Mammalian Two-hybrid System Research Articles

Evidence of non-canonical NOTCH signaling: Delta-like 1 homolog (DLK1) directly interacts with the NOTCH1 receptor in mammals

Canonical NOTCH signaling, known to be essential for tissue development, requires the Delta-Serrate-LAG2 (DSL) domain for NOTCH to interact with its ligand. However, despite lacking DSL, Delta-like 1 homolog (DLK1), a protein that plays a significant role in mammalian development, has been suggested to interact with NOTCH1 and act as an antagonist. This non-canonical interaction is, however controversial, and evidence for a direct interaction, still lacking in mammals. In this study, we elucidated the putative DLK1-NOTCH1 interaction in a mammalian context. Taking a global approach and using Dlk1+/+ and Dlk1−/− mouse tissues at E16.5, we demonstrated that several NOTCH signaling pathways indeed are affected by DLK1 during tissue development, and this was supported by a lower activation of NOTCH1 protein in Dlk1+/+ embryos. Likewise, but using a distinct Dlk1-manipulated (siRNA) setup in a mammalian cell line, NOTCH signaling was substantially inhibited by DLK1. Using a mammalian two-hybrid system, we firmly established that the effect of DLK1 on NOTCH signaling was due to a direct interaction between DLK1 and NOTCH1. By careful dissection of this mechanism, we found this interaction to occur between EGF domains 5 and 6 of DLK1 and EGF domains 10–15 of NOTCH1. Thus, our data provide the first evidence for a direct interaction between DLK1 and NOTCH1 in mammals, and substantiate that non-canonical NOTCH ligands exist, adding to the complexity of NOTCH signaling.

Molecular cloning of canine co-chaperone small glutamine-rich tetratricopeptide repeat-containing protein α (SGTA) and investigation of its ability to suppress androgen receptor signalling in androgen-independent prostate cancer

Although the morbidity of canine prostate cancer is low, the majority of cases present with resistance to androgen therapy and poor clinical outcomes. These pathological conditions are similar to the signs of the terminal stage of human androgen-independent prostate cancer. The co-chaperone small glutamine-rich tetratricopeptide repeat-containing protein α (SGTA) is known to be overexpressed in human androgen-independent prostate cancer. However, there is little information about the structure and function of canine SGTA. In this study, canine SGTA was cloned and analysed for its ability to suppress androgen receptor signalling.The full-length open reading frame (ORF) of the canine SGTA gene was amplified by RT-PCR using primers designed from canine-expressed sequence tags that were homologous to human SGTA. The canine SGTA ORF has high homology with the corresponding human (89%) and mouse (81%) sequences. SGTA dimerisation region and tetratricopeptide repeat (TPR) domains are conserved across the three species. The ability of canine SGTA to undergo homodimerisation was demonstrated by a mammalian two-hybrid system and a pull-down assay. The negative impact of canine SGTA on androgen receptor (AR) signalling was demonstrated using a reporter assay in androgen-independent human prostate cancer cell lines. Pathological analysis showed overexpression of SGTA in canine prostate cancer, but not in hyperplasia. A reporter assay in prostate cells demonstrated suppression of AR signalling by canine SGTA. Altogether, these results suggest that canine SGTA may play an important role in the acquisition of androgen independence by canine prostate cancer cells.

The interaction between the Hepatitis C proteins NS4B and NS5A is involved in viral replication

Hepatitis C virus (HCV) replicates in membrane associated, highly ordered replication complexes (RCs). These complexes include viral and host proteins necessary for viral RNA genome replication. The interaction network among viral and host proteins underlying the formation of these RCs is yet to be thoroughly characterized. Here, we investigated the association between NS4B and NS5A, two critical RC components. We characterized the interaction between these proteins using fluorescence resonance energy transfer and a mammalian two-hybrid system. Specific tryptophan residues within the C-terminal domain (CTD) of NS4B were shown to mediate this interaction. Domain I of NS5A, was sufficient to mediate its interaction with NS4B. Mutations in the NS4B CTD tryptophan residues abolished viral replication. Moreover, one of these mutations also affected NS5A hyperphosphorylation. These findings provide new insights into the importance of the NS4B–NS5A interaction and serve as a starting point for studying the complex interactions between the replicase subunits.

Identification of a selective glucocorticoid receptor ligand for the treatment of chronic inflammation in type 2 diabetes mellitus.

