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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 - published pdf attached
 
 
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Journal of Virology, September 2010, p. 9359-9368, Vol. 84, No. 18
 
Yun Zhu,1,{dagger} Lu Lu,2,{dagger} Liling Xu,1 Hengwen Yang,1 Shibo Jiang,2* and Ying-Hua Chen1*
 
Laboratory of Immunology, School of Life Sciences, Tsinghua University, Beijing Key Laboratory for Protein Therapeutics, Protein Science Laboratory of MOE, Beijing, People's Republic of China,1 Lindsley F. Kimball Research Institute, New York Blood Center, New York, New York2
 
ABSTRACT
 
Human immunodeficiency virus type 1 (HIV-1) gp41 plays a critical role in the viral fusion process, and its N- and C-terminal heptad repeat domains serve as important targets for developing anti-HIV-1 drugs, like T-20 (generic name, enfuvirtide; brand name, Fuzeon). Here, we conducted a yeast two-hybrid screening on a human bone marrow cDNA library using the recombinant soluble gp41 ectodomain as the bait and identified a novel gp41 core-binding molecule, designated P20. P20 showed no homology with a current HIV fusion inhibitor, T-20, but had sequence homology to a human protein, troponin I type 3 interacting kinase (TNNI3K)-like protein. While it could bind to the six-helix bundle core structure formed by the N- and C-terminal heptad repeats, P20 did not interrupt the formation of the six-helix bundle. P20 was effective in blocking HIV-1 Env-mediated syncytium formation and inhibiting infection by a broad spectrum of HIV-1 strains with distinct subtypes and coreceptor tropism, while it was ineffective against other enveloped viruses, such as vesicular stomatitis virus and influenza A virus. P20 exhibited no significant cytotoxicity to the CD4+ cells that were used for testing antiviral activity. Among the 11 P20 mutants, four analogous peptides with a common motif (WGRLEGRRT) exhibited significantly reduced anti-HIV-1 activity, suggesting that this region is the critical active site of P20. Therefore, this peptide can be used as a lead for developing novel HIV fusion inhibitors and as a probe for studying the membrane-fusogenic mechanism of HIV.
 
INTRODUCTION
 
Human immunodeficiency virus type 1 (HIV-1) is an enveloped virus, and its envelope protein (Env) complex controls the key processes by which HIV-1 delivers its replicative material into target cells. Specifically, the Env surface subunit, gp120, binds the cellular receptor CD4 and a coreceptor, CCR5 or CXCR4, which triggers conformational changes of the transmembrane subunit, gp41 (8). The N-terminal heptad repeat (NHR) in the gp41 ectodomain interacts with its C-terminal heptad repeat (CHR) to form a trimer of hairpins, or six-helix bundle (6-HB; also known as the gp41 fusion core) (38, 51), which brings the viral and target cell membranes into close proximity and promotes membrane fusion (3, 51). Therefore, the gp41 6-HB core plays an important role in viral fusion and may serve as an attractive target for the development of HIV fusion/entry inhibitors (20).
 
In the early 1990s, a number of peptides derived from the gp41 NHR and CHR regions were discovered to exhibit highly potent anti-HIV-1 activity by binding to the corresponding region of gp41 at the fusion-intermediate state (22, 23, 38, 52, 53) and blocking gp41 6-HB core formation (4, 9, 32, 47). One of the CHR-peptides, T-20 (generic name, enfuvirtide; brand name, Fuzeon), was licensed by the FDA as the first member of a new class of anti-HIV drugs, the HIV fusion inhibitors (33, 53). Although T-20 is very effective in inhibiting infection by a broad spectrum of HIV-1 strains, especially those resistant to current antiretroviral therapies (26), T-20 itself also can easily induce drug resistance in T-20-treated patients, resulting in virologic failure (36, 46, 50, 55). Therefore, it is essential to identify and develop novel HIV-1 fusion inhibitors having a mechanism of action or target different from that for T-20 and with improved drug resistance profiles.
 
