A bivalent recombinant protein inactivates HIV-1 by targeting the gp41 prehairpin fusion intermediate induced by CD4 D1D2 domains
© Lu et al.; licensee BioMed Central Ltd. 2012
Received: 3 September 2012
Accepted: 11 November 2012
Published: 7 December 2012
Most currently approved anti-HIV drugs (e.g., reverse transcriptase inhibitors, protease inhibitors and fusion/entry inhibitors) must act inside or on surface of the target cell to inhibit HIV infection, but none can directly inactivate virions away from cells. Although soluble CD4 (sCD4) can inactivate laboratory-adapted HIV-1 strains, it fails to reduce the viral loads in clinical trials because of its low potency against primary isolates and tendency to enhance HIV-1 infection at low concentration. Thus, it is essential to design a better HIV inactivator with improved potency for developing new anti-HIV therapeutics that can actively attack the virus in the circulation before it attaches to and enter into the target cell.
We engineered a bivalent HIV-1 inactivator, designated 2DLT, by linking the D1D2 domain of CD4 to T1144, the next generation HIV fusion inhibitor, with a 35-mer linker. The D1D2 domain in this soluble 2DLT protein could bind to the CD4-binding site and induce the formation of the gp41 prehairpin fusion-intermediate (PFI), but showed no sCD4-mediated enhancement of HIV-1 infection. The T1144 domain in 2DLT then bound to the exposed PFI, resulting in rapid inactivation of HIV-1 virions in the absence of the target cell. Beside, 2DLT could also inhibit fusion of the virus with the target cell if the virion escapes the first attack of 2DLT.
This bivalent molecule can serve as a dual barrier against HIV infection by first inactivating HIV-1 virions away from cells and then blocking HIV-1 entry on the target cell surface, indicating its potential for development as a new class of anti-HIV drug.
KeywordsHIV-1 gp41 Peptide Six helix bundle Inactivation HIV-1 fusion inhibitor
Thus far, thirty-two anti-HIV drugs (including five fixed-dose combinations) have been licensed by the United States Food and Drug Administration (FDA) for treatment of HIV infection/AIDS (http://www.hivandhepatitis.com/hiv_and_aids/hiv_treat.html). Most of these drugs act inside the host cell to inhibit viral replication by targeting HIV reverse transcriptase, protease, or integrase. Two of them, enfuvirtide (also known as T20)  and Marviroc , act on surface of the target cell to block viral fusion and entry by interacting with the HIV-1 gp41 N-terminal heptad repeat (NHR) and the coreceptor, CCR5, respectively. However, none of these anti-HIV drugs can inactivate virions in the absence of the target cell.
Some of the non-ionic surfactants, such as Nonoxynol-9 (N-9), could effectively inactivate HIV-1 virions by lysing the viral envelope membrane . However, because of its high cytotoxicity, it cannot be used as an anti-HIV drug in clinics since it can also damage cellular membranes . Therefore, it is essential to design an HIV inactivator using a protein or peptide that can actively attack the virion before it attaches to and enters into the host cells, with low or no toxic effect on the host cells.
Monomeric soluble CD4 (sCD4) that can specifically binds to the HIV-1 gp120 and then inactivate the virion was one of the first anti-HIV-1 agents tested in clinical trial. Unfortunately, it failed to reduce the viral loads in HIV-1-infected individuals [12, 13]. However, sCD4 and CD4-mimetics could efficiently induce the formation of the gp41 PFI with the exposed grooves on the NHR-trimer , which is the target of peptidic HIV fusion inhibitors, such as SJ-2176 , T20 , C34 [17, 18] and T1144 [19, 20]. These results suggest that a molecule containing a CD4 or CD4-mimetic and a gp41 PFI-binding domain (such as T1144) can inactivate HIV-1 more efficiently than sCD4 or CD4-mimetic since T1144 can bind to the exposed gp41 grooves induced by binding of sCD4 or CD4-mimetic to gp120 to speed the virus inactivation. Based on this hypothesis, we engineered a bivalent protein, designated 2DLT, in which the D1D2 domains of CD4 were linked to T1144 by a 35-mer flexible linker to allow the free movement of the two functional domains in the bivalent molecule (Figure 1B). The D1D2 fragment in this bivalent protein is expected to bind specifically with gp120 on the surface of HIV virions or HIV-infected cells (Figure 1C) and trigger formation of the gp41 PFI with the exposed hydrophobic grooves (Figure 1D), while the T1144 domain can bind to the exposed grooves on the gp41 NHR-trimer, resulting in rapid inactivation of the cell-free HIV-1 before its attachment to the target cell. Indeed, the 2DLT protein could effectively bind to both gp120 and gp41, block gp41 6-HB formation, inactivate cell-free HIV-1 and inhibit HIV-1 Env-mediated cell-cell fusion, but without the sCD4-mediated enhancing effects on HIV-1 infection. Therefore, this engineered bivalent molecule has substantial potential for development as an anti-HIV therapeutic for treatment of patients who fail to respond to the current anti-HIV drugs and as a topical microbicide for preventing sexual transmission of HIV.
Construction, expression and characterization of the bivalent fusion protein 2DLT
The expression plasmids pD1D2-PDI and p2DLT-PDI were constructed by linking the DNA fragment encoding D1D2 with those coding the 35-mer linker (GGGGS)7 and T1144 sequentially by three-step overlapping PCR using the corresponding primer pairs. The nucleotide sequences of the vectors were confirmed by DNA sequencing. The recombinant bivalent protein 2DLT and the control protein D1D2 (Figure 1B) were expressed in E. coli. To avoid the formation of inclusion bodies, we used the protein disulfide isomerase (PDI) chaperone-expression system since we and others have shown that PDI, as a fusion partner, could significantly increase the soluble expression of recombinant proteins in the cytoplasm of E. coli[21, 22], and we successfully obtained soluble D1D2 and 2DLT proteins. After purification, we analyzed these proteins with SDS-PAGE and Western blot. On the gels, two proteins migrated near the expected position of the proteins (Figure 1Ea, D1D2: ~23KD; 2DLT: ~29KD). Both displayed specific interaction with anti-CD4 polyclonal antibodies (pAb) (Figure 1Eb). While the anti-T1144 pAb bound with 2DLT at the predicted size, no band was revealed in the D1D2 lane (Figure 1Ec). Similarly, anti-CD4 pAb (T4-4) could react with both D1D2 and 2DLT, while anti-T1144 pAb was able to recognize 2DLT only in the ELISA. Notably, both D1D2 and 2DLT could react with the CD4-specific and conformation-dependent monoclonal antibody (mAb) Sim.4 (Figure 1F), suggesting that the soluble D1D2 and 2DLT may have a correctly folded conformation.
