-bl cells in which lane 1 shows the expression of β-gal upon HIV-1 infection in control cells. Lanes below indicate the prevention of infection by T-20 (1 μg/ml-positive control) and peptides, P-2 and P-2.1 (100 μg/ml) which was reflected in terms of reduction in β-gal expression. The same assay was repeated with FACS to count the number of cells fluoresced which were shown in panel (B). Scrambled peptide which is used as negative control did not stop the infection.
inhibited the viral infection and the subsequent expression of β-gal (Fig. 8, panel A). The results of FACS analysis presented in Fig. 8 (panel B) confirm that peptides possess affinity to gp120 and inhibit HIV-1 infection. Here the scrambled peptide could not prevent the HIV infection confirming the specificity of peptides.
4.8. Action of peptides on gp120 mediated cell fusion HL2/3 cells and SupT1 cells were incubated in the presence and absence of peptides (100 μg/ml) for 2 h. After extensive washing with PBS the cells were analyzed using confocal microscopy. The
788
C. Bhaskar et al. / Biochimica et Biophysica Acta 1834 (2013) 780–790
results show that peptides P-2 and P-2.1 indeed bound to HL2/3 cells and inhibited the fusion of SupT1 cells and HL2/3 cells, whereas the scrambled peptide failed to prevent the fusion (Fig. 9).
4.9. Action of peptides on multiple strains of HIV-1 The peptides, P-2 and P-2.1 exhibited neutralization activity against viral strains namely CEM 50, 93RW024, MN and NL-43 of HIV-1. At 50 μg/ml concentration, the peptides showed 40–80% antiviral activity against the HIV-1 isolates analyzed (Fig. 10).
5. Discussion Even though Epap-1 is a human protein, its immunological properties were unexplored. The stability of this 90 kDa protein and its bioavailability are the major concerns if it was promoted as a lead molecule for therapeutic application. In previous studies, Epap-1 was shown to interact with V3 and C5 regions of gp120 and the identification of specific region in Epap-1 interacting with these regions will decipher the mode of action of naturally occurring Epap-1. Since the crystal structure of Epap-1 was unavailable, we have modeled its structure and docked it with gp120 to obtain the exact region that will help in virus neutralization. Molecular analysis of
Fig. 9. Inhibition of cell fusion by peptides: Top 2 panels showed the interaction of CD4+ SupT1 cells (blue) and gp120+ HL2/3 cells (yellow) with each other in the absence of an inhibitor and presence of scrambled peptide which results in the successful fusion of cells (arrows in red shows the fusion of cells). Whereas the presence of peptides (100 μg/ml) prevented the interaction between both the cells, indicated by the inhibition of dye transfer (lower panels).
C. Bhaskar et al. / Biochimica et Biophysica Acta 1834 (2013) 780–790
789
One of the major limitations in the present study is the inability in predicting the C5 binding region of the Epap-1 with the existing molecular model. The other is the higher IC-50 value of the peptides. The identification of the C5 binding domain in the Epap-1 may result in getting potent peptides which can act in a concerted manner with the existing peptides that can enhance the possibility of reducing the IC-50 value to the nanomolar level. The important and immediate focus is to reduce the IC-50 value of the peptide by measuring the binding strength between the peptides and gp120. Further studies are under way to assess the activity after creating mutations in the potent peptides by scanning mutagenesis approach. 6. Conclusion
Fig. 10. Antiviral action of peptides on different strains of HIV: Here the peptides show antiviral action on the different viral strains namely CEM 50, 93RW024, MN and NL-43 of HIV-1. At 50 μg/ml of final concentration the peptides showed 40–80% antiviral action on all the strains (n = 3,*p b 0.05, **p b 0.001).
