Folia Microbiol DOI 10.1007/s12223-015-0387-x
Preparation and characterization of polyclonal antibody against Kaposi’s sarcoma-associated herpesvirus lytic gene encoding RTA Weifei Fan 1 & Qiao Tang 2 & Chenyou Shen 3 & Di Qin 3 & Chun Lu 3 & Qin Yan 3
Received: 30 July 2014 / Accepted: 15 March 2015 # Institute of Microbiology, Academy of Sciences of the Czech Republic, v.v.i. 2015
Abstract Replication and transcription activator (RTA) is a critical lytic protein encoded by Kaposi’s sarcoma-associated herpesvirus (KSHV). To prepare rabbit polyclonal antibody against RTA, three antigenic polypeptides of KSHV RTA were initially synthesized. The fragment of RTA was cloned into p3FlagBsd to construct the recombinant plasmid, pRTA-Flag. 293 T and EA.hy926 cells were transfected with pRTA-Flag to obtain RTA-Flag fusion protein, which was detected using anti-Flag antibody. Next, New Zealand white rabbits were immunized with keyhole limpet hemocyanin-conjugated peptides to generate polyclonal antibodies against RTA. Enzymelinked immunosorbent assays were performed to characterize the polyclonal antibodies, and the titers of the polyclonal antibodies against RTA were greater than 1:11,000. Western blotting and immunofluorescence assay revealed that the prepared antibody reacted specifically with the RTA-Flag fusion protein as well as the native viral protein in KSHV-infected primary effusion lymphoma cells. Collectively, our work successfully constructed the recombinant expression vector, pRTA-Flag, and prepared the polyclonal antibody against RTA, which was valuable for investigating the biochemical and biological functions of the critical KSHV lytic gene. Qin Yan holds a PhD, Nanjing Medical University. * Qin Yan [email protected] 1
Department of Oncology, Jiangsu Province Official Hospital, 65 Jiangsu Road, Nanjing 210024, People’s Republic of China
2
Department of Clinical Laboratory, The Affiliated Nanjing First Hospital of Nanjing Medical University, Nanjing 210029, People’s Republic of China
3
Department of Microbiology, Nanjing Medical University, Nanjing, Jiangsu 210029, People’s Republic of China
Introduction Kaposi’s sarcoma-associated herpesvirus (KSHV), also known as human herpesvirus 8 (HHV-8), was initially discovered from the Kaposi’s sarcoma (KS) lesion of an AIDS patient in 1994 (Chang et al. 1994). KSHV is a large doublestranded DNA oncogenic virus belonging to the gamma-2herpesvirus subfamily (Lee et al. 2012). In addition to KS, KSHV is also causally linked to the development of primary effusion lymphoma (PEL) and multicentric Castleman’s disease (Ye et al. 2011). As well as other gamma-2-herpesviruses, KSHV displays two distinct and alternative phases of the life cycle: latent and lytic replication (Boshoff and Chang 2001). Latent phase makes KSHV evade the host immune surveillance and establish a persistent infection to promote tumor development (Lee et al. 2010; Ye et al. 2011), while lytic replication facilitates the spread of the virus and causes the associated diseases (Mercader et al. 2000; Ye et al. 2011). The KSHV genome encodes more than 90 open reading frames (ORFs) and at least 25 mature microRNAs (Ziegelbauer 2011). The lytic genes are classified into three major groups based on expression kinetics, as follows: immediate early (IE) genes, early genes, and late genes. The IE genes are expressed immediately after primary infection or upon reactivation from latency. The IE genes encode for proteins with regulatory functions in activating the cascade of downstream genes and initiating viral transcription. After the IE genes, but before viral DNA synthesis, early genes are made, including K1 (Bowser et al. 2006), viral interferon factor-1 (Chen et al. 2000), ORF57 (Han and Swaminathan 2006), polyadenylated nuclear (PAN) RNA (Song et al. 2002), and viral interleukin-6 (vIL-6) (Deng et al. 2002). Late genes, such as K8.1 and ORF65, usually do not appear until 30 h after induction (Sarid et al. 1998; Sun et al. 1999).
Folia Microbiol
ORF50, which encodes replication and transcription activator (RTA), is the best-characterized IE gene of KSHV, and is necessary and sufficient to drive KSHV lytic replication and the production of viral progeny (Greene et al. 2007). As the first expressed lytic gene, RTA is followed by early genes and late genes. After the expression of viral lytic genes, viral DNA replication, capsid packaging, and virion release completes the viral lytic replication cycle (Ye et al. 2011). RTA can autoactivate its own promoter and transactivate downstream lytic genes (Deng et al. 2000). Moreover, as a transcriptional activator, RTA not only activates promoters by binding DNA directly, but also interacts with cellular proteins, such as octamer-1 (Carroll et al. 2007), KSHV RTA binding protein (Wang et al. 2001b), and RBP-Jκ (Liang et al. 2002). Therefore, many studies indicate that RTA plays a central role in the physiologic mechanisms controlling reactivation of KSHV from latency. To further investigate the biochemical and biological functions, the rabbit polyclonal antibody against KSHV lytic gene encoding protein RTA were generated and evaluated in this study. These results suggest that the prepared antibody will be useful for studying the role of RTA on KSHV infection and pathogenesis.