2DLT inactivated cell-free HIV-1 virions
Inactivation of cell-free HIV-1 R5 and X4 strains by 2DLT
HIV-1 strain (subtype, tropism)
Bal (B, R5)
157.2 ± 34.2
24.5 ± 2.25
IIIB (B, X4)
51.2 ± 9.21
17.3 ± 1.62
92US657 (B, R5)
319.2 ± 56.1
48.7 ± 14.5
93 MW959 (C, R5)
478.2 ± 89.3
78.6 ± 7.04
92TH009 (E/A, R5)
209.5 ± 6.1
69.9 ± 14.4
Like sCD4 and D1D2, 2DLT could bind to gp120 on the cell surface
Like T1144, 2DLT bound to the groove on the gp41 PFI N-trimer and subsequently blocked gp41 6-HB formation
The gp41 6-HB formation is a critical step during HIV-1 fusion with the target cell. The peptides derived from the gp41 CHR, e.g. C34 and T1144 are able to bind with viral gp41 N-trimer to block the 6-HB core formation [19, 27]. Here, we used a sandwich ELISA and fluorescence native polyacrylamide gel electrophoresis (FN-PAGE) to determine if 2DLT, like T1144, possessed inhibitory activity on gp41 6-HB formation in a model system mimicking the gp41 6-HB core formation by mixing the gp41 N36 and C34 (or FAM-labeled C34) peptides at equal molar concentration [17, 28]. In the ELISA, 2DLT, like T1144, inhibited the 6-HB formation in a dose-dependent manner with an IC50 of 0.5 ±0.06 μM, while D1D2 protein at 10 μM exhibited no significant inhibition (Figure 4B). Similarly, 2DLT could effectively block 6-HB formation in a dose-dependent manner when it was tested at 5, 10, and 20 μM as shown in the FN-PAGE (Figure 4Ca and Cc, lanes 5 to 7), whereas D1D2 protein at the same concentrations showed no significant inhibition (Figure 4Cb and Cd, lines 5 to 7). The D1D2 and 2DLT bands were not observable on the gels because they carry net positive charges, like the N-peptide N36 (lane 1 in Figure 4C) and run in a reversed direction under the native gel condition as previously described [27, 29]. These results indicate that 2DLT can interact with the gp41 N-trimer and block the 6-HB core formation between viral gp41 NHR and CHR domains.
2DLT could disrupt the function of the CD4-induced gp41 PFI, and caused no significant enhancement of HIV-1 infection in CD4-/CCR5+cells
2DLT inhibited HIV-1 infection and HIV-1-mediated cell-cell fusion
Inhibitory activity of the recombinant proteins and peptides on HIV-1-mediated cell-cell fusion and HIV-1 replication
HIV-1 IIIB-mediated cell fusion
IIIB (B, X4)
Bal (B, R5)
92US657 (B, R5)*
93 MW959 (C, R5)*
92TH009 (E/A, R5)*
Effect of pre-incubation of the virus with inactivators or inhibitors on inhibition of HIV-1 replication
Preincubation time (min)
IC50 ( nM ) for inhibiting HIV-1 Bal infection
137.10 ± 6.66
11.85 ± 2.23
32.35 ± 6.86
2.92 ± 1.01
129.25 ± 7.22
7.52 ± 3.65
27.35 ± 3.26
2.71 ± 0.98
103.27 ± 6.26
3.22 ± 1.20
20.13 ± 4.21
2.61 ± 0.58
86.69 ± 6.13
2.38 ± 0.59
20.51 ± 3.22
2.22 ± 0.82
Soluble CD4 (sCD4) was previously recognized as a potential HIV-1 inactivator since it can bind to gp120 for inducing inactivation of HIV-1 virions. However, a very high concentration of sCD4 is required to neutralize infection by primary HIV-1 isolates [14, 31, 32]. Even worse, sCD4 at low concentration can enhance HIV-1 infection of CD4-/CCR5+ cells . To improve sCD4-mediated HIV-1 neutralizing activity, several groups have constructed hybrid proteins by fusing CD4 or D1D2 with human IgG (CD4-IgG2 or PRO 542) [33, 34] or with a monoclonal antibody (mAb) specific for the CD4-induced epitope, such as 17b (sCD4-17b) . In a clinical trial, CD4-IgG2 treatment led to about 0.5 log10 reduction in viral load . However, CD4-IgG fusion proteins have two limitations. First, the molecule is too large to be expressed in a large quantity. Second, the inactivator targeting only gp120 may not be very effective in rapid decay of the gp41 PFI, a critical step of inactivating HIV-1 . Therefore, the CD4-induced gp41 PFI is expected to be a novel target for development of HIV inactivators with improved efficacy.
Similar to several gp120-specific mAbs, sCD4 was shown to modestly enhance the infectivity of HIV-1 at suboptimal concentrations in CD4-/CCR5+ cells , which is more prominent in some simian immunodeficiency virus (SIV) and SIV-related HIV-2 strains [14, 42–45]. This phenomenon was explained by the hypothesis that sCD4 can efficiently replace cell-surface CD4 to drive virus infection in CD4-negative and CCR5-positive cells . In the present study, we found that the soluble D1D2 molecule could enhance infectivity of a CCR5-using HIV-1 strain Bal in Cf2Th-CCR5 (CD4-/CCR5+) cells at the concentration from 5 to 200 nM, while 2DLT and T1144 exhibited no enhancement of HIV-1 infection at the same concentration range. This suggests that unlike the soluble D1D2 protein, the D1D2 domains in 2DLT may not be able to replace CD4 on cell surface to drive HIV-1 infection in the CD4-/CCR5+ cells or the rapid decay of the gp41 fusion-intermediate mediated by 2DLT results in the abolishment of the potential enhancement of HIV-1 infection.
We have demonstrated that like the isolated T1144 peptide, the T1144 domain in 2DLT could also effectively inhibit HIV-1 infection and HIV-1-mediated cell-cell fusion (Figure 6 and Table 2). The dual functions of 2DLT make it superior to the other kind of inactivators (such as N9 or C31G) since if the HIV-1 virions are not completely inactivated, 2DLT is able to block the viral fusion and entry with its T1144 domain.