The present study identified Epap-1 derived peptide with multiple interactions in V3 domain of gp120 which can block the viral entry and thereby leading to the neutralization of virus infection. Supplementary data to this article can be found online at http:// dx.doi.org/10.1016/j.bbapap.2013.01.017. Role of the funding source
peptides in the presence of gp160 helped in the identification of a 32 mer peptide with significant activity. Further, molecular analysis yielded 15 mer peptide with significant HIV-1 neutralizing activity. Epap-1 derived 32 mer peptides principally interact with gp120 in a region surrounding the V3 loop involving Glu 268 to Lys 357, while V3 region spans from aa 296 to 331 depending upon the viral isolate. Most importantly residues Alanine and Argenine of Epap-1 are involved in interaction with gp120. These results can be confirmed though our observation of the region in gp120 is blocked by the binding of peptides to the epitopes recognized by 257D and F425 monoclonal antibodies. It was shown that F425 is a broadly neutralizing antibody that binds to the tip of V3 and the presence of Ile309, Arg315 and Phe317 is important for high-affinity binding [32]. On the other hand, mAb 257D binds strongly to two adjacent peptides, RKRIHI and KRIHIG, showing that it recognizes the smallest reactive peptide KRIHI which is located to the left of the conserved tip of the V3 loop. The flanking N- and C-terminal Arginine and Glycine residues of V3 play a role in the binding to this mAb [33]. The V3 loop region is of high importance since it is involved in many aspects of HIV-1 viral infectivity. The V3 loop was termed as the “principal neutralizing determinant” [34] because peptides containing the V3 region can induce neutralizing antibodies [35–37] and is arguably a potential target for HIV vaccine design [38]. The possibility of generating an effective vaccine based on intact gp120 or peptides containing the V3 loop region was decreased because even though the constructs induced a strong immune response toward V3, the antibodies generated happened to neutralize only laboratory-adapted isolates (TCLA) [39,40] and are specific for only a small number of strains [41]. This paradox is due to the differential exposure of V3 on various strains, and at different stages of the viral entry thereby decreasing the accessibility of the V3 region to antibody. Primary viral isolates, which utilize the CCR5 coreceptor are extremely resistant to neutralization by the majority of antibodies, including directed against the V3 loop. This may be due to the inaccessibility of V3 loop in primary isolates, either due to carbohydrate masking or because of interactions with the gp120 complex. Recent studies have shown that V3 may be shielded by the V1/V2 region [42,43], thus supporting the results from earlier investigations into the functional properties of V1/V2 [44,45]. Even if V3 region is not exposed on the surface, an interaction with adjoining residues of V3 loop such as V2 region may initiate nucleation of peptide interaction followed by alignment at V3 domain. So the identification of the potential regions in Epap-1, especially peptides may provide insight into a mechanism that will assist in addressing the above difficulties to develop a therapy with broadly neutralizing effect.