The present study aimed to identify a new selective glucocorticoid receptor (GR) ligand for the treatment of chronic inflammation in type 2 diabetes mellitus. The IN Cell Analyzer 1000 platform was employed to screen for compounds that may promote GR nuclear translocation. A mammalian two-hybrid system and transactivation assay-were used to analyze the selected GR ligands and evaluate their activities for GR transcription and the recruitment of co-activators. A novel selective GR ligand, compound Q40, was identified that was able to promote GR nuclear translocation in a short period of time. It increased the ability of GR to recruit co-activators in a concentration-dependent manner, but had no positive effect on GR transcriptional activity. In conclusion, an increase in the expression levels of gluconeogeneic genes, induced by the transcriptional activation of GR, is the predisposing factor most commonly associated with the side-effects of glucocorticoids. The results suggest that compound Q40 is a ligand of the GR and exerts an agonistic action on the recruitment of co-activators without sugar dysmetabolism-related side-effects. Thus, compound Q40 has the potential to be used as an anti-inflammatory adjuvant therapy with minimal side-effects in patients with type 2 diabetes mellitus.

Characterization of L1 ORF1p Self-Interaction and Cellular Localization Using a Mammalian Two-Hybrid System

Long INterspersed Element-1 (LINE-1, L1) is an active retrotransposon that mobilizes using a ribonucleoprotein particle (RNP) intermediate composed of the full-length bicistronic L1 mRNA and the two proteins (ORF1p and ORF2p) encoded by that mRNA. ORF1p and ORF2p demonstrate cis-preference for their encoding mRNA. Previous studies of ORF1p, purified from bacterial and insect cells demonstrated that this protein forms trimers in vitro. While valuable for understanding ORF1p function, these in vitro approaches do not provide any information on ORF1p self-interaction in the context of mammalian cells. We used a mammalian two-hybrid (M2H) system in order to study L1 ORF1p self-interaction in human and mouse cells. We demonstrate that the M2H system successfully detects human and mouse ORF1p self-interactions in transiently transfected mammalian cells. We also generated mouse and human ORF1p-specific antibodies to characterize the expression of ORF1p fusion proteins used in the M2H system. Using these antibodies, we demonstrate that ORF1p interaction in trans leads to the formation of heterodimers that are expected to produce a positive signal in the M2H system. Although the role for L1 ORF1p cis-preference in L1 mobilization is established, the impact of ability of ORF1pto interact in trans on the L1 replication cycle is not known. Furthermore, western blot analysis of ORF1p generated by a full-length L1, wild type ORF1, or a codon-optimized ORF1 expression vector is detected in the nucleus. In contrast, the addition of a tag to the N-terminus of the mouse and human ORF1 proteins can significantly alter the subcellular localization in a tag-specific manner. These data support that nuclear localization of ORF1p may contribute to L1 (and potentially the SINE Alu) RNP nuclear access in the host cell.

Transcriptional Activity and Expression of Liver X Receptor in the Ascidian Halocynthia roretzi

Liver X receptors, LXRs, are ligand-activated transcription factors that belong to the group H nuclear receptor (NR) superfamily. In this study, an LXR (HrLXR) cDNA was cloned from the ascidian Halocynthia roretzi hepatopancreas and characterized to examine the functional conservation of ancestral LXRs in chordates. A phylogenetic analysis of HrLXR showed that it belongs to the tunicate (urochordate) LXR subgroup, which is distinct from vertebrate LXRs. Quantitative real-time PCR analysis revealed that HrLXR mRNA was expressed predominantly in the gills, and highly expressed in unfertilized eggs followed by decrease at later embryonic and larval stages. Unexpectedly, HrLXR was not activated by GW3965, whereas a synthetic ligand for a farnesoid X receptor, GW4064, activated HrLXR. This activation was abolished by the deletion of 51 amino acids from the N-terminus. In a mammalian two-hybrid system, HrLXR interacted with HrRXR in the presence of GW4064 or 9-cis retinoic acid. The injection of GW3965 and GW4064 in vivo increased the ATPbinding cassette sub-family G member 4 and HrLXR mRNA levels in the hepatopancreas and gills. These results suggest that the mRNA expression and transcriptional properties of HrLXR are different from those of vertebrate LXRs, although HrLXR is likely responsive to the related NR ligand, GW4064.