Materials and methods Cells culture, transfection, and animals EA.hy926, BJAB, and PEL cell lines, including BCBL-1 and JSC-1 cells, were cultured in RPMI-1640 containing 10 % heat-inactivated fetal bovine serum (FBS), 2 mmol/L L-glutamine, 100 U/mL of penicillin, and 100 μg/mL of streptomycin at 37 °C in a humidified, 5 % CO2 atmosphere. The human embryonic kidney fibroblast cell line, 293 T, was maintained in Dulbecco's modified Eagle's medium supplemented with 10 % FBS. Transfections were conducted with LipofectamineTM 2000 (Invitrogen, NY, USA) according to the manufacturer’s instructions. Male New Zealand white rabbits (3 months old, weighing 3 kg) were purchased from BaiQi Biotechnology (Suzhou, China). The protocol was approved by the Animal Care and Ethical Committee of Nanjing Medical University. All procedures were in accordance with the guidelines of the Institute for Laboratory Animal Research of the Nanjing Medical University. Plasmid construction The expression vector, p3FlagBsd, which fused with a triple FLAG epitope on the N-terminal of pcDNA3.1, was kindly provided by Dr. Shou-Jiang Gao (University of Southern California, Los Angeles, CA, USA) (Chen 2006). The fragment
of KSHV RTA (NCBI Reference Sequence: YP_001129401) was amplified from total BCBL-1 cDNA by polymerase chain reaction (PCR) with the following primers, which contained the Nhe I and Xho I restriction sites (underlined): 5′-CTA GCT AGC TAG GCC ACC ATG GCG CAA GAT GAC AAG GGT A-3′ (sense); and 5′-CCG CTC GAG TAG GGT TTC TTA CGC CGG CAT-3′ (anti-sense). The amplified PCR fragment was inserted into p3FlagBsd and sequenced using a commercial service (Invitrogen Biotechnology Company, Shanghai, China). Finally, following transfection 293 T and EA.hy926 cells with pRTA-Flag, RTA-Flag fusion protein was detected by Western blotting. Polypeptides composition and polyclonal antibodies production SUMOplot software was used to analyze the sequences of RTA on the basis of a series of parameters, including antigen hydrophilicity, N-mer, beta-turn, flexibility, and accessibility (Xue et al. 2006). Combined with the hydrophilic prediction by ProtScale, three RTA polypeptides coupled with keyhole limpet hemocyanin (KLH) were separately designed. Then, the designed polypeptides were synthesized and purified by reverse-phase high-performance liquid chromatography using a commercial service (BaiQi Biotechnology, Suzhou, China). Anti-recombinant RTA rabbit serum was then obtained from six adult male New Zealand white rabbits immunized with 400 μg/animal of purified recombinant KLH-conjugated polypeptides emulsified in complete Freund’s adjuvant (Sigma-Aldrich, MO, USA) by subcutaneous injection on the dorsum (each polypeptide was used to immunize two rabbits, e.g., RB1A and RB1B were immunized with polypeptide no. 1). Each rabbit was booster-immunized with 200 μg of RTA-KLH polypeptides emulsified in incomplete Freund’s adjuvant by subcutaneous injection every 2 weeks. Blood was obtained from ear marginal veins of each rabbit to prepare non-immune serum before immunization. After four booster immunizations, about 80–100 mL blood was harvested again by carotid artery, and then 30–40 mL antiserum was taken apart for final collection. The immunization efficiency was subsequently analyzed by enzyme-linked immunosorbent assays (ELISAs) and Western blottings, and serum samples were stored at −80 °C. Protein purification Anti-RTA antisera was purified with caprylic acid-ammonium sulfate precipitation, according to previously published protocols (Ruan et al. 2005; Yang et al. 2012). One volume of antiserum was diluted with four volumes of 0.06 mol/L acetic acid buffer (17.4 mol/L glacial acetic acid was diluted to 0.06 mol/L with distilled water) and adjusted to pH 4.5 with 5 mol/L NaOH. Caprylic acid was added drop-by-drop at
Folia Microbiol
room temperature to a final concentration of 25 μL caprylic acid per milliliter sample. After vigorous mixing for 30 min, the sample was centrifuged (10,000×g for 10 min at 4 °C) to remove floaters and the precipitate. The supernatant was filtered through filter paper three times. The solution was then adjusted to pH 7.4 and kept in an ice bath for 10 min. Solid ammonium sulfate (0.277 g per mL) was added to the solution. After stirring for 30 min at 4 °C, the solution was centrifuged (10,000×g for 10 min). The supernatant was discarded. The precipitate was dissolved in phosphate-buffered saline (PBS) and dialyzed against PBS until no NH4+ was present. Finally, the purity of the antibody was monitored by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE).