Here, we sought to screen a human bone marrow cDNA library in a yeast two-hybrid screening assay using the recombinant soluble gp41 ectodomain (rsgp41e) as the bait in hopes of identifying a novel HIV fusion inhibitor with sequence homology to a human protein and low immunogenicity to humans to avoid its rapid clearance by specific human antibodies (1). We identified a 32-mer peptide, designated P20, with sequence homology to human troponin I type 3 interacting kinase (TNNI3K)-like protein. P20 could specifically bind to the gp41 6-HB core and strongly blocked HIV-1 Env-mediated membrane fusion. It potently inhibited infection by a number of laboratory-adapted HIV-1 strains, including T-20-resistant variants, and a broad spectrum of primary HIV-1 isolates. These results suggest that P20 has the potential to be developed further as a novel anti-HIV-1 therapeutic and can be used as a probe to study the role of the HIV-1 gp41 6-HB core in the membrane fusion process.
 
DISCUSSION
 
Recently, a number of human proteins that are required for HIV infection have been identified through genomewide screenings (2, 27, 56). Some human proteins, such as apolipoprotein B mRNA-editing enzyme 3G (APOBEC3G) and tripartite motif protein 5 alpha (Trim5{alpha}), serve as restriction factors targeting HIV replication (12, 48). The sequences in these proteins responsible for binding with the HIV-1 molecules, e.g., gp41, may be used for designing novel anti-HIV therapeutics.
 
Using a yeast two-hybrid system with rsgp41e as the bait to screen a human bone marrow cDNA library, we have identified a novel gp41 core-binding molecule, P20, which is effective in blocking HIV-1 Env-mediated cell fusion and inhibiting infection by a broad spectrum of HIV-1 strains with distinct subtypes and coreceptor tropism, but it exhibits no inhibitory activity against other enveloped viruses, such as VSV and influenza virus. P20 peptide has a sequence partly homologous to human TNNI3K-like protein or some other unknown human protein, although the potential interaction of this human protein with HIV-1 gp41 has not been defined yet. In addition to its low molecular mass, P20 may have advantages for drug development over the HIV-1 gp41-based HIV-1 fusion inhibitors like T-20, since it may not induce strong antibody responses when it is used in humans, thus avoiding rapid clearance by specific human antibodies. Furthermore, it was highly soluble for easy expression and purification via the prokaryotic expression system.
 
Unlike the current HIV fusion inhibitors derived from the HIV-1 gp41 NHR and CHR domains, such as T-20, which target the corresponding CHR and NHR regions, respectively, P20 may inhibit HIV entry by targeting the gp41 core conformation, since it bound neither the N peptide N36 nor the C peptide C34 but interacted with the 6-HB core formed by N36 and C34. Furthermore, P20 could not block 6-HB formation, while most of the N and C peptides are effective in inhibiting the formation of the gp41 6-HB core. It was reported that some gp41 6-HB-specfic human MAbs (167-D and 1281), mouse MAbs (9F2 and 2G8), and some protein inhibitors also could inhibit HIV-1 Env-mediated cell-cell fusion at the suboptimal temperature 31.5 or 37íC (13, 14, 17, 19, 30, 57). In contrast, the human MAbs that recognize the cluster I epitopes in gp41, such as 240-D and 181-D (54), did not block HIV-1 Env-mediated membrane fusion at both 31.5 and 37íC (13). Most recently, Roymans et al. (45) demonstrated that TMC353121, a potent respiratory syncytial virus (RSV) fusion inhibitor, bound with the amino acid residues in both NHR and CHR domains, resulting in the stabilization of an alternate 6-HB conformation and causing a local disturbance of the natural 6-HB conformation rather than completely blocking 6-HB formation. These findings provide clues for unraveling the mechanism of action of P20 and other 6-HB-binding molecules in inhibiting viral fusion.
 