We have not tested the ability of 2DLT to induce drug-resistant mutations in HIV-1 variants because it will be difficult to interpret the results from the HIV entry inhibitor-induced mutation studies. For example, people may generally believe that sCD4 should induce drug-resistant mutations in the CD4-binding site in viral gp120. However, the mutation sites in the sCD4-resistant HIV-1 variants were mainly located in the V1, V2, and V3 loops in gp120 and the NHR region in gp41 . Similarly, the mutation sites in the HIV-1 variants resistant to T2635, an analogous peptide of T1144, were not located in the hydrophobic pocket of the gp41 NHR domain, which is the main target site of T2635, T1144 and other C-peptides with the pocket-binding domain, but rather in other regions in gp120 and gp41 . This could be possibly explained by the fact that both the CD4 binding site in gp120 and the hydrophobic pocket in gp41 are highly conserved, and the viruses with mutations at these sites may not survive due to the loss of the critical viral functions. Therefore, both D1D2 and T1144 may possess high genetic barrier to resistance. We expect that 2DLT may have a higher genetic barrier to resistance than D1D2 and T1144 when each of them is used alone since 2DLT, like the combination of D1D2 and T1144, is able to simultaneously interact with two target sites, while D1D2 or T1144 can bind to only one target site.
We have designed and engineered a bivalent protein 2DLT, which can speed the T1144 domain-mediated decay of the gp41 PFI induced by binding of D1D2 domain to gp120, resulting in rapid inactivation of HIV-1 virions. It may also inhibit HIV-1 fusion and entry through its T1144 domain in case when the HIV-1 virion escapes from 2DLT-mediated inactivation. Both of its binding sites in gp120 and gp41 are highly conserved, and it can actively attack the virus in the absence of host cells, making it a promising candidate for further development as a therapeutic for the treatment of HIV/AIDS or as a topical microbicide for preventing sexual transmission of HIV.
CHO cells stably transfected with either the HIV-1HXB2 Env-expressing vector pEE14 (CHO-WT) or control pEE14 vector (CHO-EE) were cultured in Glutamine-deficient minimal essential medium (GMEM-S) containing 400 μM Methionine sulfoximine (Sigma, St. Louis, MO). Cf2Th/syn CCR5 cells stably expressing CCR5 receptors were cultured in DMEM complemented medium with 10% FBS, pen/strep, 500 μg/ml G418, 500 μg/ml zeocin and 3 μg/ml puromycin (Invitrogen, Carlsbad, CA). MT-2 and TZM-b1 cells, as well as laboratory-adapted HIV-1 strains IIIB and Bal, and primary HIV-1 isolates were obtained from the AIDS Research and Reference Reagent Program of NIH. The N-peptide N36 (aa 546–581), and C-peptides C34 (aa 628–661), T1144, and T20 (aa 638–673), used in this study were derived from the NHR and CHR, respectively, of the HIV-1HXB2 gp41 (Figure 1A). These peptides (> 95% purity) were synthesized by a standard solid-phase FMOC method using an Applied Biosystems model 433A peptide synthesizer.
Construction of vectors encoding D1D2 and 2DLT
To create the expression plasmid pD1D2-PDI and p2DLT-PDI, DNA fragments encoding D1D2 (aa 1–185 of CD4), the 35-mer linker (GGGGS)7, and T1144 were linked together by three-step overlapping PCR. We took p2DLT-PDI as an example. First, the D1D2 (FD1D2his: 5′-CGCGGATCCCATCACCATCACCATCATAAGAAAGTGGTGCTG-3′, RD1D2: 5′-CACTTCCTCCTCCTCTATGCTGGAGGCCTTCTGGAA-3′), L35 (FL35: 5′-GGAGGAGGA GGAAGTGGCGGCGGCGGCTCGGGTGGTGGTGGTTCTGGAGGTGGCGGTAGCGGAGGTGGAGGTAGTGGAGGC-3′, RL35: 5′-GCTACCTCCGCCTCCCGAACCTCCGCCTCCA CTACCTCCACCTCCGCTACCGCCACCTCCAGAACCACCACCACCCGAG-3′) and T1144 (FT1144: 5′-GAGGCGGAGGTAGCACGACCTGGGAAGCATGGGACAGAGCTATTGCTG AATACGCAGCTAGGATAGAAGCTTTACTCAGAGCTTTA-3′, RT1144: 5′-CGGAGAT CTCTATAATTCCCTTAAGGCTGCTTCATTCTTTTCTTGCTGTTCTTGTAAAGCTCTGAGTAAAGCTTCTATCC-3′) DNA fragments were genera-ted by overlapping PCR using the corresponding primer pairs. Second, the DNA fragments coding for L35 and T1144 were linked by overlapping PCR with the primers FL35 and RT1144. Third, the two DNA fragments encoding D1D2 and L35-T1144 were linked by overlapping PCR with the DNA fragment D1D2 and the primers FD1D2his and RT1144. Finally, the amplified DNA fragment coding for 2DLT was digested by BamHI and EcoR I and inserted into the expression vector pGEX-6p-1 to generate the p2DLT plasmid. In order to prevent the formation of the inclusion bodies in E. coli, we inserted a protein disulfide isomerase (PDI) [21, 22] DNA sequence (aa 18–508) with PreScission protease cutting site (called ppase site) in the N terminus into the EcoR I and Xho I sites located at the C terminus of His-2DLT gene in the plasmid p2DLT to extend the GST-his-2DLT reading frame, resulting in the generation of chimeric GST-his-2DLT-ppase-PDI. This plasmid is called p2DLT-PDI. The sequences were confirmed by DNA sequencing.
Protein expression and purification
To express D1D2 and 2DLT fusion proteins, Escherichia coli strain Rosetta 2 (DE3) pLysS (Novagen) was transformed with pD1D2-PDI and p2DLT-PDI, respectively, cultured at 37°C to OD600 = 0.4, then induced at 16-22°C for 8–12 h with 0.4 mM IPTG. The cells were harvested and lysed by sonication in the presence of protease inhibitor mixture (Roche). After centrifugation, supernatants containing the fusion protein were collected. The protein was purified with Glutathione-Sepharose 4B affinity columns and cleaved with PreScissionTM Protease (GE Healthcare). These fusion proteins were purified by His·Bind® Purification Kit (Novagen) and fast protein liquid chromatography (FPLC), and then analyzed by SDS-PAGE.