Funding source has no role in design, execution and interpretation of the results incorporated in the manuscript. Acknowledgements We acknowledge the financial assistance under DBT sponsored research project. CB was ICMR SRF and currently UoH-DBT-CREBB postdoctoral fellow. Infrastructure developed through UGC funded CAS and XI plan used for the work. We acknowledge contributors of antibodies and viral strains from NIH–AIDS reagent and reference program. Antibodies: C5 region: 670-30D (Dr. S. Zolla-Pazner); V3 loop: V3-21, 298–315 (Dr. J. Laman); V3 loop: F425 B 4a.1 (cf F425) and gp41: F240 (Dr. M. Posner and Dr. L. Cavacini); CD4 Mab: SIM4 (Dr. J.E.K. Hildreth); C3 reactive: B32-FFY, 382–384 (Dr. G.W. Lewis). Viruses: HIV-193IN101 (Dr. R. Bollinger), HIV-1MN is of biotype-SI (X4) (Dr. R. Gallo), HIV-1CEM-50 (Dr. Robin Mukhopadhyaya) HIV-1 93RW024, and HIV-1 94UG103 (The UNAIDS Network for HIV Isolation and Characterization, and the DAIDS, NIAID). References [1] G.M. Lucas, R.E. Chaisson, R.D. Moore, Highly active antiretroviral therapy in a large urban clinic: risk factors for virologic failure and adverse drug reactions, Ann. Intern. Med. 131 (1999) 81–87. [2] A. Carr, D.A. Cooper, Adverse effects of antiretroviral therapy, Lancet 356 (2000) 1423–1430. [3] S.C. Piscitelli, C. Flexner, J.R. Minor, M.A. Polis, H. Masur, Drug interactions in patients infected with human immunodeficiency virus, Clin. Infect. Dis. 23 (1996) 685–693. [4] D. Boden, A. Hurley, L. Zhang, Y. Cao, Y. Guo, E. Jones, J. Tsay, J. Ip, C. Farthing, K. Limoli, N. Parkin, M. Markowitz, HIV-1 drug resistance in newly infected individuals, JAMA 282 (1999) 1135–1141. [5] D. Finzi, J. Blankson, J.D. Siliciano, J.B. Margolick, K. Chadwick, T. Pierson, Latent infection of CD4+ T cells provides a mechanism for lifelong persistence of HIV-1, even in patients on effective combination therapy, Nat. Med. 5 (1999) 512–517. [6] S.A. Wegner, S.K. Brodine, J.R. Mascola, S.A. Tasker, R.A. Shaffer, M.J. Starkey, A. Barile, G.J. Martin, N. Aronson, W.W. Emmons, K. Stephan, S. Bloor, J. Vingerhoets, K. Hertogs, B. Larder, Prevalence of genotypic and phenotypic resistance to anti-retroviral drugs in a cohort of therapy-naive HIV-1 infected US military personnel, AIDS 14 (2000) 1009–1015. [7] S. Yerly, L. Kaiser, E. Race, J.P. Bru, F. Clavel, L. Perrin, Transmission of antiretroviral-drug-resistant HIV-1 variants, Lancet 354 (1999) 729–733. [8] Q.J. Sattentau, J.P. Moore, Human immunodeficiency virus type 1 neutralization is determined by epitope exposure on the gp120 oligomer, J. Exp. Med. 182 (1995) 185–196. [9] E. Emini, P. Nara, W. Schlief, J. Lewis, J. Davide, D. Lee, J. Kessler, S. Conley, M. Matsushita, S. Putney, R. Gerety, J. Eichberg, Antibody-mediated in vitro neutralization of human immunodeficiency virus type 1 abolishes infectivity for chimpanzees, J. Virol. 64 (1990) 3674–3678.
790
C. Bhaskar et al. / Biochimica et Biophysica Acta 1834 (2013) 780–790
[10] D. Ho, et al., Conformational epitope on gp120 important in CD4 binding and human immunodeficiency virus type 1 neutralization identified by a human monoclonal antibody, J. Virol. 65 (1991) 489–493. [11] M. Posner, et al., An IgG human monoclonal antibody which reacts with HIV-1 gp120, inhibits virus binding to cells, and neutralizes infection, J. Immunol. 146 (1991) 4325–4332. [12] M. Thali, et al., Discontinuous, conserved neutralization epitopes overlapping the CD4 binding region of the HIV-1 gp120 envelope glycoprotein, J. Virol. 