Substrate Specificity of R3 Receptor-like Protein-tyrosine Phosphatase Subfamily toward Receptor Protein-tyrosine Kinases

Receptor-like protein-tyrosine phosphatases (RPTPs) are involved in various aspects of cellular functions, such as proliferation, differentiation, survival, migration, and metabolism. A small number of RPTPs have been reported to regulate activities of some cellular proteins including receptor protein-tyrosine kinases (RPTKs). However, our understanding about the roles of individual RPTPs in the regulation of RPTKs is still limited. The R3 RPTP subfamily reportedly plays pivotal roles in the development of several tissues including the vascular and nervous systems. Here, we examined enzyme-substrate relationships between the four R3 RPTP subfamily members and 21 RPTK members selected from 14 RPTK subfamilies by using a mammalian two-hybrid system with substrate-trapping RPTP mutants. Among the 84 RPTP-RPTK combinations conceivable, we detected 30 positive interactions: 25 of the enzyme-substrate relationships were novel. We randomly chose several RPTKs assumed to be substrates for R3 RPTPs, and validated the results of this screen by in vitro dephosphorylation assays, and by cell-based assays involving overexpression and knock-down experiments. Because their functional relationships were verified without exception, it is probable that the RPTKs identified as potential substrates are actually physiological substrates for the R3 RPTPs. Interestingly, some RPTKs were recognized as substrates by all R3 members, but others were recognized by only one or a few members. The enzyme-substrate relationships identified in the present study will shed light on physiological roles of the R3 RPTP subfamily.

CYP24A1 and CYP27B1 Polymorphisms Modulate Vitamin D Metabolism in Colon Cancer Cells

Vitamin D is a well-studied agent for cancer chemoprevention and treatment. Its chief circulating metabolite, 25-hydroxyvitamin D, is converted into the active hormone 1,25-dihydroxyvitamin D (1,25D) by the cytochrome P450 enzyme CYP27B1 in kidney and other tissues. 1,25D is then deactivated by CYP24A1 and ultimately catabolized. Colorectal carcinoma cells express CYP27B1 and CYP24A1 that locally regulate 1,25D with potential implications for its impact on carcinogenesis. While 1,25D inhibits cancer growth, the effects of polymorphic variations in genes encoding proteins involved in 1,25D homeostasis are poorly understood. Using an RXR-VDR mammalian two-hybrid (M2H) biologic assay system, we measured vitamin D metabolite uptake and activation of the vitamin D receptor (VDR) pathway in colon cancer cells that expressed one of five CYP27B1 single-nucleotide polymorphisms (SNP) or four CYP24A1 SNPs. Compared with the wild-type control, four of five CYP27B1 SNPs reduced enzymatic activity, whereas one (V166L) increased activity. For CYP24A1, all tested SNPs reduced enzyme activity. Quantitative real-time PCR analyses supported the results of M2H experiments. The observed SNP-directed variation in CYP functionality indicated that vitamin D homeostasis is complex and may be influenced by genetic factors. A comprehensive understanding of 1,25D metabolism may allow for a more personalized approach toward treating vitamin D-related disorders and evaluating risk for carcinogenesis.

A Second CRM1-Dependent Nuclear Export Signal in the Influenza A Virus NS2 Protein Contributes to the Nuclear Export of Viral Ribonucleoproteins

Influenza A virus NS2 protein, also called nuclear export protein (NEP), is crucial for the nuclear export of viral ribonucleoproteins. However, the molecular mechanisms of NEP mediation in this process remain incompletely understood. A leucine-rich nuclear export signal (NES2) in NEP, located at the predicted N2 helix of the N-terminal domain, was identified in the present study. NES2 was demonstrated to be a transferable NES, with its nuclear export activity depending on the nuclear export receptor chromosome region maintenance 1 (CRM1)-mediated pathway. The interaction between NEP and CRM1 is coordinately regulated by both the previously reported NES (NES1) and now the new NES2. Deletion of the NES1 enhances the interaction between NEP and CRM1, and deletion of the NES1 and NES2 motifs completely abolishes this interaction. Moreover, NES2 interacts with CRM1 in the mammalian two-hybrid system. Mutant viruses containing NES2 alterations generated by reversed genetics exhibit reduced viral growth and delay in the nuclear export of viral ribonucleoproteins (vRNPs). The NES2 motif is highly conserved in the influenza A and B viruses. The results demonstrate that leucine-rich NES2 is involved in the nuclear export of vRNPs and contributes to the understanding of nucleocytoplasmic transport of influenza virus vRNPs.

