A Three-Fragment Fluorescence Complementation for Imaging of

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A Three-Fragment Fluorescence Complementation for Imaging of Ternary Complexes Under Physiological Conditions Minghai Chen, Wei Li, Zhi-Ping Zhang, Jingdi Pan, Yuhan Sun, Xiaowei Zhang, Xian-En Zhang, and Zongqiang Cui Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b02661 • Publication Date (Web): 26 Oct 2018 Downloaded from http://pubs.acs.org on October 28, 2018

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Analytical Chemistry

A Three-Fragment Fluorescence Complementation for Imaging of Ternary Complexes Under Physiological Conditions Minghai Chen,†,§,1 Wei Li,†,1 Zhi-Ping Zhang,† Jingdi Pan,†,§ Yuhan Sun,†,§ Xiaowei Zhang,† Xian-En Zhang,‡ and Zongqiang Cui†,*

†State

Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy

of Sciences, Wuhan 430071, China ‡National

Laboratory of Biomacromolecules, CAS Center for Excellence in

Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China §University

1These

of Chinese Academy of Sciences, Beijing 100049, China

authors contributed equally to this work

*To whom correspondence should be addressed: Tel: +86 (0)27 87199115; Fax: +86 (0)27 87199492; E-mail: [email protected]

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ABSTRACT Protein–protein interactions (PPIs) occur in a vast variety of cellular processes, and many processes are regulated by multiple protein interactions. Identification of PPIs is essential for the analysis of biological pathways and to further understand underlying molecular mechanisms. However, visualization and identification of multi-protein complexes including ternary complexes in living cells under physiological conditions remains challenging. In this work, we reported a three-fragment fluorescence complementation (TFFC) by splitting the Venus fluorescent protein for visualizing ternary complexes in living cells under physiological conditions. With this Venus-based TFFC system, we identified the multi-interaction of weak-affinity ternary complexes under physiological conditions. The TFFC system was further applied to the analysis of multi-interactions during the

HIV-1

integration

process,

revealing

the

important

role

of

the

barrier-to-autointegration factor protein in HIV-1 integration. This TFFC system provides a useful tool for visualizing and identifying ternary complexes in living cells under physiological conditions.


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INTRODUCTION Many biological processes require multiple proteins to interact with each other. For example, the interactions of protein complexes during the integration process of retroviruses play a pivotal role in mediating the completion of the virus life cycle.1,2 Monitoring multi-protein interactions in living cells should aid our understanding of the mechanisms of physiological and biochemical processes. Fluorescence-based methods including fluorescence resonance energy transfer (FRET),3,4 fluorescence cross-correlation spectroscopy (FCCS)5 and bimolecular fluorescence complementation (BiFC)6-8 have been used for studying PPIs in living cells. These methods, however, are usually applied to analyze the interaction between two proteins. To visualize ternary complexes in cells, three chromophore-based FRET system9 and the BiFC-based FRET assay10 have been developed. However, because these assays require sophisticated instruments and complex data analysis, they were not widely used. We recently developed a kind of TFFC assay for visualizing ternary complexes in living cells.11 The TFFC assay is based on the reconstruction of a fluorescent protein from its three non-fluorescent splits that have been created by dividing the protein at appropriate points in the sequence. These three non-fluorescent fragments can be reconstituted to emit fluorescence 3 ACS Paragon Plus Environment


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when three proteins each fused to a different non-fluorescent fragment simultaneous associate to form a stable ternary complex of interest. However, chromophore