SDS-PAGE and Western blot analysis
Purified fusion proteins were analyzed by SDS-PAGE as previously described . Briefly, D1D2 or 2DLT was mixed with 4X SDS sample buffer (Novagen, Gibbstown, NJ) and boiled for 5 minutes or kept at room temperature (RT) before loading onto a 10-20% Tricine-Glycine gel (Invitrogen, Carlsbad, CA). The electrophoresis was conducted in SDS-PAGE running buffer with 125 V constant voltage at 4°C. In Western blot, the anti-human CD4 and anti-T1144 polyclonal antibodies were used.
Enzyme-linked immunosorbent assay (ELISA)
D1D2 and 2DLT fusion proteins were characterized by ELISA as previously described . Briefly, they were coated onto a 96-well polystyrene plate (Costar, Corning Inc., Corning, NY) (10 μg/ml in 0.1 M Tris–HCl, pH 8.8), which was blocked with 2% non-fat milk in PBS. The polyclonal antibodies against CD4, conformation-dependent monoclonal antibody against CD4 (Sim.4) and anti-T1144 polyclonal antibody, respectively, were added to the plate. After incubation at 37°C for 60 min, horseradish peroxidase (HRP)-labeled antibodies (ZYMED Laboratories, S. San Francisco, CA) and the substrate TMB (Sigma) were added, sequentially. The binding of D1D2 and 2DLT to gp120 or gp41 NHR, and their inhibitory activity on gp41 6-HB formation were determined by ELISA as previously described .
The binding of D1D2 and 2DLT to gp120/gp41 expressed on the cell surface was detected by flow cytometry as previously described [49, 51]. Briefly, the cultured CHO-WT (with Env) and CHO-EE (with no Env) cells were detached from plate and washed with wash buffer (PBS containing 5% GBS) three times and incubated with the testing protein for 1 h at 4°C. After three washes, anti-CD4 or anti-T1144 polyclonal antibody was added for 1 h at 4°C. After three washes, FITC-conjugated anti-rabbit or mouse antibody was added and incubated for 1 h at 4°C. After three washes, the cells were examined by flow cytometry and the fluorescence intensity was recorded by FACSCalibur (Becton Dickinson).
Surface plasmon resonance (SPR) assay
The binding affinity of D1D2 and 2DLT to gp120 was measured by SPR using the BIAcore3000 system (Pharmacia, Piscataway, NJ), following the Manual of the Biomolecular Interaction Analysis (BIA) Technology as described previously . Briefly, gp120 (100 μg/ml) was immobilized onto the CM3 sensor chip by amine coupling, and the unreacted sites were blocked with ethanolamine. The dissociation reaction was done by washing with running buffer (10 mM HEPES pH7.4 containing 0.15 M NaCl, 3.4 mM EDTA and 0.005% v/v surfactant) for at least 2 min.
Fluorescence native polyacrylamide gel electrophoresis (FN-PAGE)
FN-PAGE for detecting 6-HB formation as described before . Briefly, a testing peptide or protein (100 μM) was pre-incubated with N36 (100 μM) at 37°C for 30 min, followed by addition of C34-FAM (100 μM) at 37°C for 30 minutes. The mixtures were added into Tris-glycine native sample buffer (Invitrogen, Carlsbad, CA). The samples (20 μl) were then loaded onto Tris-glycine gels (18%; Invitrogen, Carlsbad, CA), which were run under 120 V constant voltage at room temperature for 1 h. The gels were stained and visualized with the FluorChem 8800 Imaging System (Alpha Innotech Corp., San Leandro, CA) using a transillumination UV light source with excitation wavelength at 520 nm and then with Coomassie Blue.
Inhibition of HIV-1 infection
Inhibitory activities of D1D2 and 2DLT on HIV-1 infection were determined as previously described [53, 54]. For inhibition of HIV-1 IIIB (subtype B, X4) infection, 100 TCID50 of the virus was added to 1 × 104/ml MT-2 cells in RPMI medium 1640 containing 10% FBS in the presence or absence of the test peptide or protein overnight. The culture supernatants were removed, and fresh media were added. On the fourth day post-infection, culture supernatants were collected for detection of p24 antigen by ELISA. For inhibition of infection by the HIV-1 strain Bal (subtype B, R5), TZM-bl cells (1 × 105/ml) were pre-cultured overnight and infected with Bal at 100 TCID50 in the presence or absence of the test peptide or protein overnight. The cells were harvested and lysed on the fourth day post-infection with lysing reagent. The luciferase activity was analyzed using a luciferase kit (Promega, Madison, WI) and a luminometer (Ultra 386, Tecan, Durham, NC) according to the manufacturer’s instructions. For testing the effect of pre-incubation times on the inhibitory activity of 2DLT, the TZM-b1 assay was performed after pre-incubation of the HIV-1 Bal virus with the inhibitors for 0, 15, 60 and 240 min. For inhibition of primary HIV-1 isolate infection, peripheral blood mononuclear cells (PBMCs) were isolated from the blood of healthy donors. The PHA-stimulated cells were infected with a primary HIV-1 isolate at a multiplicity of infection (MOI) of 0.01 in the absence or presence of peptide or protein at graded concentrations. The supernatants were collected on the 7th day post-infection and tested for p24 antigen by ELISA as previously described [54, 55]. The percent inhibition of p24 production or luciferase activity was calculated.
Inactivation of HIV-1 virions
The virus inactivation by D1D2 and 2DLT was determined as previously described [25, 56, 57]. Briefly, 100 μl of the protein or peptide at graded concentration were added to 100 μl of an HIV-1 strain (200 TCID50/ml), followed by incubation at 4°C for 1 h. Then, PEG-6000 was added to the treated virus at final concentration of 3%, 4°C for 1 h. The mixture was centrifuged on a microfuge at 15,000 rpm for 30 min. The supernatants were removed and the pellet was washed with 3% PEG in PBS containing 10 mg/ml BSA. The viral pellet was then resuspended in 100 μl of PBS, before addition of 100 μl MT-2 or TZM-bl cells (1 × 105/ml). After cultur at 37°C for 3 days, p24 production in MT-2 cell culture or luciferase activity in TZM-bl cell culture was tested as previously described [58–60].