ELISA Indirect ELISA was performed to determine the titer of anti-RTA. Briefly, the purified RTA protein was diluted to 1 mg/L in 50 mmol/L carbonate-bicarbonate buffer (pH 9.6) and coated on plates at 100 μL per well in 96-well immunoplates at 4 °C overnight. The wells were washed three times with PBS buffer. The coated wells were blocked with 100 μL of 10 % fetal calf serum for 2 h at 37 °C, then incubated with 100 μL of anti-RTA with serial 1:2 dilutions (normal serum was the negative control). After incubation for 1 h at 37 °C, the wells were washed and incubated with 100 μL of horseradish peroxidase (HRP)-conjugated goat antirabbit IgG (dilution, 1:5,000; Santa Cruz Biotechnology, Inc., CA, USA) for 1 h at 37 °C. The wells were then washed five times with PBS buffer and incubated with 100 μL of enzyme substrates (tetramethyl benzidine) for 30 min at 37 °C. Color development was then stopped, and the absorbance was measured at 450 nm using a microplate reader. The relational reagents of ELISA assay were purchased from TIANGEN Biotechnology. Fig. 1 Amplification of KSHV RTA and construction of recombination plasmid pRTAFlag. a Amplification of the fragment of RTA by PCR. b PCR amplification of recombinant plasmid pRTA-Flag
Western blotting The 293 T and EA.hy926 cell lysates, which were transfected with pRTA-Flag for 48 h, or BJAB, BCBL-1, and JSC-1 cell lysates were separated by 10 % SDS-PAGE, transferred to an Immobilon P (polyvinylidene difluoride) membrane, and blocked with 5 % powdered milk in TBST (50 mmol/L Tris, pH 7.5, 150 mmol/L NaCl, 0.01 % Tween 20). The membrane was then incubated with primary antibodies diluted in 2.5 % powdered milk in TBST, washed extensively, and incubated with HRP-conjugated species-specific secondary antibodies (Santa Cruz Biotechnology, Inc). The primary antibodies used in Western blotting were the prepared antibodies, anti-Flag M2 rabbit monoclonal antibody (MAb) from Cell Signaling Technology (Beverly, MA, USA), anti-vIL-6 rabbit polyclonal antibody (PAb) from Advanced Biotechnologies, Inc. (Columbia, MD, USA), and anti-GAPDH mouse MAb from Santa Cruz Biotechnology, Inc. Proteins were visualized with enhanced chemiluminescence reagents (Cell Signaling Technology), according to the manufacturer’s instructions. The data shown were repeated at least three times to confirm the results. Immunofluorescence assay (IFA) BJAB cells and BCBL-1 cells treated with or without 12-Otetradecanoylphorbol-13-acetate (TPA; 20 ng/mL, Sigma, USA) for 48 h (107/mL) were washed and smeared on chamber slides. Slides were incubated with anti-RTA antibody from RB3A rabbit immunized with polypeptide no. 3 or antiKSHV ORF59 (clone 11D1; IgG2b) mouse MAb from Advanced Biotechnologies, Inc. (Columbia, MD, USA) at 1:100 dilution. Alexa Fluor 647 (Invitrogen)-conjugated goat antirabbit antibody (1:200 dilution) was used as a secondary antibody for detection of specific signal. The cells were counterstained with 4’, 6’-diamidino-2-phenylindole (DAPI). Images were observed and recorded with a Zeiss Axiovert 200 M epifluorescence microscope (Carl Zeiss, Inc.).
Folia Microbiol
Fig. 2 Detection of recombination RTA protein in 293 T and EA.hy926 cells with the anti-Flag antibody. The expression of the recombinant RTA protein in 293 T (a) or EA.hy926 (b) cells transfected with pRTA-Flag or empty vector was detected with the anti-Flag antibody in Western blotting
Results Amplification of KSHV RTA and construction of recombination plasmid pRTA-Flag The RTA fragment was cloned by PCR amplification and confirmed with 1.0 % agarose gel (w/v) electrophoresis by detection of the expected 3,538 bp fragment (Fig. 1a). A fragment consistent with RTA was inserted between the Nhe I and Xho I sites of the expression vector, p3FlagBsd. After verification by DNA sequencing, recombinant plasmid pRTA-Flag showed an identical sequence to that previously registered in GenBank (accession number YP_001129401; data not shown; Fig. 1b). Expression of recombination RTA protein in 293 T and EA.hy926 cells Western blotting was performed to detect the expression of full-length RTA protein with anti-Flag as the primary antibody. 