reconstruction

from

the

split

mIrisFP

fragments

requires

preincubation of cells at a low temperature.11 This could potentially limit the application of this approach under physiological conditions. In addition, a higher TFFC complementary efficiency has the advantage of detecting weak and transient interactions. Therefore, the availability of new fluorescent protein fragments with higher complementary efficiency for analyzing ternary complexes under physiological conditions will undoubtedly increase future applications of the TFFC assay. Integration of the viral genome is a key step of Human immunodeficiency virus (HIV-1) infection because it ensures both expression of viral genes, and thus production of new progeny viruses, and also the stable maintenance of viral genetic information in infected cells.12,13 Integration is performed by integrase (IN) and several cellular factors also play important roles in this process.2 For example, Lens epithelium-derived growth factor (LEDGF/p75) is critical for tethering IN and the viral pre-integration complex (PIC) to host chromatin. LEDGF/p75 interacts with IN and subsequently co-localizes with cellular chromosomes.14 Transient and stable knockdown of LEDGF/p75 affects the HIV-1 integration step.15,16 The cellular protein barrier-to-autointegration factor (BAF) consists of 89 amino acids,17 is highly conserved among metazoans and was originally identified as a 4 ACS Paragon Plus Environment

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factor that can inhibit intramolecular integration of retroviruses.18 BAF was later shown to be a component of proviral pre-integration complexes.19 Recently it has been revealed that BAF also assembles on transposon DNA, protecting these mobile genetic elements from suicidal autointegration.20,21 Whether these two cellular factors have other specific functions during the HIV-1 integration process remains unclear. Here, we report a TFFC system based on splitting the Venus fluorescent protein for imaging ternary complexes under physiological conditions in living cells. The Venus-TFFC system was also used to study the integration process of HIV-1. The results revealed that a ternary complex forms among LEDGF/p75, BAF and IN during the integration process, and BAF may also remodel the structure of chromosomes, thus influencing the HIV-1 integration process.

EXPERIMENTAL SECTION Plasmid Construction. The Venus fluorescent protein was divided at two positions between amino acids 154/155 and 172/173 to construct the TFFC system. The three non-fluorescent fragments were named VN154, VN(155–172) and VC173, which correspond to the amino acids sequences of 1–154, 155–172 and 173–238, respectively. For the construction of pVN154-NFAT1 and pVN154-mNFAT1 plasmids, VN154 was amplified using the Venus sequence as the template by PCR. The forward primer contains Kozak sequences and a Flag 5 ACS Paragon Plus Environment


tag. The VN154 was then inserted into the pcDNA3.1 (+) with restriction enzymes NheI and BamHI. NFAT1 and mNFAT1 were amplified using the plasmids pmIN150-NFAT1 and pmIN150-mNFAT111 as the template by PCR and then inserted into the pcDNA3.1-VN154 by using restriction enzymes EcoRI and NotI . The linker sequence GGGGSGGGGS was used to connect the coding regions. To construct the pbJun-VN(155–172) plasmid, bJun was amplified using the pbJun-iRN97 plasmid as the template.8 The forward primer also contains Kozak sequences. The bJun was then inserted into the pcDNA3.1 (+) by using restriction enzymes NheI and HindIII VN(155–172) was amplified from the Venus sequence and then inserted into the pcDNA3.1-bJun by using restriction enzymes KpnI and NotI. GGGGSGGGGS was used as the linker to connect the coding regions. For the construction of the pbFos-VC173 plasmid, bFos was amplified using the piRC98-bFos plasmid as the template.8 The forward primer also contains Kozak sequences and a Flag tag. The bFos was then inserted into the pcDNA3.1 (+) by using restriction enzymes NheI and HindIII. VC173 was amplified using the Venus sequence and then inserted into the pcDNA3.1-bFos by using restriction enzymes KpnI and NotI. GGGGSGGGGS was used to connect the coding regions. The pbFos-VC173, bearing unique NheI and HindIII restriction sites, was exchanged with the mbFos to construct the pmbFos-VC173 plasmid. mbFos was amplified using the piRC98-mbFos plasmid as the template.8 Construction of plasmids with different linkers was achieved using a similar process as described above, except 6 ACS Paragon Plus Environment