Thus far, HIV Env-mediated membrane fusion is a largely unknown process, especially whether or not membranes fuse immediately after 6-HB formation. Miyauchi et al. provided compelling evidence to show that HIV enters cells primarily by endocytosis (39), which proves that the virus and target cell membranes do not start to fuse immediately after gp41 changes conformation. Instead, a series of complex molecular events, which facilitate viral entry by endocytosis or other means, occur during this short period. Therefore, it is possible that cell fusion takes place inside the target cell only moments later. Our previous study demonstrated that gp41 6-HB could interact with epsin (18), which is an essential accessory factor of endocytosis and docks to the plasma membrane by interacting with the lipid. Here, we found that P20 interacted with the gp41 6-HB and blocked HIV-1-mediated membrane fusion. It bound neither the effector cells nor the target cells, but it interacted with the syncytium formed by the fusion between the effector and target cells (Fig. 5).
 
To dissect the specific role of each amino acid in P20, we have designed 11 mutants and tested their biological and biochemical functions. Interestingly, the anti-HIV-1 activity of P20 and its mutants is well correlated with their binding affinities to the gp41 6-HB (Fig. 8). Mutations of the first three amino acids both increase the binding ability of P20 to the 6-HB and its antiviral efficacy, while the mutations in P20-E, P20-G, P20-H, and P20-I peptides, which share the motif WGRLEGRRT, show a significant decrease of gp41 6-HB-binding affinity and antiviral efficacy. This suggests that these amino acids compose the active site of P20 that plays a vital role in the inhibition of viral fusion. Further research should focus on these important sites to further optimize the antiviral efficacy of P20.
 
T-20 is a first-generation fusion inhibitor antiviral drug that has been successfully used in the last few years. However, T-20 is increasingly found to be ineffective against resistant viruses. Thus, it is necessary to replace it with a next-generation drug with an improved resistance profile. Both P20 and P20-A inhibited the T-20-resistant strain, suggesting that their mechanism of action and target are different from those of T-20.
 
In summary, by yeast two-hybrid screening on a human bone marrow cDNA library using rsgp41e as the bait, we have successfully identified a novel HIV-1 fusion inhibitor, P20, which has sequence homologous to that of a human protein and is able to bind to the gp41 6-HB core. P20 can be further developed as a novel anti-HIV-1 therapeutic and used as a probe for studying the possible role of the gp41 6-HB core in the late stages of membrane fusion.
 
RESULTS
 
Identification of a gp41-binding molecule with sequence homology to human TNNI3K-like protein.
 
To identify a gp41-binding molecule with sequence homology to a human protein, we conducted a yeast two-hybrid screening on a human bone marrow cDNA library using rsgp41e as the bait. Out of more than 1 x 106 clones screened, a blue clone on a -Try/-Leu/-His/-Ade/X-Gal plate was identified, and the corresponding plasmid was recovered and sequenced. Since it was the 20th protein on the plate, it was named P20; it consists of 32 amino acids (Fig. 1A). Blasting the human protein database at NCBI showed that it had similarity to human TNNI3K-like protein (GenBank accession no. CAE45949.1) (Fig. 1A), confirming that P20 is partly derived from a human protein-coding clone in the human bone marrow cDNA library. To demonstrate the specificity of the interaction, pGADT7-P20 and the control plasmid pGADT7-T were transformed into yeast strain AH109, which contains the bait plasmid pGBKT7-rsgp41e or the control bait plasmid pGBKT7-P53. Only the yeasts containing pGADT7-P20/pGBKT7-rsgp41e and pGADT7-T/pGBKT7-P53 displayed the His+/Ade+/Mel+ phenotype (Fig. 1B), suggesting that P20 can specifically interact with rsgp41e, as the control T protein specifically interacts with P53 (29). The recombinant P20 peptide was expressed in Escherichia coli and purified with affinity chromatography. We found that P20 was highly soluble and easily purified, with an estimated molecular mass of 3.5 kDa (Fig. 1C), making it easier to pursue further research and applications.
 