293 T and EA.hy926 cells were transfected with pRTA-Flag for 48 h, and the lysates of cells were detected by Western blotting. A single band at about 110 kDa was detected with anti-Flag, which stood for RTA-Flag fusion protein (Fig. 2a and b). Preparation, purification, and identification of RTA-KLH fusion protein On the basis of a series of parameters, we separately designed three antigenic polypeptides of KSHV RTA (Table 1), and then RTA protein was prepared and purified according to a routine protocol. Polyclonal antibodies against RTA were generated in six adult male New Zealand white rabbits by immunization with purified recombinant RTA-KLH polypeptides Table 1
(anti-RTA). Next, according to the normal protocol, the antiRTA IgG was purified with caprylic acid–ammonium sulfate precipitation and the purity of the prepared antibodies was monitored by SDS-PAGE to observe the expected RTA immunoglobulin heavy and light chains without irrelevant bands (data not shown). Detection of the RTA antisera titer by ELISA The polyclonal antibodies against full-length of RTA were generated by immunizing rabbits with RTA-KLH (anti-RTAKLH antisera). With indirect ELISA, the anti-RTA antisera were assayed at different dilutions ranged from 1:1,000–1:8, 000 for reactivity with RTA or the control proteins (Table 2). The titers of anti-RTA antisera were determined to be 1:11,000. Detection of full-length RTA protein by prepared antibodies To determine whether the anti-RTA antibodies detect fulllength RTA, the whole cell lysates of 293 T cells transfected with pRTA-Flag were subjected to SDS-PAGE. Only the prepared antibodies from RB3A and RB3B immunized with polypeptide no. 3 specifically recognized the full-length RTA expressed in transfected 293 T cells (Fig. 3a–f). PEL cells, including BCBL-1 (KSHV+ EBV−) and JSC-1 (KSHV+ EBV+) cells were used to determine whether the prepared antibodies were suitable for RTA detection, while the human B cell lymphoma BJAB cells (KSHV− EBV−) were acted as the negative control. The anti-RTA antibody from RB3A was preferred for realistic applications because of the superior performance, as shown in Fig. 3. Western blotting revealed that the anti-RTA antibody from RB3A clearly detected full-length RTA protein in JSC-1 and BCBL-1 cells
Epitope candidates designed for RTA (no. 1–3)
Sequence no.
Epitope
Len
Start
End
Rabbit no.
1 2 3
NH2-AQDDKGKKLRRSC-CONH2 NH2-KKRKALTVPEADT-CONH2 NH2-ATPANEVQESGTLYQLHQWRNYFRD-CONH2
13 13 25
2 527 667
14 539 691
RB1A-B RB2A-B RB3A-B
Folia Microbiol Table 2 Detection of antibody titer in serum by indirect ELISA No.
Antigen
Blank
Negative 1:1,000
Negative 1:4,000
Positive 1:1,000
Positive 1:4,000
Positive 1:8,000
RB1A RB1B RB2A RB2B RB3A RB3B
1 1 2 2 3 3
0.079 0.080 0.082 0.090 0.083 0.093
0.085 0.109 0.132 0.166 0.159 0.168
0.080 0.088 0.091 0.103 0.094 0.110
2.561 3.528 3.741 3.892 3.485 3.560
1.288 1.564 1.965 2.141 1.613 1.823
0.612 0.996 1.124 0.870 1.092 1.057
Blank normal serum collected from each rabbit before immunization; Negative immunized serum collected from each rabbit when A450 value1.0 with 1:4,000 dilution.
after treatment with TPA for 48 h, but not in BJAB cells (Fig. 4). Furthermore, the expression of KSHV RTA was found since 6 h after TPA treatment in BCBL-1 cells and gradually increased with time going, but not in BJAB cells (Fig. 5). Simultaneously, the commercially anti-KSHV vIL-6 rabbit polyclonal antibody was used as the positive control (Figs. 4 and 5). To determine the most appropriate application concentration, different dilution rate of anti-RTA antibody from RB3Awas used in Western blottings. As shown in Figs. 4 and 5, the prepared antibody could react specifically with RTA at dilution of 1:2,000 and 1:1,000. Moreover, IFA was performed to evaluate whether the antiRTA antibody could recognize the native protein in the KSHV-infected PEL cells. We found that native RTA was obviously detected by anti-RTA antibody from RB3A in BCBL-1 cells treated without or with TPA for 48 h, but not
in KSHV-negative BJAB cells (Fig. 6a). The commercially anti-KSHV ORF59 mouse monoclonal antibody was also used as the positive control in IFA (Fig. 6b).