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the GGGGSGGGGS sequence was replaced with other linker sequences. For the construction of the pVN154-p65 plasmid, the gene coding for p65 was amplified by PCR using the random-primed HeLa cDNA as the template and then fused to the fragment VN154, whereas construction of the pVN154-p65 25 plasmid was achieved by deleting the coding sequence of amino acids 187–211 of p65 from the pVN154-p65 plasmid. The accession number of p65 is 1MY5. The construction

of

pVN154-IN,

pVN154-IN(R166A),

pVN(155–172)-BAF

and

pp75-VC173 plasmids was conducted as follows. IN was amplified using the piRC98-IN

as

the

template,8

IN(R166A)

was

amplified

using

the

piRC98-IN(R166A) as the template,8 BAF was amplified by PCR using the random-primed HeLa cDNA as the template, p75 was amplified using the pp75-iRN97 as the template,8 and these PCR products were fused to VN154, VN(155–172) and VC173, respectively. The accession number of BAF is 1CI4. The primers designed in this work are listed in Table S1. DNA sequencing was used to verify the constructions in this work. Cell Culture and Plasmid Transfection. HEK 293T, HeLa, Vero and TZM-bl cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin and 100 μg/ml streptomycin. All of the cells were incubated at 37 °C with 5% CO2 in a humidified incubator. The cells were cultured on 35-mm glass bottom cover slips. When the cells were at 70–80% confluence, cells were transfected with 2 μg of 7 ACS Paragon Plus Environment


each of plasmids by Lipofectamine 2000 (Invitrogen; United States). The transfected cells were then cultured in complete DMEM medium for another 24 h. Fluorescent Imaging and Quantitative Analysis. Fluorescent imaging was carried out with the UltraVIEW VOX Confocal system by using a 60X oil immersion objective lens (1.4 numerical aperture) (PerkinElmer, Co.). The yellow fluorescence of the Venus was excited with a 514 nm laser. The cyan fluorescence of the ECFP was excited with a 445 nm laser. Hoechst 33342 was used to stain cell nuclei, and excited with a 405 nm laser. In this work, all of the TFFC fluorescence signals were detected in live cells. For quantitative analysis of fluorescence images, fluorescence intensities were obtained from the original yellow or cyan images by subtracting the background fluorescence, respectively. The background fluorescence were defined as the average fluorescence intensity of a region (50 × 50 pixel2) without a yellow signal in the Venus channel or corresponding region in the cyan fluorescence channel. The yellow-to-cyan fluorescent intensity ratio was calculated by dividing the intensity of the yellow fluorescence by that of the cyan fluorescence. For each combination, the average fluorescence intensity of about sixty fluorescent cells which were randomly selected from 10–15 images, was used to calculate the relative TFFC efficiency. Western Blotting. To determine the expression levels of fusion proteins, cells were subjected to western blot analysis. The cell lysates were cleared by 8 ACS Paragon Plus Environment

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centrifugation at 10,000×g for 10 min and subjected to sodium dodecyl sulfate-polyacrylamide

gel

electrophoresis,

and

transferred

onto

the

polyvinylidene fluoride membranes. After blocking at 4 °C overnight with phosphate-buffered saline (PBS) supplemented with 5% (w/v) skim milk, the membranes were incubated with specific antibodies (Abcam). The membranes were then incubated with horseradish peroxidase-conjugated goat anti-mouse IgG (Abcam) at 37 °C for 2 h, and detected using a chemiluminescent detection system (BioRad). Virus Production, Infection and Cellular DNA Extraction. To produce the HIV-1 virus, 100 mm plates of virus-producing HEK 293T cells were transfected with appropriate plasmids using the Lipofectamine 2000 reagent. The plasmid transfections and virus experiments were performed in BSL-2 or BSL-3 laboratories. Briefly, HIV-1 viruses were produced by transfecting 10.0 μg of pAD8. Subsequently, DMEM (Gibco) with 10% fetal bovine serum (FBS) was added to the cells after 4−6 h transfection. Forty-eight hours after transfection, virions were collected, cellular debris was cleared by low-speed centrifugation and the supernatant was filtered through a 0.45 μm filter. The virus infection process was conducted as follows: 3.0 × 105 TZM-bl cells were plated per well in a 6-well plate in 2.0 mL DMEM with 10% FBS and incubated overnight in a tissue culture incubator. Infected cells, which had been transfected

with

30

nM

siRNA

for

48


h

(siRNAs

sequences:


5'-GGCCUAUGUUGUCCUUGGCdTdT-3'