To measure the effects of D1D2 and 2DLT on induction of short-lived PFI of HIV-1 Env, we used a cell-based ELISA as previously described . Briefly, CHO-WT cells steadily expressing HIV-1 Env were seeded in 96-well plates (5 × 104/well). Cells were then harvested and washed twice with blocking buffer (35 mg/ml BSA, 10 mg/ml non-fat dry milk, 1.8 mM CaCl2, 1 mM MgCl2, 25 mM Tris, pH 7.5 and 140 mM NaCl). For pulse activation experiments, the cells were incubated with D1D2 (2.5 μM) or 2DLT (2.5 μM) suspended in blocking buffer for three minutes, washed three times with blocking buffer and incubated with the C34-biotin (2 μM). To study the temperature dependence of NHR groove exposure, the D1D2- or 2DLT-pulsed cells were incubated at the requisite temperature for different lengths of time. The cells were subsequently returned to room temperature for incubation with C34-biotin. Cells were then washed four times with blocking buffer and four times with washing buffer (140 mM NaCl, 1.8 mM CaCl2, 1 mM MgCl2 and 20 mM Tris, pH 7.5). A horseradish peroxidase-conjugated streptavidin (ZYMED Laboratories, S. San Francisco, CA) was then incubated with the samples for 45 minutes at room temperature. Cells were washed 5 times with blocking buffer and 5 times with washing buffer. HRP enzyme activity was determined after the addition of 33 μl per well of a 1:1 mixture of Western Lightning oxidizing and luminal reagents (Perkin Elmer Life Sciences) supplemented with 150 mM NaCl. Light emission was measured.
HIV-1 infectivity in CD4-/CCR5+cells treated by D1D2 or 2DLT
The effects of D1D2 and 2DLT on HIV-1 infection of CD4-/CCR5+ target cells were evaluated as previously described . Briefly, HIV-1 Bal (100 TCID50/well was cultured with Cf2Th-CCR5 cells (1 × 106 cells per well) at room temperature for 1 h, then was resuspended, followed by addition of D1D2 or 2DLT at different concentrations. After a short centrifugation, the mixture was incubated for 8–12 h at room temperature. The viral infectivity was measured three days later.
Inhibition of HIV-1-mediated cell-cell fusion
HIV-1 mediated cell-cell fusion was measured with a dye transfer assay as previously described [27, 29]. In brief, the HIV-1IIIB chronically infected H9 (H9/HIV-1IIIB) cells were labeled with Calcein-Am (Molecular Probes, Inc., Eugene, OR). After washes, the fluorescence-labeled H9/HIV-1IIIB cells were incubated with MT-2 cells at 37°C for 2 h in the absence or presence of an inhibitor at a graded concentration. The percentage of fused cells was counted under a fluorescence microscope (Zeiss, Germany), and the 50% inhibitory concentration of each drug was calculated with the Calcusyn software program [27, 29].
C-terminal heptad repeat
N-terminal heptad repeat
Native polyacrylamide gel electrophoresis
50% cytotoxicity concentrations.
MT-2, TZM-b1, CHO-WT, CHO-EE, and Cf2Th/syn CCR5 cells, the HIV-1IIIB chronically infected H9 cells, and HIV-1 strains were obtained from the AIDS Research and Reference Reagent Program, NIH, USA. This work was supported by grants from the National Natural Science Foundation of China (#81173098 to SJ and #81102476 to LL), 973 Programme of China (#2012CB519001 to SJ) and “Chen Guang” Project of SMEC and SEDF (11CG03) to LL.
- Lalezari JP, Henry K, O’Hearn M, Montaner JS, Piliero PJ, Trottier B, Walmsley S, Cohen C, Kuritzkes DR, Eron JJ, Chung J, DeMasi R, Donatacci L, Drobnes C, Delehanty J, Salgo M: Enfuvirtide, an HIV-1 fusion inhibitor, for drug-resistant HIV infection in north and south America. N Engl J Med. 2003, 348: 2175-2185. 10.1056/NEJMoa035026.View ArticlePubMedGoogle Scholar
- Dorr P, Westby M, Dobbs S, Griffin P, Irvine B, Macartney M, Mori J, Rickett G, Smith-Burchnell C, Napier C, Webster R, Armour D, Price D, Stammen B, Wood A, Perros M: Maraviroc (UK-427,857), a potent, orally bioavailable, and selective small-molecule inhibitor of chemokine receptor CCR5 with broad-spectrum anti-human immunodeficiency virus type 1 activity. Antimicrob Agents Chemother. 2005, 49: 4721-4732. 10.1128/AAC.49.11.4721-4732.2005.PubMed CentralView ArticlePubMedGoogle Scholar
- Polsky B, Baron PA, Gold JW, Smith JL, Jensen RH, Armstrong D: In vitro inactivation of HIV-1 by contraceptive sponge containing nonoxynol-9. Lancet. 1988, 1: 1456-View ArticlePubMedGoogle Scholar
- Phillips DM, Taylor CL, Zacharopoulos VR, Maguire RA: Nonoxynol-9 causes rapid exfoliation of sheets of rectal epithelium. Contraception. 2000, 62: 149-154. 10.1016/S0010-7824(00)00156-6.View ArticlePubMedGoogle Scholar
- Chan DC, Kim PS: HIV entry and its inhibition. Cell. 1998, 93: 681-684. 10.1016/S0092-8674(00)81430-0.View ArticlePubMedGoogle Scholar
- Jiang S, Debnath AK: Development of HIV entry inhibitors taregeted to the coiled coil regions of gp41. Biochem Biophys Res Commun. 2000, 269: 641-646. 10.1006/bbrc.1999.1972.View ArticlePubMedGoogle Scholar
- Melikyan GB: Common principles and intermediates of viral protein-mediated fusion: the HIV-1 paradigm. Retrovirology. 2008, 5: 111-10.1186/1742-4690-5-111.PubMed CentralView ArticlePubMedGoogle Scholar