Fig. 3 Detection of full-length RTA protein in 293 T cells with the prepared antibodies. a, b Recombinant KSHV RTA protein was detected with anti-RTA antibodies from RB1A and RB1B (rabbits immunized with polypeptide no. 1); c, d Recombinant KSHV RTA protein was
detected with anti-RTA antibodies from RB2A and RB2B (rabbits immunized with polypeptide no. 2); e, f Recombinant KSHV RTA protein was detected with anti-RTA antibodies from RB3A and RB3B (rabbits immunized with polypeptide no. 3)
Discussion KSHV infection undergoes latent and lytic phases, and viral lytic replication plays a critical role in the process of KSHV pathogenesis. The switch from latent to lytic gene expression is governed by an immediate–early gene product (RTA), which is encoded by ORF50. The RTA protein consists of 691 amino acids and is predicted to have a molecular mass of 73.7 kDa; however, the expressed protein, when analyzed by Western blotting, appears to be approximately 110 kDa, which may due to post-translation modification,
Folia Microbiol
Fig. 4 Detection of RTA in PEL cell lines with prepared antibody. BJAB cells and PEL cell lines, including BCBL-1 and JSC-1 cells, were collected at 48 h after treatment with or without TPA (20 ng/mL), and total proteins were analyzed by Western blotting with anti-RTA antibody from RB3A rabbit immunized with polypeptide no. 3 (1:2,000 or 1:1,000 dilution) or anti-KSHV vIL-6 rabbit polyclonal antibody. M: protein ladder (#SM0671, Fermentas)
phophorylation, or other mechanisms (Lukac et al. 1999). The RTA protein contains an N-terminal DNA-binding domain, a central dimerization domain, a C-terminal acidic activation domain, and two nuclear localization signals (Chen et al. 2000; Lukac et al. 1999). The DNA-binding domain of RTA is located at the amino terminus (aa 1–530), while the activation domain is located at the carboxyl terminus of the protein (aa 486–691) (Lukac et al. 1999). The activation domain also contains four repeated units of a highly hydrophobic domain, known as activation domains 1-4 (AD1-AD4) (Lukac et al. 1999; Wang et al. 2001a), with sequence homology to other transcriptional factors. These features suggest that RTA is a member of transcriptional factors. RTA is necessary and sufficient to trigger the KSHV lytic cascade and the production of viral progeny. RTA has been shown to autoregulate its own expression and transactivate a multitude of downstream viral genes, including PAN (Song et al. 2002), ORF57 (Han and Swaminathan 2006), ORF21 (Zhang et al. 1998), ORF59 (Liu et al. 2008), ORF73 (Staudt and Dittmer 2006), ORF74 (Jeong et al. 2001), K1 (Bowser et al. 2006), K2 (Deng et al. 2002), K5 (St Swierzko et al.
Fig. 5 Detection of the dynamic expression of native RTA in BCBL-1 cells with prepared antibody. BCBL-1 cells were collected at 6, 24, or 48 h after treatment with or without TPA (20 ng/mL), while BJAB cells were also collected at 6 h after treatment with or without TPA. Total proteins were analyzed by Western blotting with anti-RTA antibody from RB3A rabbit immunized with polypeptide no. 3 (1:2,000 or 1:1,000 dilution) or anti-KSHV vIL-6 rabbit polyclonal antibody. M: protein ladder (#SM0671, Fermentas)
2000), K8/K-bZIP (Byun et al. 2002), K9 (Ueda et al. 2002), K12 (Chang et al. 2002), and K15 (Wong and Damania 2006). The mechanism of RTA transactivation is either by direct recognition of RTA response elements in the promoters of the targets genes or interaction with cellular and/or viral co-factors bound to the promoters (Chang et al. 2005; Song et al. 2003). Although RTA is a significant lytic protein of KSHV, previous studies usually used Tag-antibody to detect the recombination protein or homemade antibody to detect the native protein without the detailed information (Chen et al. 2000; Lin et al. 2011). For instance, H. Katano et al. developed a rabbit anti-RTA protein polyclonal antibody to react with the RTA protein in HHV-8–infected cells lines, while the expression vector produced almost the full-length of the RTA protein (631 amino acids) as a fusion protein (Katano et al. 2001), and Gary Hayward et al. also generated the rabbit antiserum against a synthetic peptide representing the KSHV RTA segment between amino acids 527 and 539 (Wang et al. 2003). In comparison with these studies, we prepared and purified rabbit polyclonal antibody against the partial activation domain of RTA (aa 667–691), which may get more pure protein for antibody production, make less obstruction on binding to DNA comparing with other viral or cellular factors, and finally detect the native viral protein successfully (Qin et al. 2010). In the current study, we generated anti-RTA antibodies (RA1A, RA1B, RA2A, RA2B, RA3A, and RA3B) from six
Folia Microbiol Fig. 6 Detection of native RTA in BCBL-1 and BJAB cells with prepared antibody by IFA. BJAB cells and BCBL-1 cells treated with or without TPA (20 ng/mL) for 48 h were examined by IFA with anti-RTA antibody from RB3A rabbit immunized with polypeptide no. 3 (a) or anti-ORF59 mouse monoclonal antibody (b). DAPI (blue) stains nuclei (original magnifications, ×10)