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(siBAF)

and

5’-AACGUACGCGGAAUACUUCGA-3’ (siNC)) and had a final viral p24 concentration of 500 ng/mL in a final volume of 500 μL DMEM-FBS were cultured for 2 h in a tissue culture incubator. One-and-a-half milliliter of DMEM-FBS was added and the incubation continued. Cells were collected 48 h after transfection and DNA was extracted using the DNeasy Tissue kit (Qiagen, Valencia, CA). For the knockdown-rescue experiment, 3 μg of pBAF was transfected into the knockdown cells. Nested Alu-PCR Process. To amplify the integrated HIV-1 DNA forms, the nested Alu-PCR process was conducted as follows. The first round of PCRs was conducted with the Alu-LTR-F and Alu-LTR-R primers. The PCR run parameters were one cycle: 94 °C for 2 min; 30 cycles: 94 °C for 15 sec, 55 °C for 30 sec, 68 °C for 45 sec; and one cycle: 68 °C for 10 min. The second round of PCR reactions was conducted with the NI-2-F and NI-2-R primers. The thermocycler parameters used for the second PCR round were the same as described for the first round.

RESULTS AND DISCUSSION Splitting Venus for the Construction of the TFFC System. Guided by the crystal structure of Venus (PDB ID: 3AKO)22 and sequence alignment with the mIrisFP fluorescent protein,23 two split sites between amino acids 154/155 and 10 ACS Paragon Plus Environment

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172/173 within the loops of the barrel-like structure were selected to create the Venus-based TFFC system (Figure 1a). The schematic principles of the TFFC system are shown in Figure 1b. These three non-fluorescent fragments can reconstitute to form an active protein that emits fluorescence when three protein units each fused to one of the non-fluorescent fragments successfully associate to form a ternary complex. These split Venus fragments were used to construct the plasmids pVN154, pVN(155–172) and pVC173. The interaction of the bFos-bJun-NFAT1 complex was chosen as a model complex to test the TFFC system.10 The NFAT1, bJun and bFos coding sequences were inserted into pVN154, pVN(155–172) and pVC173, respectively. When VN154-NFAT1, bJun-VN(155–172) and bFos-VC173 proteins were co-expressed in cells, a strong yellow TFFC signal from the reconstitution of the Venus fragments was observed and exhibited a specific nucleoli localization pattern (Figure 2a). As a control, when mNFAT1 that R466A/I467A/T533G were mutated to abolish NFAT1 interaction with the bJun-bFos heterodimer,10 or mbFos that deletion of a leucine zipper to eliminate bFos-bJun interaction, but do not affect bFos interaction with other partners,6 was fused to VN154 or VC173, respectively, and co-expressed with the other two fused fragments, no TFFC signal could be detected. In addition, there was also no complementary fluorescence signal when the short middle fragment was absent (Figure 2a). During these experiments, pECFP-C1 was also co-transfected into the cells as an 11 ACS Paragon Plus Environment


internal control. The statistical analysis of the complementary efficiency based on the fluorescence intensity ratio of TFFC/ECFP (yellow/cyan) produced in these assays demonstrated that the complementary fluorescence of VN154-NFAT1, bJun-VN(155–172) and bFos-VC173 was significantly higher than that of the control groups (Figure 2b). Western blot analysis showed that the level of expression of NFAT1 and mNFAT1 was the same (Figure 2c). Moreover, western blot analysis of bFos and mbFos production revealed that both proteins were expressed at the same level (Figure 2c). The TFFC-reconstituted Venus also had similar photophysical properties compared with the native Venus (Figure S1). Unlike our previously reported mIrisFP-derived TFFC assay, which required low temperature pretreatment (e.g., 27 °C), the Venus-based TFFC system could regain native fluorescence under physiological conditions. These results demonstrated that Venus could be split at 154/155 and 172/173 sites to develop the TFFC system for visualizing ternary complex interactions under physiological conditions in living cells. We then tested the influence of the type and length of the linker on the complementary fluorescence of this Venus-based TFFC system. Here, we denoted no linker as G0, GGGGS as G1, (GGGGS)2 as G2 and (GGGGS)3 as G3. RSIAT was also tested here as the linker, which was used in other BiFC constructs.6 Here, RSIAT was denoted as R1, (RSIAT)2 denoted as R2 and (RSIAT)3 denoted as R3. The linker sequences are shown in Figure 3a. All of the 12 ACS Paragon Plus Environment