Random Mutagenesis MAPPIT Analysis Identifies Binding Sites for Vif and Gag in Both Cytidine Deaminase Domains of Apobec3G

The mammalian two-hybrid system MAPPIT allows the detection of protein-protein interactions in intact human cells. We developed a random mutagenesis screening strategy based on MAPPIT to detect mutations that disrupt the interaction of one protein with multiple protein interactors simultanously. The strategy was used to detect residues of the human cytidine deaminase Apobec3G that are important for its homodimerization and its interaction with the HIV-1 Gag and Vif proteins. The strategy is able to identify the previously described head-to-head homodimerization interface in the N-terminal domain of Apobec3G. Our analysis further detects two new potential interaction surfaces in the N-and C-terminal domain of Apobec3G for interaction with Vif and Gag or for Apobec3G dimerization.

Identification of in vivo interaction between rabbit hemorrhagic disease virus capsid protein and minor structural protein

We investigated the ability of the rabbit hemorrhagic disease virus (RHDV) capsid protein (VP60) to interact specifically with the minor structural protein VP10, using an in vivo cell-based CheckMate™ Mammalian Two-Hybrid System. RHDV VP60 protein interacted specifically with VP10. Immunofluorescence analysis and co-immunoprecipitation with specific antibodies revealed the existence of biologically important VP60/VP10 complexes. However, when VP60 was divided into two fragments, the interaction between VP60 and VP10 was impaired dramatically. These results will be helpful for further investigating the mechanism of RHDV particle assembly.

Bornavirus Closely Associates and Segregates with Host Chromosomes to Ensure Persistent Intranuclear Infection

Bornaviruses are nonsegmented negative-strand RNA viruses that establish a persistent infection in the nucleus and occasionally integrate a DNA genome copy into the host chromosomal DNA. However, how these viruses achieve intranuclear infection remains unclear. We show that Borna disease virus (BDV), a mammalian bornavirus, closely associates with the cellular chromosome to ensure intranuclear infection. BDV generates viral factories within the nucleus using host chromatin as a scaffold. In addition, the viral ribonucleoprotein (RNP) interacts directly with the host chromosome throughout the cell cycle, using core histones as a docking platform. HMGB1, a host chromatin-remodeling DNA architectural protein, is required to stabilize RNP on chromosomes and for efficient BDV RNA transcription in the nucleus. During metaphase, the association of RNP with mitotic chromosomes allows the viral RNA to segregate into daughter cells and ensure persistent infection. Thus, bornaviruses likely evolved a chromosome-dependent life cycle to achieve stable intranuclear infection.

AR-Q640X, a model to study the effects of constitutively active C-terminally truncated AR variants in prostate cancer cells

A recently identified mechanism allowing prostate cancer (PCa) cells to grow in the absence of androgens is the expression of constitutively active, C-terminally truncated androgen receptor (AR) variants lacking vast parts of the ligand-binding domain. These AR variants termed ARΔLBD are either products of alternative splicing, point mutations leading to premature stop codons or proteolytic cleavage of the AR. Some controversies exist about the requirement of additional full-length AR for the full transcriptional activity of the ARΔLBD. On basis of a mutated, C-terminally truncated AR termed Q640X, we developed an experimental model for the study of ARΔLBD in PCa cells. Activation of AR-dependent promoters was analyzed by reporter gene assays. Dimerization studies were conducted using a mammalian two-hybrid system. Although Q640X/Q640X homodimers were able to induce the expression of certain AR target genes, Q640X/AR heterodimers were necessary to activate the full panel of androgen-dependent genes under androgen-deprived conditions. The following study supports the hypothesis that castration-resistant prostate cancer (CRPC) cells are able to activate specific androgen-dependent genes by selective modulation of the ratio between ARΔLBD and their putative dimerization partners like the full-length AR or other ARΔLBD in the absence of androgens. The present data suggest that AR-mutant Q640X is a powerful experimental tool for the functional analysis of ARΔLBD in CRPC.