rabbits immunized with three different synthesized antigen peptides coupled with KLH. Western blotting revealed that RA3A and RA3B anti-RTA antibodies from rabbits immunized with no. 3 antigen peptides detected the 110 kDa fulllength RTA protein expressed in 293 T cells. Unfortunately, the remaining antibodies did not recognize the recombination
RTA protein expressed in 293 T cells. The possible explanation for this observation is that the algorithms for structure prediction are poor for immunogenicity. Although the selection of peptides on the basis of the algorithms may be an obvious choice for identification of potential peptides (Ponomarenko and Bourne 2007), the algorithms for structure
Folia Microbiol
prediction were originally designed to determine antigenicity and the proposed sequences may not be immunogenic (Trier et al. 2012). Also, if the peptide template is located internally, the peptide antibody will most likely not be able to interact with the corresponding protein because the peptide is buried in the structure. Another possible explanation is due to the impact of the FLAG-tag on the three-dimensional conformation of recombination protein (Bucher et al. 2002). The binding affinity of most antibodies is influenced by conformational determinants, and antibodies may not bind the same protein in a denatured state (Lipman et al. 2005). The switch from latent to lytic viral replication of KSHV is initiated by an abundance of stimuli that induce the expression of RTA, such as TPA, which cause the demethylation of RTA promoter (Yu et al. 1999). The expression of RTA precedes the expression of all other cycloheximide-resistant IE genes and cycloheximide-sensitive early genes (Sun et al. 1999). Using the prepared antibody, our study confirmed that the expression of RTA strikingly increased in BCBL-1 and JSC-1 cells after TPA treatment for 48 h. In addition, the expression of RTA was markedly raised in BCBL-1 cells from 6 to 24 h following TPA induction. The corresponding results indicated that the produced antibody was able to detect the veritable dynamic expression of this KSHV lytic gene, RTA. In summary, the rabbit polyclonal antibody with specificity for KSHV lytic gene RTA were generated in this study to reveal its expression pattern and biochemical properties, which is functional at least in three different assays: Western blottings, IFA, and ELISA. The other usage of the antibody remains to be identified in future. Because RTA is a critical protein of the KSHV lytic cycle, the prepared antibody may provide a preparative work for the investigation of biological functions of RTA on KSHV infection and pathogenesis. (To facilitate statistics of antibody titer and calculation for practical application, the prepared antibodies were diluted to the final concentration of 1 mg/mL). Acknowledgments We are grateful to Dr. Shou-Jiang Gao (University of Southern California) for generously providing plasmid. This work was supported by grants from the Natural Science Youth Foundation of Jiangsu Province (BK20140908) and Science Development Foundation of Nanjing Medical University (2013NJMU007). Conflict of interest disclosure The authors declare no competing financial interest.
References Boshoff C, Chang Y (2001) Kaposi’s sarcoma-associated herpesvirus: a new DNA tumor virus. Annu Rev Med 52:453–470 Bowser BS, Morris S, Song MJ, Sun R, Damania B (2006) Characterization of Kaposi’s sarcoma-associated herpesvirus (KSHV) K1 promoter activation by Rta. Virology 348:309–327
Bucher MH, Evdokimov AG, Waugh DS (2002) Differential effects of short affinity tags on the crystallization of Pyrococcus furiosus maltodextrin-binding protein. Acta Crystallogr D Biol Crystallogr 58:392–397 Byun H, Gwack Y, Hwang S, Choe J (2002) Kaposi’s sarcoma-associated herpesvirus open reading frame (ORF) 50 transactivates K8 and ORF57 promoters via heterogeneous response elements. Mol Cell 14:185–191 Carroll KD, Khadim F, Spadavecchia S, Palmeri D, Lukac DM (2007) Direct interactions of Kaposi’s sarcoma-associated herpesvirus/ human herpesvirus 8 ORF50/Rta protein with the cellular protein octamer-1 and DNA are critical for specifying transactivation of a delayed–early promoter and stimulating viral reactivation. J Virol 81:8451–8467 Chang H, Dittmer DP, Shin YC, Hong Y, Jung JU (2005) Role of Notch signal transduction in Kaposi’s sarcoma-associated herpesvirus gene expression. J Virol 79:14371–14382 Chang PJ, Shedd D, Gradoville L, Cho MS, Chen LW, Chang J, Miller G (2002) Open reading frame 50 protein of Kaposi’s sarcomaassociated herpesvirus directly activates the viral PAN and K12 genes by binding to related response elements. J Virol 76:3168– 3178 Chang Y, Cesarman E, Pessin MS, Lee F, Culpepper