66 (1992) 5635–5641. [13] M. Thali, et al., Characterization of conserved human immunodeficiency virus type 1 gp120 neutralization epitopes exposed upon gp120-CD4 binding, J. Virol. 67 (1993) 3978–3988. [14] A. Trkola, et al., Human monoclonal antibody 2G12 defines a distinctive neutralization epitope on the gp120 glycoprotein of human immunodeficiency virus type 1, J. Virol. 70 (1996) 1100–1108. [15] L. Musey, Y. Hu, L. Eckert, M. Christensen, T. Karchmer, M.J. McElrath, HIV-1 induces cytotoxic T lymphocytes in the cervix of infected women, J. Exp. Med. 185 (2) (1997) 293–303. [16] B. Rosok, P. Voltersvik, B.M. Larsson, J. Albert, J.E. Brinchmann, B. Asjö, CD8+ T cells from HIV type 1-seronegative individuals suppress virus replication in acutely infected cells, AIDS Res. Hum. Retroviruses 13 (1) (1997) 79–85. [17] C. Hayano, Accumulation of CD16+ cells with secretion of Ksp37 in decidua at the end of pregnancy, Am. J. Reprod. Immunol. 48 (1) (2002) 57–62. [18] J.S. Hunt, M.G. Petroff, T.G. Burnett, Uterine leukocytes: key players in pregnancy, Semin. Cell Dev. Biol. 11 (2) (2000) 127–137. [19] K. Ohtani, Y. Suzuki, S. Eda, T. Kawai, T. Kase, H. Keshi, Y. Sakai, A. Fukuoh, T. Sakamoto, H. Itabe, T. Suzutani, M. Ogasawara, I. Yoshida, N. Wakamiya, The membrane-type collectin CL-P1 is a scavenger receptor on vascular endothelial cells, J. Biol. Chem. 276 (2001) 44222–44228. [20] B.K. Patterson, H. Behbahani, W.J. Kabat, Y. Sullivan, M.R. O'Gorman, A. Landay, Z. Flener, N. Khan, R. Yogev, J. Andersson, Leukemia inhibitory factor inhibits HIV-1 replication and is upregulated in placentae from non transmitting women, J. Clin. Invest. 107 (2001) 287–294. [21] P. Sedlmyr, A. Blaschitz, R. Wintersteiger, M. Semlitsch, A. Hammer, C.R. MacKenzie, W. Walcher, O. Reich, O. Takikawa, G. Dohr, Localization of indoleamine 2,3-dioxygenase in human female reproductive organs and the placenta, Mol. Hum. Reprod. 8 (2002) 385–391. [22] P. Tiensiwakul, Stromal cell-derived factor (SDF) 1-3′A polymorphism may play a role in resistance to HIV-1 infection in seronegative high-risk Thais, Intervirology 47 (2) (2004) 87–92. [23] A.K. Kondapi, M.A. Hafiz, T. Sivaram, Anti-HIV activity of a glycoprotein from first trimester placental tissue, Antivir. Res. 54 (2002) 47–57. [24] K.P. Roda Rani, Dheeraj Pelluru, Anand K. Kondapi, A conserved molecular action of native and recombinant Epap-1 in inhibition of HIV-1 gp120 mediated viral entry, Arch. Biochem. Biophys. 456 (1) (2006) 79–92. [25] J. Liu, A. Bartesaghi, M.J. Borgnia, G. Sapiro, S. Subramaniam, Molecular architecture of native HIV-1 gp120 trimers, Nature 455 (7209) (2008) 109–113. [26] D.W. Ritchie, Recent progress and future directions in protein–protein docking, Curr. Protein Pept. Sci. 9 (1) (2008) 1–15. [27] G. Macindoe, L. Mavridis, V. Venkatraman, M.D. Devignes, D.W. Ritchie, HexServer: an FFT-based protein docking server powered by graphics processors, Nucleic Acids Res. 38 (2010) W445–W449. [28] A.E. Torda, J.B. Procter, T. Huber, Wurst: a protein threading server with a structural scoring function, sequence profiles and optimized substitution matrices, Nucleic Acids Res. 32 (2004) W532–W535, (Web Server issue). [29] D. Van Der Spoel, E. Lindahl, B. Hess, G. Groenhof, A.E. Mark, H.J. Berendsen, GROMACS: fast, flexible, and free, J. Comput. Chem. 26 (16) (2005) 1701–1718. [30] X. Huang, W. Miller, A time-efficient linear-space local similarity algorithm, Adv. Appl. Math. 12 (1991) 337–357.