- Bhakta SJ, Shang L, Prince JL, Claiborne DT, Hunter E: Mutagenesis of tyrosine and di-leucine motifs in the HIV-1 envelope cytoplasmic domain results in a loss of Env-mediated fusion and infectivity. Retrovirology. 2011, 8: 37-10.1186/1742-4690-8-37.PubMed CentralView ArticlePubMedGoogle Scholar
- Dimonte S, Mercurio F, Svicher V, D’Arrigo R, Perno CF, Ceccherini-Silberstein F: Selected amino acid mutations in HIV-1 B subtype gp41 are associated with specific gp120v(3) signatures in the regulation of co-receptor usage. Retrovirology. 2011, 8: 33-10.1186/1742-4690-8-33.PubMed CentralView ArticlePubMedGoogle Scholar
- D’Souza MP, Cairns JS, Plaeger SF: Current evidence and future directions for targeting HIV entry: therapeutic and prophylactic strategies. JAMA. 2000, 284: 215-222. 10.1001/jama.284.2.215.View ArticlePubMedGoogle Scholar
- Huang CC, Lam SN, Acharya P, Tang M, Xiang SH, Hussan SS, Stanfield RL, Robinson J, Sodroski J, Wilson IA, Wyatt R, Bewley CA, Kwong PD: Structures of the CCR5 N terminus and of a tyrosine-sulfated antibody with HIV-1 gp120 and CD4. Science. 2007, 317: 1930-1934. 10.1126/science.1145373.PubMed CentralView ArticlePubMedGoogle Scholar
- Daar ES, Li XL, Moudgil T, Ho DD: High concentrations of recombinant soluble CD4 are required to neutralize primary human immunodeficiency virus type 1 isolates. Proc Natl Acad Sci U S A. 1990, 87: 6574-6578. 10.1073/pnas.87.17.6574.PubMed CentralView ArticlePubMedGoogle Scholar
- Schooley RT, Merigan TC, Gaut P, Hirsch MS, Holodniy M, Flynn T, Liu S, Byington RE, Henochowicz S, Gubish E: Recombinant soluble CD4 therapy in patients with the acquired immunodeficiency syndrome (AIDS) and AIDS-related complex. A phase I-II escalating dosage trial. Ann Intern Med. 1990, 112: 247-253.View ArticlePubMedGoogle Scholar
- Haim H, Si Z, Madani N, Wang L, Courter JR, Princiotto A, Kassa A, DeGrace M, Gee-Estrada K, Mefford M, Gabuzda D, Smith AB, Sodroski J: Soluble CD4 and CD4-mimetic compounds inhibit HIV-1 infection by induction of a short-lived activated state. PLoS Pathog. 2009, 5: e1000360-10.1371/journal.ppat.1000360.PubMed CentralView ArticlePubMedGoogle Scholar
- Jiang S, Lin K, Strick N, Neurath AR: HIV-1 inhibition by a peptide. Nature. 1993, 365: 113-View ArticlePubMedGoogle Scholar
- Wild CT, Shugars DC, Greenwell TK, McDanal CB, Matthews TJ: Peptides corresponding to a predictive alpha-helical domain of human immunodeficiency virus type 1 gp41 are potent inhibitors of virus infection. Proc Natl Acad Sci USA. 1994, 91: 9770-9774. 10.1073/pnas.91.21.9770.PubMed CentralView ArticlePubMedGoogle Scholar
- Lu M, Blacklow SC, Kim PS: A trimeric structural domain of the HIV-1 transmembrane glycoprotein. Nat Struct Biol. 1995, 2: 1075-1082. 10.1038/nsb1295-1075.View ArticlePubMedGoogle Scholar
- Lu M, Kim PS: A trimeric structural subdomain of the HIV-1 transmembrane glycoprotein. J Biomol Struct Dyn. 1997, 15: 465-471. 10.1080/07391102.1997.10508958.View ArticlePubMedGoogle Scholar
- Delmedico M, Bray B, Cammack N, Davison D, Dwyer J, Frick L, Tvermoes N, Wring S, Zhang H, Greenberg M: Next generation HIV peptide fusion inhibitor candidates achieve potent, durable suppression of virus replication in vitro and improved pharmacokinetic properties. 13th Conference on Retroviruses and Opportunistic Infections. 2006, Abstract No. 48Google Scholar
- Dwyer JJ, Wilson KL, Davison DK, Freel SA, Seedorff JE, Wring SA, Tvermoes NA, Matthews TJ, Greenberg ML, Delmedico MK: Design of helical, oligomeric HIV-1 fusion inhibitor peptides with potent activity against enfuvirtide-resistant virus. Proc Natl Acad Sci USA. 2007, 104: 12772-12777. 10.1073/pnas.0701478104.PubMed CentralView ArticlePubMedGoogle Scholar
- Liu Y, Zhao TJ, Yan YB, Zhou HM: Increase of soluble expression in Escherichia coli cytoplasm by a protein disulfide isomerase gene fusion system. Protein Expr Purif. 2005, 44: 155-161. 10.1016/j.pep.2005.03.030.View ArticlePubMedGoogle Scholar
- Lu L, Zhu Y, Diao J, Wang Z, Chen YH: V3 CTL epitope density in a single recombinant molecule antigen differentially affects the number and activity of primary and memory CD8+ T cells. Vaccine. 2008, 26: 845-852. 10.1016/j.vaccine.2007.11.022.View ArticlePubMedGoogle Scholar
- Kutner RH, Zhang XY, Reiser J: Production, concentration and titration of pseudotyped HIV-1-based lentiviral vectors. Nat Protoc. 2009, 4: 495-505. 10.1038/nprot.2009.22.View ArticlePubMedGoogle Scholar
- Ibegbu CC, Kennedy MS, Maddon PJ, Deen KC, Hicks D, Sweet RW, McDougal JS: Structural features of CD4 required for binding to HIV. J Immunol. 1989, 142: 2250-2256.PubMedGoogle Scholar
- Neurath AR, Strick N, Li YY: Anti-HIV-1 activity of anionic polymers: a comparative study of candidate microbicides. BMC Infect Dis. 2002, 2: 27-10.1186/1471-2334-2-27.PubMed CentralView ArticlePubMedGoogle Scholar
- Neurath AR, Strick N, Jiang S, Li YY, Debnath AK: Anti-HIV-1 activity of cellulose acetate phthalate: synergy with soluble CD4 and induction of “dead-end” gp41 six-helix bundles. BMC Infect Dis. 2002, 2: 6-10.1186/1471-2334-2-6.PubMed CentralView ArticlePubMedGoogle Scholar