J, Knowles DM, Moore PS (1994) Identification of herpesvirus-like DNA sequences in AIDS-associated Kaposi’s sarcoma. Science 266:1865–1869 Chen J (2006) Serial analysis of binding elements for human transcription factors. Nat Protoc 1:1481–1493 Chen J, Ueda K, Sakakibara S, Okuno T, Yamanishi K (2000) Transcriptional regulation of the Kaposi’s sarcoma-associated herpesvirus viral interferon regulatory factor gene. J Virol 74:8623– 8634 Deng H, Song MJ, Chu JT, Sun R (2002) Transcriptional regulation of the interleukin-6 gene of human herpesvirus 8 (Kaposi’s sarcomaassociated herpesvirus). J Virol 76:8252–8264 Deng H, Young A, Sun R (2000) Auto-activation of the Rta gene of human herpesvirus-8/Kaposi’s sarcoma-associated herpesvirus. J Gen Virol 81:3043–3048 Greene W, Kuhne K, Ye F, Chen J, Zhou F, Lei X, Gao SJ (2007) Molecular biology of KSHV in relation to AIDS-associated oncogenesis. Cancer Treat Res 133:69–127 Han Z, Swaminathan S (2006) Kaposi’s sarcoma-associated herpesvirus lytic gene ORF57 is essential for infectious virion production. J Virol 80:5251–5260 Jeong J, Papin J, Dittmer D (2001) Differential regulation of the overlapping Kaposi’s sarcoma-associated herpesvirus vGCR (orf74) and LANA (orf73) promoters. J Virol 75:1798–1807 Katano H, Sato Y, Itoh H, Sata T (2001) Expression of human herpesvirus 8 (HHV-8)-encoded immediate early protein, open reading frame 50, in HHV-8-associated diseases. J Hum Virol 4:96–102 Lee HR, Brulois K, Wong L, Jung JU (2012) Modulation of immune system by Kaposi’s sarcoma-associated herpesvirus: lessons from viral evasion strategies. Front Microbiol 3:44 Lee HR, Lee S, Chaudhary PM, Gill P, Jung JU (2010) Immune evasion by Kaposi’s sarcoma-associated herpesvirus. Future Microbiol 5: 1349–1365 Liang Y, Chang J, Lynch SJ, Lukac DM, Ganem D (2002) The lytic switch protein of KSHV activates gene expression via functional interaction with RBP-Jkappa (CSL), the target of the Notch signaling pathway. Genes Dev 16:1977–1989 Lin X, Liang D, He Z, Deng Q, Robertson ES, Lan K (2011) miR-K12-75p encoded by Kaposi’s sarcoma-associated herpesvirus stabilizes the latent state by targeting viral ORF50/RTA. PLoS ONE 6:e16224 Lipman NS, Jackson LR, Trudel LJ, Weis-Garcia F (2005) Monoclonal versus polyclonal antibodies: distinguishing characteristics, applications, and information resources. ILAR J 46:258–268
Folia Microbiol Liu Y, Cao Y, Liang D, Gao Y, Xia T, Robertson ES, Lan K (2008) Kaposi’s sarcoma-associated herpesvirus RTA activates the processivity factor ORF59 through interaction with RBP-Jkappa and a cis-acting RTA responsive element. Virology 380:264–275 Lukac DM, Kirshner JR, Ganem D (1999) Transcriptional activation by the product of open reading frame 50 of Kaposi’s sarcomaassociated herpesvirus is required for lytic viral reactivation in B cells. J Virol 73:9348–9361 Mercader M, Taddeo B, Panella JR, Chandran B, Nickoloff BJ, Foreman KE (2000) Induction of HHV-8 lytic cycle replication by inflammatory cytokines produced by HIV-1-infected T cells. Am J Pathol 156: 1961–1971 Ponomarenko JV, Bourne PE (2007) Antibody–protein interactions: benchmark datasets and prediction tools evaluation. BMC Struct Biol 7:64 Qin Y, Liu Z, Zhang T, Wang Y, Li X, Wang J (2010) Generation and application of polyclonal antibody against replication and transcription activator of Kaposi’s sarcoma-associated herpesvirus. Appl Biochem Biotechnol 160:1217–1226 Ruan GP, Ma L, He XW, Meng MJ, Zhu Y, Zhou MQ, Hu ZM, Wang XN (2005) Efficient production, purification, and application of egg yolk antibodies against human HLA-A*0201 heavy chain and light chain (beta2m). Protein Expr Purif 44:45–51 Sarid R, Flore O, Bohenzky RA, Chang Y, Moore PS (1998) Transcription mapping of the Kaposi’s sarcoma-associated herpesvirus (human herpesvirus 8) genome in a body cavity-based lymphoma cell line (BC-1). J Virol 72:1005–1012 Song MJ, Deng H, Sun R (2003) Comparative study of regulation of RTA-responsive genes in Kaposi’s sarcoma-associated herpesvirus/ human herpesvirus 8. J Virol 77:9451–9462 Song MJ, Li X, Brown HJ, Sun R (2002) Characterization of interactions between RTA and the promoter of polyadenylated nuclear RNA in Kaposi’s sarcoma-associated herpesvirus/human herpesvirus 8. J Virol 76:5000–5013 St Swierzko A, Kirikae T, Kirikae F, Hirata M, Cedzynski M, Ziolkowski A, Hirai Y, Kusumoto S, Yokochi T, Nakano M (2000) Biological activities of lipopolysaccharides of Proteus spp. and their interactions with polymyxin B and an 18-kDa cationic antimicrobial protein (CAP18)-derived peptide. J Med Microbiol 49:127–138 Staudt MR, Dittmer DP (2006) Promoter switching allows simultaneous transcription of LANA and K14/vGPCR of Kaposi’s sarcomaassociated herpesvirus. Virology 350:192–205