P20 could not significantly interact with the individual NHR and CHR peptides but strongly bound to the gp41 6-HB core.
 
Since the gp41 NHR and CHR regions are the critical functional domains involved in the fusion core formation and the viral fusion process and serve as important targets for HIV fusion inhibitors, we tested the binding activity of P20 with the NHR and CHR peptides and the 6-HB core formed by the NHR and CHR peptides in different assays. As shown in Fig. 2A and B, P20 exhibited weak binding activity with gp41 NHR and CHR peptides (e.g., N36 and C34, respectively), but it strongly bound to 3NC polypeptide, an in vitro model of the gp41 6-HB core that was formed by the NHR and CHR peptides (37), in a dose-dependent manner. The interaction between P20 and 3NC was further confirmed by the SPR analysis (Fig. 2C). Experimental curves (solid line) were shown overlaid with fitted curves (dashed line) obtained with the 1:1 binding-with-drifting baseline model, suggesting a 1:1 stoichiometry of P20 binding to the gp41 6-HB. These results indicate that P20 specifically binds to the conformational gp41 core structure formed by the gp41 NHR and CHR domains but cannot interact with the linear NHR and CHR sequences.
 
P20 was ineffective in blocking gp41 6-HB core formation between the NHR and CHR peptides.
 
We subsequently tested whether P20 could inhibit the formation of 6-HB formed by the peptides N36 and C34 by ELISA and FN-PAGE. ADS-J1, a small-molecule HIV-1 fusion inhibitor that was proven to block gp41 core formation (24, 49), was used as a control. In ELISA, ADS-J1 strongly blocked the gp41 6-HB formation with an IC50 of 3 μM, while P20 peptide at concentrations of up to 20 μM exhibited no significant inhibition on 6-HB formation (Fig. 3 A). In FN-PAGE (Fig. 3B), C34-FITC alone showed a clear fluorescence band at the lower position in the gel (lane 2). The mixture of N36 and C34-FITC displayed a fluorescence band at the upper position corresponding to the gp41 6-HB (34) and, at the same time, a lower band corresponding to the C34-FITC disappeared (lane 3), suggesting that N36 and C34-FITC associate to form a 6-HB. The addition of ADS-J1 to the mixture of N36 and C34-FITC resulted in the disappearance of the upper band and the reappearance of the lower band (line 4), confirming that ADS-J1 is able to block 6-HB formation. However, after the addition of P20 to the N36/C34-FITC mixture, the lower band did not reappear, but instead a new band at a position higher than that of the 6-HB band appeared. These results indicate that P20 does not block 6-HB formation but rather binds to the N36/C34-FITC 6-HB to form a complex with sizes different from that of 6-HB.
 
P20 inhibited HIV-1 Env-mediated membrane fusion.
 
We then investigated whether the P20 peptide could inhibit HIV-1 Env-mediated syncytium formation. Like the C peptide C34 and small-molecule HIV-1 fusion inhibitor ADS-J1, P20 potently inhibited syncytium formation in a dose-dependent manner with an IC50 of 0.108 μM (Fig. 4A). To further confirm whether P20 interacts with a fusion-active conformation of HIV-1 Env, we used flow cytometry to determine the binding activity of P20 to the effector and target cells in the fusion and nonfusion states. Our previous studies have shown that only the sodium butyrate-treated CHO-WT cells (7) are able to fuse with 3T3.T4.CXCR4 cells, resulting in syncytium formation. Here, we found that P20 bound with neither the 3T3.T4.CXCR4 cells (Fig. 4B, graph a) nor the CHO-WT cells (Fig. 4B, graph b). P20 also could not bind to the cocultured 3T3.T4.CXCR4 cells and CHO-WT cells not treated with sodium butyrate (with no syncytium formation) (Fig. 4B, graph c), nor could it bind to the sodium butyrate-treated CHO-WT cells (Fig. 4B, graph d). However, it significantly bound to the cocultured 3T3.T4.CXCR4 cells and sodium butyrate-treated CHO-WT cells (with syncytium formation) (Fig. 4B, graph e). These results suggest that P20 does not bind to the native HIV-1 Env expressed on the effector cells but is effective in binding to the Env in the fusion-active state, possibly to the gp41 fusion core at the late stage of membrane fusion.
 