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split probes in a combination have the same linker. We transfected these plasmids into Vero cells and the results showed that construction with the G2 linker produced the highest complementary fluorescence when compared with the other constructs (Figure 3b,c). Some constructs did not produce any fluorescence, which may be due to the composition and length of the linker, which affects the spatial configuration of the reconstituted Venus fluorescent protein. These results showed that the type and length of the linker influenced the reconstitution of the TFFC system, and the G2 linker is the most suitable linker for use in the Venus-based TFFC system. Imaging the Ternary Complex Interactions in Living Cells with the Venus-Based TFFC System. We then selected the ternary complex of bJun-bFos-p65 to assess the validity and reliability of the Venus-based TFFC system for studying weak interactions (i.e., weak affinity) of multi-proteins in living cells.6,10 The results in Figure 4a show that when the fusion proteins VN154-p65, bJun-VN(155–172) and bFos-VC173 were co-expressed in Vero cells, yellow complementary TFFC signals could be detected. As a negative control, mutant bFos was also fused to VC173 and co-expressed with the other two fused fragments. No obvious TFFC fluorescence signal could be detected even though mbFos-VC173 has a comparable expression level to that of bFos-VC173 (Figure 2c). p65△25, which has amino acids 187–211 deleted to abolish the interaction of p65 with the bJun-bFos heterodimer,10 was used as an additional negative control. 13 ACS Paragon Plus Environment


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No clear complementary fluorescence was detected when p65△25 was combined with the bJun-bFos heterodimer (Figure 4a). pECFP-C1 was also co-transfected into cells as an internal control in these experiments. The statistical analysis the fluorescence intensity ratio of TFFC/ECFP (yellow/cyan) produced in the assays demonstrated

that

the

complementary

fluorescence

of

VN154-p65,

bJun-VN(155–172) and bFos-VC173 was significantly higher than that of the control groups (Figure 4b). These results further verified that the newly constructed Venus-based TFFC system is suitable for studying proteins that form weak-affinity ternary complexes in live cells. Imaging a Ternary Complex During HIV-1 Integration Using the Venus-Based TFFC System. The integration of reverse transcribed viral cDNA into the host chromosome is an essential step during the life cycle of HIV-1. Identifying and visualizing protein interactions during the HIV-1 integration process is important, because such information should aid the development of therapeutics against HIV-1. Here, by using the Venus-based TFFC system, we studied an important protein complex during the HIV-1 integration process. We aimed to image the ternary complex formed by IN, BAF and LEDGF/p75 during the HIV-1 integration process. As shown in Figure 5a, the co-expression of VN154-IN, VN(155–172)-BAF and LEDGF/p75-VC173 yielded a bright yellow TFFC fluorescence signal. pECFP-C1 was also co-transfected into the cells as an internal control. The TFFC signal from IN-BAF-LEDGF/p75 in the cell nuclei is 14 ACS Paragon Plus Environment

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mostly co-localized with cellular chromosomes stained with Hoechst 33342, indicating that IN, BAF and LEDGF/p75 interact with the host chromosome in living cells. As a negative control, IN with a R166A point mutation, which does not interact with LEDGF/p75 but remains enzymatically active, was fused to the C-terminus of VN154 and tested for its interaction with LEDGF/p75 and BAF. As shown in the Figure 5a, IN(R166A), BAF and LEDGF/p75 in the Venus TFFC system did not produce a complementary TFFC signal. The result further verified the specific interaction of IN, BAF and LEDGF/p75 in the TFFC system. A series of selected optical sections of a TZM-bl cell along the z-axis using confocal microscopy (spacing 0.4 μm) is depicted in Figure 5b. LEDGF/p75 has been reported to tether the HIV-1 integrase to the host chromosome and co-localizes with the cell chromosome.24 Here, our result showed that BAF may also play an important role in the integration process. Next, we further examined the role of BAF in the HIV-1 integration process. First, siRNA against BAF was transfected into TZM-bl cells. As shown in Figure 6a, the protein expression level of BAF was significantly reduced by siRNA. Then, cells were infected with HIV-1. Forty-eight hours after infection, cells were collected and DNA was extracted using the DNeasy Tissue kit. DNA was subjected to a modified nested Alu-PCR procedure that specifically detects integrated HIV-1 DNA forms.25 The principle of the Alu-PCR technique is shown in Figure 6b and the integrated HIV-1 DNA can be specifically amplified. The results 15 ACS Paragon Plus Environment