English

Restin, a novel member of the melanoma-associated antigen super family, is cloned from differentiated leukemia cells induced by retinoic acid. Restin may interact with some transcription factors. The present study investigated the effect of restin on activating transcription factor 3 (ATF3), an important protein in numerous physiological situations, using the mammalian two-hybrid system. The results show that ATF3 strongly interacted with restin. To determine the effect of restin on ATF3 activity, an ATF3 reporter plasmid with a luciferase gene was constructed. Hela and NIH3T3 cells transfected with both restin and ATF3 exhibited accelerated ATF3 activity compared with those transfected with only ATF3. This result suggests the role of restin in facilitating ATF3 activity. Restin was also shown to up-regulate ATF3 activity by its interaction with the ATF3 DNA binding domain. Overall, these data suggests the involvement of restin in important cellular modulation processes in Hela and NIH3T3 cells by enhancing ATF3 activity. Key words: Restin, MAGE, ATF3, two-hybrid system, ATF3 reporter, luciferase.

Raloxifene inhibits menin-dependent estrogen receptor activation in breast cancer cells

Menin is a tumor suppressor encoded by Men1 that is mutated in the human-inherited tumor syndrome--multiple endocrine neoplasia type 1. Menin binds to estrogen receptors (ER) to enhance estrogen activity in breast cancer cells. Our clinical study showed that the outcome in the case of menin-positive tumors was worse than in the case of menin-negative tumors. We examined the role of raloxifene on the cell growth in a menin-positive breast cancer cell line. To examine the mechanism of raloxifene on menin-dependent activation of ER, we employed the mammalian two-hybrid system. We have established a breast cancer cell line that stably expresses menin. Using these cells, we have examined the effect of raloxifene and tamoxifen on cell growth of menin-transfected cells. The expression of activation function (AF)-2 enhanced menin-mediated luciferase expression in the mammalian two-hybrid assay. Raloxifene attenuated the effect of menin on estrogen response element-luciferase activation, indicating that raloxifene inhibited the binding of menin to AF-2. Raloxifene significantly inhibited the growth of menin-transfected cells in a dose-dependent manner. Tamoxifen also inhibited menin-transfected MCF-7 cells; however, this inhibition was much less than that of raloxifene. Raloxifene inhibits the binding of menin to the AF-2 domain of ERα, suggesting that raloxifene is one of the therapeutic options for menin-positive breast cancer.

Tumor suppressor REIC/Dkk-3 interacts with the dynein light chain, Tctex-1

REIC/Dkk-3 is a member of the Dickkopf family proteins known as Wnt-antagonists, and REIC/Dkk-3 expression is downregulated in a broad range of cancer types. REIC/Dkk-3 acts as a tumor suppressor in multiple cancer cell lines by inducing apoptosis through endoplasmic reticulum (ER) stress signaling. However, the intracellular interaction partners of REIC/Dkk-3 have not been fully elucidated. By employing yeast two-hybrid screening, we identified the human dynein light chain, Tctex-1, as a novel interaction partner of REIC/Dkk-3. We further disclosed that the interaction involves the 136–157 amino acid region of REIC/Dkk-3 by using the mammalian two-hybrid system. Interestingly, this binding region of REIC/Dkk-3 with Tctex-1 contains an amino acid sequence motif [-E-X-G-R-R-X-H-] which was previously reported as the Tctex-1 binding domain of dynein intermediate chain (DIC). Immunocytochemistry demonstrated that both REIC/Dkk-3 and Tctex-1 were localized around the ER of human fibroblasts, and the similar distribution pattern of the proteins suggests that their interaction occurs around the ER. This is the first study showing the interaction of a Dickkopf family protein with a dynein motor complex protein. The link between REIC/Dkk-3 and Tctex-1 may be of significance for understanding the molecular functions of the proteins in ER stress signaling and intracellular dynein motor dynamics, respectively.

Alantolactone Inhibits Cell Proliferation by Interrupting the Interaction between Cripto-1 and Activin Receptor Type II A in Activin Signaling Pathway

It has been suggested that deregulation of activin signaling contributes to tumor formation. Activin signaling is blocked in cancer cells due to the complex formed by Cripto-1, activin, and activin receptor type II (ActRII). In this study, the authors used a mammalian two-hybrid system to construct a drug screening model to obtain a small molecular inhibitor capable of interrupting the interaction between Cripto-1 and ActRII. They screened 300 natural components and identified alantolactone. Data suggested that alantolactone induced activin/SMAD3 signaling in human colon adenocarcinoma HCT-8 cells. The authors also found that alantolactone exhibited antiproliferative function specific to tumor cells, with almost no toxicity to normal cells at a concentration of 5 µg/mL. Furthermore, they proved that the antiproliferative function of alantolactone was activin/SMAD3 dependent. These results suggest that alantolactone performs its antitumor effect by interrupting the interaction between Cripto-1 and the activin receptor type IIA in the activin signaling pathway. Moreover, screening for inhibitors of Cripto-1/ActRII is a potentially beneficial approach to aid in discovering novel cancer treatment.

