Bovine HEXIM1 inhibits bovine immunodeficiency virus replication through regulating BTat-mediated transactivation
- Hong-yan Guo†1,
- Yong-gang Ma†2,
- Yuan-ming Gai1,
- Zhi-bin Liang1,
- Jing Ma1,
- Yang Su1,
- Qi-cheng Zhang1,
- Qi-min Chen1 and
- Juan Tan1Email author
© Guo et al.; licensee BioMed Central Ltd. 2013
Received: 26 September 2012
Accepted: 5 March 2013
Published: 27 March 2013
The bovine immunodeficiency virus (BIV) transactivator (BTat) recruits the bovine cyclin T1 (B-cyclin T1) to the LTR to facilitate the transcription of BIV. Here, we demonstrate that bovine hexamethylene bisacetamide (HMBA)-induced protein 1 (BHEXIM1) inhibits BTat-mediated BIV LTR transcription. The results of in vivo and in vitro assays show direct binding of BHEXIM1 to the B-cyclin T1. These results suggest that the repression arises from BHEXIM1-BTat competition for B-cyclin T1, which allows BHEXIM1 to displace BTat from B-cyclin T1. Furthermore, we found that the C-terminal region and the centrally located region of BHEXIM1 are required for BHEXIM1 to associate with B-cyclin T1. Knockdown of BHEXIM1 enhances BIV replication. Taken together, our study provides the first clear evidence that BHEXIM1 is involved in BIV replication through regulating BTat-mediated transactivation.
Bovine immunodeficiency virus (BIV) belongs to the Lentivirus genus of the Retroviridae family. It was initially isolated from the leucocytes of an 8-year old Louisiana dairy cow, R-29, that had persistent lymphocytosis, lymphadenopathy, central nervous system lesions and wasting. BIV infection has been detected in many countries around the world using serological or molecular assays[2–7]. Although most of these reports show BIV infection is asymptomatic, BIV seropositivity is variably associated with weight loss, secondary diseases, and diminished milk production[5, 7, 8].
The BIV genome, like human immunodeficiency virus (HIV) and other lentiviruses, contains the structural genes gag, pol, env, and several accessory genes, including tat, rev, vif, vpw, vpy, and tmx[9, 10]. One of the most studied regulatory proteins is the Tat protein, which enhances viral gene expression. The Tat proteins of different lentiviruses are divided into two groups depending on whether they bind TAR (transactivation response region) to elevate viral gene expression. The first group includes Tat proteins from BIV, Jembrana disease virus (JDV), HIV-1 and HIV-2, simian immunodeficiency virus (SIV), and equine infectious anemia virus (EIAV)[11, 12]. These Tat proteins bind to the TAR present at the 5′ end of viral RNA transcripts and recruit the cellular positive transcription elongation factor b (P-TEFb), which consists of the cyclin-dependent kinase 9 (CDK9) and cyclin T1, to the viral promoter. Transcription elongation is enhanced through Tat-mediated recruitment and interaction with cyclin T1 and then CDK9-mediated phosphorylation of the carboxyl-terminal domain (CTD) of cellular RNA polymerase II (RNAP II)[13–19]. It is noteworthy that Tat from BIV (BTat) is distinct from other viruses of the first group because the recruitment of cyclin T1 does not contribute to TAR binding[12, 20]. Tat proteins in the second group are from maedi-visna virus (MVV), caprine arthritis-encephalitis (CAEV) and feline immunodeficiency virus (FIV). These Tat proteins weakly transactivate their homologous LTR in a TAR-independent manner.