showed that the integration efficiency of HIV-1 DNA in the siBAF group is lower than the control group with siNC (a negative control with no homologous sequences of the BAF gene) treatment (Figure 6c). Quantitative analyses also confirmed a significant difference between the siBAF and siNC groups (Figure 6d). The knockdown-rescue assay also verified that siBAF did not have an off-target effect (Figure 6c, d). These results showed that BAF affects HIV-1 integration during the infection process. Visualizing and identifying the ternary complexes under physiological conditions will facilitate PPI studies significantly. In this work, we developed a novel TFFC system by splitting Venus protein to image ternary complexes in living cells. The reconstructed form of Venus from the three split Venus fragments has the similar photophysical properties with the native Venus, which validates its feasibility for the new TFFC system. The split sites of Venus are located within loops 7–8 and 8–9 of the beta-fold of native Venus and in a similar location to those used for mIrisFP in the previous TFFC system developed. This should provide guidance in the development of new TFFC systems that employ other barrel-like fluorescent proteins. The new Venus-based TFFC system is different from our recently developed mIrisFP-based TFFC system, which requires preincubation of the cells at lower temperatures to enable the chromophore to mature.11 The Venus-based TFFC system can produce fluorescence under physiological conditions without 16 ACS Paragon Plus Environment

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preincubation, which eliminates the requirement of a low temperature step. Moreover, the high complementary efficiency enables detection of protein-protein interactions of weak affinity. This difference may be due to the lower environmental sensitivity of Venus and its improved maturation rate.22 The fusion orders between the Venus fragments and the interaction proteins may influence reconstruction of the complementary fluorescence. In general, we fused the smallest protein with the middle non-fluorescent fragment (VN(155–172)) to reduce space restrictions. Imaging weak interactions of multi-protein complexes should aid our understanding of new roles of these interactions in cellular processes. Here, with the Venus-based TFFC system, we imaged the bJun-bFos-p65 ternary complex, which has weak affinity in the transcription and regulation processes.10 The Venus-based TFFC system was also used to image multi-protein interactions during HIV-1 integration. Fluorescence imaging using the Venus TFFC system clearly showed that BAF, LEDGF/p75 and IN interact on the host chromosome in live cells. Our analysis further demonstrated that the cellular cofactor BAF influences the HIV-1 integration process. Recently, BAF was reported to have an important role in HIV-1 latency.26-28 Thus, localization of this ternary complex may have a relationship with HIV-1 latency but further work is required to confirm this relationship.



CONCLUSION We reported a TFFC system for imaging multi-protein interactions under physiological conditions. This Venus-based TFFC fluorescent system provides a useful tool to study ternary complexes in living cells. The application of the TFFC system for imaging the ternary complex during HIV-1 integration provided insights into HIV-1 infection.

ASSOCIATED CONTENT Supporting Information The supporting information is available free of charge on the ACS Publications website at DOI: The supporting results and the sequences of primers. AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]. Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes The authors declare no competing financial interest. ACKNOWLEDGMENTS 18 ACS Paragon Plus Environment

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This work was supported by the National Key Research and Development Program of China (2018ZX10301405), the Strategic Priority Research Program of the Chinese Academy of Sciences (No. XDPB03), the Natural Science Foundation of China (No. 31470269, No.91743108 and No. 21727816), Chinese Academy of Sciences (CAS) (Youth Innovation Promotion Association, and Key Research Project of Frontier Science). We thank Ding Gao of the Core Facility and Technical Support, Wuhan Institute of Virology for his support in real time confocal

imaging.