[31] P. Rice, I. Longden, A. Bleasby, EMBOSS: the European Molecular Biology Open Software Suite, Trends Genet. 16 (6) (2000) 276–277. [32] R. Pantophlet, R.O. Aguilar-Sino, T. Wrin, L.A. Cavacini, D.R. Burton, Analysis of the neutralization breadth of the anti-V3 antibody F425-B4e8 and reassessment of its epitope fine specificity by scanning mutagenesis, Virology 364 (2007) 441–453. [33] J.Y. Xu, M.K. Gorny, T. Palker, S. Karwowska, S. Zolla-Pazner, Epitope mapping of two immunodominant domains of gp41, the transmembrane protein of human immunodeficiency virus type 1, using ten human monoclonal antibodies, J. Virol. 65 (9) (1991) 4832–4838. [34] K. Javaherian, A.J. Langlois, C. McDanal, K.L. Ross, L.I. Eckler, C.L. Jellis, A.T. Profy, J.R. Rusche, D.P. Bolognesi, S.D. Putney, T.J. Matthews, Principal neutralizing domain of the human immunodeficiency virus type 1 envelope protein, Proc. Natl. Acad. Sci. U. S. A. 86 (1989) 6768–6772. [35] J. Goudsmit, C. Debouck, R.H. Meloen, L. Smit, M. Bakker, D.M. Asher, A.V. Wolff, C.J. Gibbs Jr., D.C. Gajdusek, Human immunodeficiency virus type 1 neutralization epitope with conserved architecture elicits early type-specific antibodies in experimentally infected chimpanzees, Proc. Natl. Acad. Sci. U. S. A. 85 (1988) 4478–4482. [36] T.J. Palker, M.E. Clark, A.J. Langlois, T.J. Matthews, K.J. Weinhold, R.R. Randall, D.P. Bolognesi, B.F. Haynes, Type-specific neutralization of the human immunodeficiency virus with antibodies to env-encoded synthetic peptides, Proc. Natl. Acad. Sci. U. S. A. 85 (1988) 1932–1936. [37] J.R. Rusche, K. Javaherian, C. McDanal, J. Petro, D.L. Lynn, R. Grimaila, A. Langlois, R.C. Gallo, L.O. Arthur, P.J. Fischinger, D.P. Bolognesi, S.D. Putney, T.J. Matthews, Antibodies that inhibit fusion of human immunodeficiency virus-infected cells bind a 24-amino acid sequence of the viral envelope, gp120, Proc. Natl. Acad. Sci. U. S. A. 85 (1988) 3198–3202. [38] S. Zolla-Pazner, Improving on nature: focusing the immune response on the V3 loop, Hum. Antibodies 14 (3–4) (2005) 69–72. [39] J.R. Mascola, J. Louwagie, F.E. McCutchan, C.L. Fischer, P.A. Hegerich, K.F. Wagner, A.K. Fowler, J.G. McNeil, D.S. Burke, Two antigenically distinct subtypes of human immunodeficiency virus type 1: viral genotype predicts neutralization serotype, J. Infect. Dis. 169 (1994) 48–54. [40] T.J. Matthews, Dilemma of neutralization resistance of HIV-1 field isolates and vaccine development, AIDS Res. Hum. Retroviruses 10 (1994) 631–632. [41] T.J. Palker, M.E. Clark, A.J. Langlois, T.J. Matthews, K.J. Weinhold, R.R. Randall, D.P. Bolognesi, B.F. Haynes, Type-specific neutralization of the human immunodeficiency virus with antibodies to env-encoded synthetic peptides, Proc. Natl. Acad. Sci. U. S. A. 85 (6) (1988) 1932–1936. [42] C.P. Krachmarov, W.J. Honnen, S.C. Kayman, M.K. Gorny, S. Zolla-Pazner, A. Pinter, Factors determining the breadth and potency of neutralization by V3-specific human monoclonal antibodies derived from subjects infected with clade A or clade B strains of human immunodeficiency virus type 1, J. Virol. 80 (14) (2006) 7127–7135. [43] A. Pinter, W.J. Honnen, Y. He, M.K. Gorny, S. Zolla-Pazner, S.C. Kayman, The V1/V2 domain of gp120 is a global regulator of the sensitivity of primary human immunodeficiency virus type 1 isolates to neutralization by antibodies commonly induced upon infection, J. Virol. 78 (10) (2004) 5205–5215. [44] J. Cao, N. Sullivan, E. Desjardin, C. Parolin, J. Robinson, R. Wyatt, J. Sodroski, Replication and neutralization of human immunodeficiency virus type 1 lacking the V1 and V2 variable loops of the gp120 envelope glycoprotein, J. Virol. 71 (12) (1997) 9808–9812. [45] R. Wyatt, N. Sullivan, M. Thali, H. Repke, D. Ho, J. Robinson, M. Posner, J. Sodroski, Functional and immunologic characterization of human immunodeficiency virus type 1 envelope glycoproteins containing deletions of the major variable regions, J. Virol. 67 (8) (1993) 4557–4565; J. Virol. 76 (18) (Sep 2002) 9035–9045.