The hexamethylene bisacetamide (HMBA)-inducible protein 1 (HEXIM1) is a component of the inactive P-TEFb. Upon binding to 7SK snRNA, HEXIM1 oligomers undergo a conformational change and then bind to cyclin T1. Following association with the P-TEFb complex, HEXIM1 inhibits the activity of CDK9[23–26]. Through this mechanism, HEXIM1 suppresses HIV-1 Tat transactivation. However, it is unknown whether HEXIM1 also interferes with BIV Tat transactivation. In this study, we show that bovine HEXIM1 (BHEXIM1) was associated with bovine cyclin T1 (B-cyclin T1) and suppressed BTat-mediated BIV LTR transcription activation. Our results further show that the nuclear localization signal and the C-terminal domain of BHEXIM1 were essential for binding to B-cyclin T1. Knockdown of BHEXIMI increased BIV replication, which further demonstrates the inhibitory effect of BHEXIM1 on BTat function.
Materials and methods
Protein A beads were purchased from Sigma (St. Louis, MO, USA). Anti-Flag M2 monoclonal antibody (MAb) was obtained from Stratagene (Agilent Technologies, Santa Clara, CA, USA). Anti-HA rabbit antibody, anti-HA mouse antibody, anti-β-actin C4 mouse antibody, horseradish peroxidase-conjugated anti-rabbit and anti-mouse secondary antibodies were obtained from Santa Cruz Biotechnology (Dallas, Texas, USA).
Plasmids and proteins
pBIV-LTR-Luc and pHIV-LTR-Luc plasmids were constructed by subcloning BIV LTR or HIV LTR into a pGL3-basic luciferase reporter vector (Promega, Madison, WI, USA) upstream of the luciferase gene. The BIV LTR and HIV LTR were provided by Dr. Charles Wood (University of Nebraska Lincoln). pCMV-HA-BHEXIM1 and pCMV-HA-HHEXIM1 were generated by inserting BHEXIM1 or HHEXIM1 cDNA into the expression vector, pCMV-HA (Clontech, Mountain View, CA, USA). BHEXIM1 deletion mutants were created by PCR and cloned into pCMV-HA. The mammalian expression plasmid, Myc-BTat, was provided by Dr Matjaz Barboric (University of California). pcDNA3.1(+)-HTat (1-86 aa) was constructed by inserting HTat (1-86 aa) into the pcDNA3.1(+) vector (Invitrogen, CA, USA). The pcDNA-B-cyclin T1 (aa residues 1-272 of bovine cyclin T1) was a kind gift from Dr. Alan Frankel (University of California) and was subcloned into the pCMV-Tag2B vector (Stratagene). The pGEX6p-1 (Amersham Bioscience, Pittsburgh, PA, USA) and pETH (Novagen, Darmstadt, Germany) vectors were used to construct bacterial expression plasmids for GST- and His-fusion proteins. Escherichia coli BL21 (DE3) strain cells were transformed with these constructs to express GST-B-cyclin T1, His-BHEXIM1, and His-BTat proteins. The proteins were purified with glutathione Sepharose 4B beads (GE healthcare, Pittsburgh, PA, USA) or His SepharoseTM 6 (GE healthcare) according to the respective manufacturer’s instructions.
Cells and viruses
293 T, HeLa, fetal bovine lung (FBL) cells were primary cells isolated from fetal bovine lung by our laboratory; canine thymus cell line (Cf2Th), which is a BIV permissive cell line, was kindly provided by Prof. Jin-Ming Gao (Peking Union Medical College); and BIV baby hamster kidney indicator cells, BIVL, which harbor the BIV LTR-luciferase reporter, were maintained in Dulbecco’s Modified Eagle Medium (DMEM, Gibco, CA, USA) supplemented with 10% fetal bovine serum (FBS) and 2 mmol/L glutamine, 50 U/mL penicillin and 50 μg/mL streptomycin sulfate. All of the cells were cultured at 37°C in 5% CO2. The R29 strain of BIV was provided by Dr. Charles Wood (University of Nebraska Lincoln). Tissue culture infectious dose endpoint (TCID50) of BIV as BIV titers was evaluated by BIVL cells. Confluent BIVL cells on 96-well plates were infected with 25 μL of 10-fold serial dilutions of stock viruses with eight parallel wells for each dilution. Then the cells were incubated at 37°C in 5% CO2 for 48 h. Examination of luciferase activity was performed and the TCID50 was calculated with the Reed-Muench method.