P20 inhibited infection by a broad spectrum of HIV-1 strains.
 
The antiviral activity of P20 against infection by laboratory-adapted HIV-1 strains was determined using different assays. We found that P20 significantly inhibited infection by the laboratory-adapted HIV-1 X4 (NL4-3 and IIIB) and R5 (Bal) strains with IC50s at low-μM levels (Table 1), while it exhibited no inhibition on VSV-G and IAV HA pseudovirus infection (Fig. 5A). P20 showed no cytotoxicity to MT-2 and TZM-bl cells at concentrations as high as 50 μM (Fig. 5B). These results suggest that P20-mediated antiviral activity is specific for HIV-1 and that P20 has no significant toxic effect on CD4+ cells that were used for testing the antiviral activity of P20. We compared the antiviral activity of T-20 to that of T-20-sensitive and -resistant strains that bear mutations in the principal determinant responsible for T-20 resistance (aa 36 to 45; GIVQQQNNLL) in the gp41 NHR domain (10, 15, 28, 36). Unlike T-20, which is much less effective against T-20-resistant strains than against T-20-sensitive strains, P20 showed similar potency against both T-20-sensitive and -resistant variants (Table 2). This result indicates that because P20 has a different target site in gp41 from that for T-20, P20 is able to inhibit infection by T-20-resistant viruses. Subsequently, we tested the inhibitory activities of P20 against a panel of 14 representative primary HIV-1 isolates with distinct subtypes (A to G and group O) and coreceptor tropism (R5 and X4R5), using T-20 as a control. As shown in Table 3, P20 could significantly inhibit infection by all of the primary HIV-1 isolates tested, with IC50s at low-μM levels. T-20, the highly potent HIV fusion inhibitor targeting gp41, inhibited infection by these primary HIV-1 isolates at the submicromolar level. These results suggest that like T-20, P20 is effective against a broad spectrum of HIV-1 strains with different genotype and phenotypes.
 
The critical functional domain is localized in a region adjacent to the C terminus of P20.
 
To identify the critical functional domain in the P20 peptide, we designed 11 triple-alanine mutants overlapping the entire sequence of P20 (Fig. 6A). All of these mutants were well expressed and purified in a manner similar to that of P20 (Fig. 6B). Results showed that the site-specific binding capacities of these P20 mutations to the 6-HB were severely affected. For example, while mutant P20-A showed enhanced affinity to the 6-HB, the mutants P20-E, P20-G, P20-H, and P20-I exhibited decreased affinity to the 6-HB (Fig. 7A). As determined by SPR analysis, the binding affinities of P20 with the 6-HB (3NC) were lower and higher than those of P20-A and P20-G, respectively (Fig. 7B). Unlike ADS-J1 but like P20, not all P20 mutants significantly inhibited gp41 6-HB formation at concentrations up to 20 μM (Fig. 7C). By analyzing the anti-HIV-1 activity of P20 and its mutants, we found that those mutants with decreased affinity of binding to 6-HB (e.g., P20-E, P20-G, P20-H, and P20-I) also were less effective than P20 and P20-A in inhibiting HIV-1 infection (Table 1 to 3). Interestingly, the 6-HB core-binding activity of P20 and its mutants is well correlated with their anti-HIV-1 activity (Fig. 8). Since those mutants with decreased biological activity share a motif (WGRLEGRRT) located in the region adjacent to the C terminus of P20, we suspect that this region is its critical functional domain. It is expected that the optimization of sequence in this region generates analog peptides with improved anti-HIV-1 activity.
 
 
 
 
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