- Liu S, Lu H, Xu Y, Wu S, Jiang S: Different from the HIV fusion inhibitor C34, the anti-HIV drug Fuzeon (T-20) inhibits HIV-1 entry by targeting multiple sites in gp41 and gp120. J Biol Chem. 2005, 280: 11259-11273. 10.1074/jbc.M411141200.View ArticlePubMedGoogle Scholar
- Liu S, Zhao Q, Jiang S: Determination of the HIV-1 gp41 postfusion conformation modeled by synthetic peptides: applicable for identification of the HIV-1 fusion inhibitors. Peptide. 2003, 24: 1303-1313.View ArticleGoogle Scholar
- Li L, He L, Tan S, Guo X, Lu H, Qi Z, Pan C, An X, Jiang S, Liu S: 3-Hydroxyphthalic anhydride-modified chicken ovalbumin exhibits potent and broad anti-HIV-1 activity: a potential microbicide for preventing sexual transmission of HIV-1. Antimicrob Agents Chemother. 2010, 54: 1700-1711. 10.1128/AAC.01046-09.PubMed CentralView ArticlePubMedGoogle Scholar
- Sullivan N, Sun Y, Binley J, Lee J, Barbas CF, Parren PW, Burton DR, Sodroski J: Determinants of human immunodeficiency virus type 1 envelope glycoprotein activation by soluble CD4 and monoclonal antibodies. J Virol. 1998, 72: 6332-6338.PubMed CentralPubMedGoogle Scholar
- Chertova E, Bess JJ, Crise BJ, Sowder RC, Schaden TM, Hilburn JM, Hoxie JA, Benveniste RE, Lifson JD, Henderson LE, Arthur LO: Envelope glycoprotein incorporation, not shedding of surface envelope glycoprotein (gp120/SU), Is the primary determinant of SU content of purified human immunodeficiency virus type 1 and simian immunodeficiency virus. J Virol. 2002, 76: 5315-5325. 10.1128/JVI.76.11.5315-5325.2002.PubMed CentralView ArticlePubMedGoogle Scholar
- McDougal JS, Kennedy MS, Orloff SL, Nicholson JK, Spira TJ: Mechanisms of human immunodeficiency virus Type 1 (HIV-1) neutralization: irreversible inactivation of infectivity by anti-HIV-1 antibody. J Virol. 1996, 70: 5236-5245.PubMed CentralPubMedGoogle Scholar
- Allaway GP, Davis-Bruno KL, Beaudry GA, Garcia EB, Wong EL, Ryder AM, Hasel KW, Gauduin MC, Koup RA, McDougal JS: Expression and characterization of CD4-IgG2, a novel heterotetramer that neutralizes primary HIV type 1 isolates. AIDS Res Hum Retroviruses. 1995, 11: 533-539. 10.1089/aid.1995.11.533.View ArticlePubMedGoogle Scholar
- Jacobson JM, Israel RJ, Lowy I, Ostrow NA, Vassilatos LS, Barish M, Tran DN, Sullivan BM, Ketas TJ, O’Neill TJ, Nagashima KA, Huang W, Petropoulos CJ, Moore JP, Maddon PJ, Olson WC: Treatment of advanced human immunodeficiency virus type 1 disease with the viral entry inhibitor PRO 542. Antimicrob Agents Chemother. 2004, 48: 423-429. 10.1128/AAC.48.2.423-429.2004.PubMed CentralView ArticlePubMedGoogle Scholar
- Dey B, Del Castillo CS, Berger EA: Neutralization of human immunodeficiency virus type 1 by sCD4-17b, a single-chain chimeric protein, based on sequential interaction of gp120 with CD4 and coreceptor. J Virol. 2003, 77: 2859-2865. 10.1128/JVI.77.5.2859-2865.2003.PubMed CentralView ArticlePubMedGoogle Scholar
- Balzarini J, Velazquez S, San-Felix A, Karlsson A, Perez-Perez MJ, Camarasa MJ, De CE: Human immunodeficiency virus type 1-specific [2′,5′-bis-O-(tert- butyldimethylsilyl)-beta-D-ribofuranosyl]-3'-spiro-5"-(4"-amino-1",2"- oxathiole-2",2"-dioxide)-purine analogues show a resistance spectrum that is different from that of the human immunodeficiency virus type 1-specific non-nucleoside analogues. Mol Pharmacol. 1993, 43: 109-114.PubMedGoogle Scholar
- Feldblum PJ, Weir SS, Cates W: The protective effect of condoms and nonoxynol-9 against HIV infection: a response to Wittkowski and colleagues. Am J Public Health. 1999, 89: 108-110.PubMed CentralView ArticlePubMedGoogle Scholar
- Feldblum PJ, Adeiga A, Bakare R, Wevill S, Lendvay A, Obadaki F, Olayemi MO, Wang L, Nanda K, Rountree W: SAVVY vaginal gel (C31G) for prevention of HIV infection: a randomized controlled trial in Nigeria. PLoS One. 2008, 3: e1474-10.1371/journal.pone.0001474.PubMed CentralView ArticlePubMedGoogle Scholar
- Krebs FC, Miller SR, Malamud D, Howett MK, Wigdahl B: Inactivation of human immunodeficiency virus type 1 by nonoxynol-9, C31G, or an alkyl sulfate, sodium dodecyl sulfate. Antiviral Res. 1999, 43: 157-173. 10.1016/S0166-3542(99)00044-3.View ArticlePubMedGoogle Scholar
- Catalone BJ, Miller SR, Ferguson ML, Malamud D, Kish-Catalone T, Thakkar NJ, Krebs FC, Howett MK, Wigdahl B: Toxicity, inflammation, and anti-human immunodeficiency virus type 1 activity following exposure to chemical moieties of C31G. Biomed Pharmacother. 2005, 59: 430-437. 10.1016/j.biopha.2005.07.008.View ArticlePubMedGoogle Scholar
- Cone RA, Hoen T, Wong X, Abusuwwa R, Anderson DJ, Moench TR: Vaginal microbicides: detecting toxicities in vivo that paradoxically increase pathogen transmission. BMC Infect Dis. 2006, 6: 90-10.1186/1471-2334-6-90.PubMed CentralView ArticlePubMedGoogle Scholar
- Schenten D, Marcon L, Karlsson GB, Parolin C, Kodama T, Gerard N, Sodroski J: Effects of soluble CD4 on simian immunodeficiency virus infection of CD4-positive and CD4-negative cells. J Virol. 1999, 73: 5373-5380.PubMed CentralPubMedGoogle Scholar