RNA isolation and RT-PCR
Total RNA was isolated from FBL cells using Trizol Reagent (Gibco) according to the manufacturer’s instructions. Fifteen micrograms of total RNA was used for cDNA synthesis using Moloney murine leukemia virus (M-MLV) reverse transcriptase (RT) (Promega). BHEXIM1 sequences were amplified by polymerase chain reaction (PCR) using the following primers: forward, 5′- GTGGAATTCAAATGGCCGAGCCACT-3′; reverse, 5′- AGCCTCGAGCTAGTCTCCAAAGTTG-3′.
Small hairpin RNA (shRNA) treatments
The pSIREN-RetroQ (Clontech) was used to construct the shRNA-BHEXIM1 plasmid, which targets the CAGCGATGAGGACTTTATG sequence. A control shRNA targeting sequence GACAGAACCAGAGGATAGA, which does not pair with any eukaryotic mRNA, was used. The lentiviral shRNA system was used in this study as previously described. Briefly, stock lentiviruses were produced by transfecting 293 T cells. Supernatants were harvested at 48 h post-transfection and were then stored at -80°C. To generate the BHEXIM1 downregulated cells, FBL cells were infected with stock lentiviruses for 12 h and then the virus was washed away. At 48 h after infection, puromycin (2 μg/mL) was added to the cultures to select for BHEXIM1-downregulated cells.
The amounts of BHEXIM1 and GAPDH mRNA in shRNA-BHEXIM1 or shRNA-control treated FBL cells were examined by real-time PCR. Real-time PCR was performed using the IQ5 Multicolor real-time PCR detection system (Bio-Rad, Hercules, CA, USA) and SYBR green real-time PCR master mix (Toyobo, Osaka, Japan) according to the manufacturers’ instructions. The sequences of the primer pairs were as follows: BHEXIM1 forward (5′-ACGACACCAGCGATGAGGAC-3′) and reverse (5′-TCCAGCCGCAGCCGATTATT-3′); BIV capsid protein-CA forward (5′-AAGGAGCCGTACACAGACTT-3′) and reverse (5′-TTCTGGAGCCGCCATACCTT-3′); and GAPDH, which was used as an internal control, forward (5′-AACGGCACAGTCAAGGCAGA-3′) and reverse (5′-TCGGCAGAAGGTGCAGAGAT-3′). The specificity of the amplification reaction was verified by melting-curve analyses. Threshold cycle (C T ) values were calculated automatically as the cycle when the sample fluorescence exceeded a threshold level corresponding to 10 standard deviations (SD) of the mean of the baseline fluorescence. The level of BHEXIM1 and CA expression was normalized to GAPDH using the 2-ΔΔCT method.
Cf2Th cells were plated in 12-well plates (1 × 105/well) and cultured at 37°C for 20 h before transfection using polyethylenimine (PEI; sigma). The total DNA in each transfection mixture was normalized using empty vector DNA. pCMV-β-gal plasmid DNA (0.1 μg) was included in each transfection. Cells were harvested at 48 h after transfection. The luciferase activity was measured using a commercial assay system (Promega). The cells were rinsed with PBS and lysed with lysis buffer (Promega). The cell lysates were then clarified from the insoluble materials by centrifugation at 12 000 rpm for 3 min. The clarified lysate (5 μL) was mixed with the luciferin reagent (50 μL) and luciferase activity was measured in a chemiluminescence measurement machine (Promega). Simultaneously, 50 μL clarified lysate was mixed with the same volume (50 μL) of o-Nitrophenyl-β-D-Galactopyranoside (ONPG) buffer (4.4 mM ONPG, 4.3 mM MgCl2-6H2O, 164 mM Na2HPO4-12H2O, 36 mM NaH2PO4-2H2O, 0.684% β-mercaptoethanol). The activity of β-galactosidase was then examined at OD405 nm with a Precision Microplate Reader (Molecular Devices, Sunnyvale, CA, USA) until the results ranged between 0.200 and 0.800. The results were used as an internal control to normalize the efficiency between transfections by the formula: (each well of luciferase activity) / (each well of β-galactosidase activity - the background of β-galactosidase activity). The luciferase activity of each well divided by the control well was expressed as the relative luciferase activity (fold). Each experiment was repeated at least three times.