Abstract 1947: FAM129B regulates cell proliferation and activates NF-κB and Nrf2 by inactivating KEAP1 in cancer cells

Abstract The Family with sequence similarity 129 (FAM129) contains three membrane proteins, FAM129A, B and C. FAM129A (also called Niban) and FAM129C (also called BCNP1) have been found to be up-regulated in thyroid tumors and chronic lymphocytic leukemia, respectively. Although phosphorylation of FAM129B has been suggested to play a role in controlling melanoma cell invasion, the molecular mechanism of FAM129B in tumorigenesis remains largely unknown. In this study, we first identified FAM129B as one of proteins with altered phosphorylation status in breast cancer stem cells by the phosphor-mass spectrum technique. Silencing of FAM129B expression in breast cancer cell line, MDA-MB-231, led to decreased proliferation, along with flattened and rounded cell morphology. By mammalian two-hybrid system and FRET (fluorescence resonance energy transfer) assay, KEAP1 (kelch-like ECH-associated protein 1) was detected to interact with FAM129B. KEAP1 has been reported to function as a substrate adaptor protein for Cul3-dependent E3 ligase complex to repress IKKβ and transcription factor Nrf2. We indeed observed that the expression of NF-κB and Nrf2 downstream genes, FN1 and NQO1, was down-regulated in FAM129B-silenced cells by qPCR analysis. On the other hand, FAM129B together with either IKKβ or Nrf2 synergistically activated NF-κB and Nrf2 promoter activity by reporter assays. Taken together, our results suggest that FAM129B might play an important role in the regulation of cell proliferation in cancers by inactivating KEAP1 which in turn activates Nrf2 and IKKβ activities. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1947. doi:10.1158/1538-7445.AM2011-1947

Phosphorylation Regulates OLIG2 Cofactor Choice and the Motor Neuron-Oligodendrocyte Fate Switch

SummaryA fundamental feature of central nervous system development is that neurons are generated before glia. In the embryonic spinal cord, for example, a group of neuroepithelial stem cells (NSCs) generates motor neurons (MNs), before switching abruptly to oligodendrocyte precursors (OLPs). We asked how transcription factor OLIG2 participates in this MN-OLP fate switch. We found that Serine 147 in the helix-loop-helix domain of OLIG2 was phosphorylated during MN production and dephosphorylated at the onset of OLP genesis. Mutating Serine 147 to Alanine (S147A) abolished MN production without preventing OLP production in transgenic mice, chicks, or cultured P19 cells. We conclude that S147 phosphorylation, possibly by protein kinase A, is required for MN but not OLP genesis and propose that dephosphorylation triggers the MN-OLP switch. Wild-type OLIG2 forms stable homodimers, whereas mutant (unphosphorylated) OLIG2S147A prefers to form heterodimers with Neurogenin 2 or other bHLH partners, suggesting a molecular basis for the switch.

A mammalian two-hybrid system-based assay for small-molecular HIV fusion inhibitors targeting gp41

gp41 is a major component of the envelope glycoprotein of human immunodeficiency virus type 1 (HIV-1) responsible for fusion of the viral envelope with the target cellular membrane. The formation of the trimer-of-hairpins core structure of gp41, via the interaction between its N-terminal heptad repeat (NHR) and its C-terminal heptad repeat (CHR) plays a key role in the membrane fusion process. Hence, inhibitors of trimer-of-hairpins formation have become a promising new class of HIV therapeutics. In the present study, based on the mammalian two-hybrid system, we developed a cell-based assay for detecting small-molecular HIV-1 fusion inhibitors targeting gp41. The optimized assay can be adapted to high-throughput screening in 96- and 384-well microplates with high signal-to-background ratios and acceptable Z′ factors. The known small-molecular gp41 inhibitors, ADS-J1, XTT formazan and tannin acid, tested positive in this assay, with half-maximal inhibitory concentration (IC 50) values of 4.9 μM, 5.6 μM and 0.8 μM, respectively. These data suggested that this novel assay is robust, sensitive and specific for identifying small-molecular HIV-1 gp41 inhibitors.