- Clapham PR, McKnight A, Weiss RA: Human immunodeficiency virus type 2 infection and fusion of CD4-negative human cell lines: induction and enhancement by soluble CD4. J Virol. 1992, 66: 3531-3537.PubMed CentralPubMedGoogle Scholar
- Allan JS, Strauss J, Buck DW: Enhancement of SIV infection with soluble receptor molecules. Science. 1990, 247: 1084-1088. 10.1126/science.2309120.View ArticlePubMedGoogle Scholar
- Moore JP, McKeating JA, Huang YX, Ashkenazi A, Ho DD: Virions of primary human immunodeficiency virus type 1 isolates resistant to soluble CD4 (sCD4) neutralization differ in sCD4 binding and glycoprotein gp120 retention from sCD4-sensitive isolates. J Virol. 1992, 66: 235-243.PubMed CentralPubMedGoogle Scholar
- Bouma P, Leavitt M, Zhang PF, Sidorov IA, Dimitrov DS, Quinnan GV: Multiple interactions across the surface of the gp120 core structure determine the global neutralization resistance phenotype of human immunodeficiency virus type 1. J Virol. 2003, 77: 8061-8071. 10.1128/JVI.77.14.8061-8071.2003.PubMed CentralView ArticlePubMedGoogle Scholar
- Eggink D, Bontjer I, Langedijk JP, Berkhout B, Sanders RW: Resistance of human immunodeficiency virus type 1 to a third-generation fusion inhibitor requires multiple mutations in gp41 and is accompanied by a dramatic loss of gp41 function. J Virol. 2011, 85: 10785-10797. 10.1128/JVI.05331-11.PubMed CentralView ArticlePubMedGoogle Scholar
- Papanikolopoulou K, Forge V, Goeltz P, Mitraki A: Formation of Highly Stable Chimeric Trimers by Fusion of an Adenovirus Fiber Shaft Fragment with the Foldon Domain of Bacteriophage T4 Fibritin. J Biol Chem. 2004, 279: 8991-8998. 10.1074/jbc.M311791200.View ArticlePubMedGoogle Scholar
- Jiang S, Lin K, Lu M: A conformation-specific monoclonal antibody reacting with fusion-active gp41 from the HIV-1 envelope glycoprotein. J Virol. 1998, 72: 10213-10217.PubMed CentralPubMedGoogle Scholar
- Huang JH, Liu ZQ, Liu S, Jiang S, Chen YH: Identification of the HIV-1 gp41 core-binding motif - HXXNPF. FEBS Lett. 2006, 580: 4807-4814. 10.1016/j.febslet.2006.07.067.View ArticlePubMedGoogle Scholar
- Zhu Y, Lu L, Xu L, Yang H, Jiang S, Chen YH: Identification of a gp41 core-binding molecule with homologous sequence of human TNNI3K-like protein as a novel human immunodeficiency virus type 1 entry inhibitor. J Virol. 2010, 84: 9359-9368. 10.1128/JVI.00644-10.PubMed CentralView ArticlePubMedGoogle Scholar
- Lu L, Zhu Y, Huang J, Chen X, Yang H, Jiang S, Chen YH: Surface Exposure of the HIV-1 Env Cytoplasmic Tail LLP2 Domain during the Membrane Fusion Process: INTERACTION WITH gp41 FUSION CORE. J Biol Chem. 2008, 283: 16723-16731. 10.1074/jbc.M801083200.PubMed CentralView ArticlePubMedGoogle Scholar
- Jiang S, Lu H, Liu S, Zhao Q, He Y, Debnath AK: N-substituted pyrrole derivatives as novel human immunodeficiency virus type 1 entry inhibitors that interfere with the gp41 six-helix bundle formation and block virus fusion. Antimicrob Agents Chemother. 2004, 48: 4349-4359. 10.1128/AAC.48.11.4349-4359.2004.PubMed CentralView ArticlePubMedGoogle Scholar
- Lu H, Zhao Q, Wallace G, Liu S, He Y, Shattock R, Neurath AR, Jiang BS: Cellulose acetate 1,2-benzenedicarboxylate inhibits infection by cell-free and cell-associated primary HIV-1 isolates. AIDS Res Hum Retroviruses. 2006, 22: 411-418. 10.1089/aid.2006.22.411.PubMed CentralView ArticlePubMedGoogle Scholar
- Rimsky LT, Shugars DC, Matthews TJ: Determinants of human immunodeficiency virus type 1 resistance to gp41-derived inhibitory peptides. J Virol. 1998, 72: 986-993.PubMed CentralPubMedGoogle Scholar
- Neurath AR, Strick N, Li YY, Debnath AK: Cellulose acetate phthalate, a common pharmaceutical excipient, inactivates HIV-1 and blocks the coreceptor binding site on the virus envelope glycoprotein gp120. BMC Infect Dis. 2001, 1: 17-10.1186/1471-2334-1-17.PubMed CentralView ArticlePubMedGoogle Scholar
- Bilello J, Belay S, Weislow O: Development of novel approaches to the evaluation of virucidal agents: separation of treated virions from test compounds. AIDS. 2001, 15 (Suppl. 1): S39-View ArticleGoogle Scholar
- Li L, Qiao P, Yang J, Lu L, Tan S, Lu H, Zhang X, Chen X, Wu S, Jiang S, Liu S: Maleic anhydride-modified chicken ovalbumin as an effective and inexpensive anti-HIV microbicide candidate for prevention of HIV sexual transmission. Retrovirology. 2010, 7: 37-10.1186/1742-4690-7-37.PubMed CentralView ArticlePubMedGoogle Scholar
- Lu L, Tong P, Yu X, Pan C, Zou P, Chen YH, Jiang S: HIV-1 variants with a single-point mutation in the gp41 pocket region exhibiting different susceptibility to HIV fusion inhibitors with pocket- or membrane-binding domain. Biochim Biophys Acta. 2012, 1818: 2950-2957. 10.1016/j.bbamem.2012.07.020.View ArticlePubMedGoogle Scholar
- Yu X, Lu L, Cai L, Tong P, Tan S, Zou P, Meng F, Chen YH, Jiang S: Mutations of Gln64 in the HIV-1 gp41 N-terminal heptad repeat render viruses resistant to peptide HIV fusion inhibitors targeting the gp41 pocket. J Virol. 2012, 86: 589-593. 10.1128/JVI.05066-11.PubMed CentralView ArticlePubMedGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.