Cf2Th cells plated in 12-well plates (1 × 105/well) were transfected with various plasmids. Fourty-eight hours post-transfection, the cells were harvested and washed twice with 1× phosphate buffered saline. The cells were then lysed with NP-40 lysis buffer (0.5% NP-40, 0.1% Triton X-100, 0.1% sodium deoxycholate, 10 mM Tris-Cl, 150 mM NaCl, 1 mM EDTA) containing 1% PMSF for 30 min on ice. After centrifugation at 10 000 × g for 10 min at 4°C, the supernatants were collected, and protein concentrations were measured using the Bio-Rad Protein Assay reagent (Catalog No. #500-0006). The samples (30 μg/lane) were separated by 12% polyacrylamide gel electrophoresis (SDS-PAGE). The proteins were then transferred onto a polyvinylidene difluoride (PVDF) membrane (Millipore, Billerica, MA, USA) by electroblotting for 1 h at 100 V, 4°C. Following incubation in 5% nonfat milk (in 1× phosphate-buffered saline) for 45 min at room temperature, the membrane was blotted with primary antibody for 90 min at room temperature and then incubated with goat anti-mouse or goat anti-rabbit secondary antibodies conjugated with peroxidase.
Co-immunoprecipitation (Co-IP) and Pulldown assays
For the co-immunoprecipitation assay, a total of 1 × 107 293 T cells in 100-mm-diameter dishes were transfected with various plasmids using the PEI reagent. Cells were harvested at 48 h after transfection; lysed in 600 μL lysis buffer (50 mM Tris-HCl [pH8.0], 150 mM NaCl, 1% NP-40, and 1 mM phenylmethylsulfonyl fluoride); sonicated; and centrifuged for 15 min at 4°C, 10 000 × g. The supernatant was incubated with antibody at 4°C for 2 h. Thirty microliters of protein A beads was then added, and the mixture was incubated for an additional 2 h at 4°C. Immunocomplexes were washed five times with the lysis buffer and then boiled in SDS loading buffer. The precipitated proteins were detected by SDS-PAGE and immunoblotting. For the GST pulldown assay, purified GST and GST-fusion proteins were immobilized on glutathione Sepharose 4B beads, and then incubated with purified His-fusion proteins. After extensive washing, the beads were boiled in SDS loading buffer and the proteins were subjected to SDS-PAGE and immunoblotting.
BHEXIM1 inhibits LTR transcriptional activation by BTat
BHEXIM1 interacts directly with B-cyclin T1
BHEXIM1 interferes with the interaction of BTat and B-cyclinT1
Identification of protein sequences involved in BHEXIM1 binding to B-cyclin T1 and inhibiting the transactivation function of BTat
Knockdown of BHEXIM1 enhances BIV replication
Viruses are obligate intracellular parasites and must employ cellular machinery for their efficient replication. Meanwhile, host cells evolve different strategies to suppress or eliminate viral infection and replication. BTat is an early regulatory protein, and it plays an essential role in the BIV life cycle. Transcriptional transactivation of the BIV LTR by essential BTat requires recruitment of cyclin T1 to the BIV TAR to enhance viral transcription elongation. HEXIM1 as a host cellular P-TEFb inhibitor may regulate Tat function and viral replication.