We

thank

Liwen

Bianji,

Edanz

Editing

China

(www.liwenbianji.cn/ac), for editing the English text of a draft of this manuscript.

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Figure 1. Scheme to construct the Venus-based TFFC system. (a) Split Venus to construct the TFFC systems. The arrows indicate the cleavage sites. (b) Schematic principle of TFFC, which is based on the simultaneous and stable association of three protein units each fused to a non-fluorescent fragment to generate a reconstituted active Venus that can emit fluorescence.


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Figure 2. Construction of the Venus-based TFFC system. (a) TFFC signals (Venus channel) were detected in Vero cells for the cleavage sites at amino acids 154/155 and 172/173 for Venus fragments because of the interaction of NFAT1-bJun-bFos. ECFP was co-expressed as the internal control. (b) Quantitative analysis of the TFFC efficiency in (a) based on the fluorescence intensity ratio of TFFC/ECFP (yellow/cyan). (c) Comparable expression of the fusion proteins in (a) were determined by western blotting with anti-Flag antibodies. α -Actin was also determined as the loading control. Nuclei were stained with Hoechst 33342. Scale bars: 20 μm. All data are given as the mean ± S.D. (n = 60). The statistical significance was evaluated using a LSD-t test post 23 ACS Paragon Plus Environment


F-test. *** indicates p < 0.001.

Figure 3. Linker optimization for construction of the Venus-based TFFC system. (a) The different linkers tested in the experiment. (b) TFFC signals (Venus channel) were detected in Vero cells for the different linkers from G0 to R3. (c) Quantitative analysis of the TFFC efficiency in (b) based on the reconstructed fluorescence intensities. Nuclei were stained with Hoechst 33342. Scale bars: 10 μm. All data are given as the mean ± S.D. (n = 20).


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Figure 4. Imaging ternary complexes in living cells using the Venus-based TFFC system. (a) Visualization of the interaction between bJun, bFos and p65 and the interactions of negative control combinations in Vero cells using the Venus-based TFFC system. ECFP was transfected as the internal control. (b) Quantitative analysis of the TFFC efficiency in (a) based on the fluorescence intensity ratio of TFFC/ECFP (yellow/cyan). Nuclei were stained with Hoechst 33342. Scale bars: 10 μm. All data are given as the mean ± S.D. (n = 60). The statistical significance was evaluated using a LSD-t test post F-test. *** indicates p < 0.001.



Figure 5. IN/BAF/p75 interactions revealed using the Venus-based TFFC system. (a) Vero cells were transfected with a combination of VN154-IN, VN(155–172)-BAF and p75-VC173 or VN154-IN(R166A), VN(155–172)-BAF and p75-VC173. ECFP was transfected as the internal control. (b) Visualization of the TFFC signals of the IN/BAF/p75 interaction in a series of optical sections through a living TZM-bl cell spaced 0.4 μm apart. ECFP was transfected as the internal control. Nuclei were stained with Hoechst 33342. Scale bars: 10 μm.


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Figure 6. HIV-1 integration analysis in cells treated by siRNA against BAF. (a) Western

blotting

of

cell

extracts

from

siNC-treated,

siBAF-treated

or

knockdown-rescued (R-group) TZM-bl cells using anti-BAF. α -Actin was also detected as the loading control. (b) Detection of integrated HIV-1 DNA by using Alu-LTR PCR. (c) Genomic DNA from siNC-treated, siBAF-treated or knockdown-rescued (R-group) TZM-bl cells was subjected to Alu-LTR PCR followed by a second round of the nested PCR. Integrated DNA was detected (~347 bp) and β-globin amplified as the control. (d) Quantitative analysis of the HIV-1 integrated cDNA in (c). All data are given as the mean ± S.D. (n = 3). The statistical significance was evaluated using a LSD-t test post F-test. ** indicates p

A Three-Fragment Fluorescence Complementation for Imaging of

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