Our study was the first one to report the role of bovine HEXIM1 in BTat transactivation and BIV replication. Our data demonstrate that BHEXIM1 inhibited BTat-mediated BIV LTR transactivation by direct interaction with B-cyclin T1. The results of the competition assay further show that BHEXIM1 disrupted BTat binding to B-cyclin T1. Finally, knockdown of BHEXIM1 increased BIV gene expression and production of BIV, indicating the regulatory role of BHEXIMI involved in BIV infection.
BHEXIM1 and HHEXIM1 share 80.50% homology in their amino acid sequences and both proteins have three main domains. Comparing to other parts of the two proteins, the N-terminal domain (NTD) exhibited a lower homology (57.05%), which is reported to be the self-inhibitory domain, because a 39 amino acid deletion was found in BHEXIM1. The central region contains an arginine-rich motif (ARM) and functions as the nuclear localization signal (NLS). This region serves as the 7SK-binding site[26, 36] and shows 92.86% homology between HHEXIM1 and BHEXIM1. Only two amino acids differ in the central regions of BHEXIM1 and HHEXIM1. The C-terminal domain is rich in acidic residues and shows 96.17% homology between HHEXIM1 and BHEXIM1. This region contains the coiled-coil domain and the conserved PYNT motif. In HHEXIM1, this region mediates direct interaction with P-TEFb. The high homology between BHEXIM1 and HHEXIM1, especially in their central and C-terminal regions, indicates their conserved structures and functions.
A recent report suggested that HTat competes with HHEXIM1 for binding to cyclin T1, displaying the reciprocal competition between HHEXIM1 and cyclin T1. The apparent discrepancy may be explained by the fact that higher amounts of BHEXIM1 compared to BTat exhibited the advantage of binding B-cyclin T1 and could displace BTat from B-cyclin T1. The central region of BHEXIM1, which may be involved in nucleus localization and 7SK RNA binding, was also proved to be important in BHEXIM1 binding B-cyclin T1 and inhibiting the transactivation function of BTat, consistent with previously demonstrated roles of HHEXIM1 on HIV[26, 36]. When the native HEXIM1 NLS was replaced with a foreign NLS sequence, the NLS-substituted HEXIM1 mutants failed to interact with P-TEFb. Thus, the arginine-rich 7SK-binding motif within the NLS of HEXIM1 was indispensable for HEXIM1’s inhibitory effect on transcription through 7SK as a molecular scaffold-mediated interaction with P-TEFb. The essential role of this domain indicated the crucial function of 7SK RNA in the interaction between BHEXIM1 and B-cyclin T1 and the potential role of BHEXIM1/7SK RNA interaction in BTat inhibition. Future studies on the function of 7SK RNA in BIV transactivation may be necessary to evaluate the proposed hypothesis.
In summary, this study demonstrates the repression of BHEXIM1 on BTat transactivation by competing with BTat for binding to B-cyclin T1, allowing it to directly displace BTat from B-cyclin T1. Furthermore, we found that the C-terminal region and the centrally located 7SK snRNA recognition region of BHEXIM1 are essential for repression. Knockdown of BHEXIM1 enhances BIV replication. Based on the data presented in this study, we propose the potential impact of BHEXIM1 on the BIV latent life cycle and the absent clinical disease signs of infected livestock. This finding may provide important information for BIV molecular biology and clinical diagnoses and prevention.
We greatly appreciate the gift of the Myc-BTat expression plasmid from Dr Matjaz Barboric (University of California, San Francisco, CA, USA) and the Bcyclin T1 expression plasmid from Alan Frankel (University of California, San Francisco). The work was supported by grants from the Chinese Ministry of Health (2012ZX 10001006), the National Natural Science Foundation of China (31070135 and 30900068), the 973 program (2010CB534907), the Tianjin Natural Science Foundation 12JCZDJC21800 and the 111 project (B08011).
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