Sun R, Lin SF, Staskus K, Gradoville L, Grogan E, Haase A, Miller G (1999) Kinetics of Kaposi’s sarcoma-associated herpesvirus gene expression. J Virol 73:2232–2242 Trier NH, Hansen PR, Houen G (2012) Production and characterization of peptide antibodies. Methods 56:136–144 Ueda K, Ishikawa K, Nishimura K, Sakakibara S, Do E, Yamanishi K (2002) Kaposi’s sarcoma-associated herpesvirus (human herpesvirus 8) replication and transcription factor activates the K9 (vIRF) gene through two distinct cis elements by a non-DNA-binding mechanism. J Virol 76:12044–12054 Wang S, Liu S, Wu M, Geng Y, Wood C (2001a) Kaposi’s sarcomaassociated herpesvirus/human herpesvirus-8 ORF50 gene product contains a potent C-terminal activation domain which activates gene expression via a specific target sequence. Arch Virol 146: 1415–1426 Wang S, Liu S, Wu MH, Geng Y, Wood C (2001b) Identification of a cellular protein that interacts and synergizes with the RTA (ORF50) protein of Kaposi’s sarcoma-associated herpesvirus in transcriptional activation. J Virol 75:11961–11973 Wang SE, Wu FY, Yu Y, Hayward GS (2003) CCAAT/enhancer-binding protein-alpha is induced during the early stages of Kaposi’s sarcoma-associated herpesvirus (KSHV) lytic cycle reactivation and together with the KSHV replication and transcription activator (RTA) cooperatively stimulates the viral RTA, MTA, and PAN promoters. J Virol 77(17):9590–9612 Wong EL, Damania B (2006) Transcriptional regulation of the Kaposi’s sarcoma-associated herpesvirus K15 gene. J Virol 80:1385–1392 Xue Y, Zhou F, Fu C, Xu Y, Yao X (2006) SUMOsp: a web server for sumoylation site prediction. Nucleic Acids Res 34:W254–W257 Yang Y, Wang B, Yang D, Lu M, Xu Y (2012) Prokaryotic expression of woodchuck cytotoxic T lymphocyte antigen 4 (wCTLA-4) and preparation of polyclonal antibody to wCTLA-4. Protein Expr Purif 81:181–185 Ye F, Lei X, Gao SJ (2011) Mechanisms of Kaposi’s sarcoma-associated herpesvirus latency and reactivation. Adv Virol 2011 Yu Y, Black JB, Goldsmith CS, Browning PJ, Bhalla K, Offermann MK (1999) Induction of human herpesvirus-8 DNA replication and transcription by butyrate and TPA in BCBL-1 cells. J Gen Virol 80(Pt 1):83–90 Zhang L, Chiu J, Lin JC (1998) Activation of human herpesvirus 8 (HHV-8) thymidine kinase (TK) TATAA-less promoter by HHV-8 ORF50 gene product is SP1 dependent. DNA Cell Biol 17:735–742 Ziegelbauer JM (2011) Functions of Kaposi’s sarcoma-associated herpesvirus microRNAs. Biochim Biophys Acta 1809:623–630
Preparation and characterization of polyclonal antibody against Kaposi’s sarcoma-associated herpesvirus lytic gene encoding RTA Weifei Fan 1 & Qiao Tang 2 & Chenyou Shen 3 & Di Qin 3 & Chun Lu 3 & Qin Yan 3
Received: 30 July 2014 / Accepted: 15 March 2015 # Institute of Microbiology, Academy of Sciences of the Czech Republic, v.v.i. 2015
Abstract Replication and transcription activator (RTA) is a critical lytic protein encoded by Kaposi’s sarcoma-associated herpesvirus (KSHV). To prepare rabbit polyclonal antibody against RTA, three antigenic polypeptides of KSHV RTA were initially synthesized. The fragment of RTA was cloned into p3FlagBsd to construct the recombinant plasmid, pRTA-Flag. 293 T and EA.hy926 cells were transfected with pRTA-Flag to obtain RTA-Flag fusion protein, which was detected using anti-Flag antibody. Next, New Zealand white rabbits were immunized with keyhole limpet hemocyanin-conjugated peptides to generate polyclonal antibodies against RTA. Enzymelinked immunosorbent assays were performed to characterize the polyclonal antibodies, and the titers of the polyclonal antibodies against RTA were greater than 1:11,000. Western blotting and immunofluorescence assay revealed that the prepared antibody reacted specifically with the RTA-Flag fusion protein as well as the native viral protein in KSHV-infected primary effusion lymphoma cells. Collectively, our work successfully constructed the recombinant expression vector, pRTA-Flag, and prepared the polyclonal antibody against RTA, which was valuable for investigating the biochemical and biological functions of the critical KSHV lytic gene. Qin Yan holds a PhD, Nanjing Medical University. * Qin Yan [email protected] 1
Department of Oncology, Jiangsu Province Official Hospital, 65 Jiangsu Road, Nanjing 210024, People’s Republic of China
2
Department of Clinical Laboratory, The Affiliated Nanjing First Hospital of Nanjing Medical University, Nanjing 210029, People’s Republic of China
3
Department of Microbiology, Nanjing Medical University, Nanjing, Jiangsu 210029, People’s Republic of China
Introduction Kaposi’s sarcoma-associated herpesvirus (KSHV), also known as human herpesvirus 8 (HHV-8), was initially discovered from the Kaposi’s sarcoma (KS) lesion of an AIDS patient in 1994 (Chang et al. 1994). KSHV is a large doublestranded DNA oncogenic virus belonging to the gamma-2herpesvirus subfamily (Lee et al. 2012). In addition to KS, KSHV is also causally linked to the development of primary effusion lymphoma (PEL) and multicentric Castleman’s disease (Ye et al. 2011). As well as other gamma-2-herpesviruses, KSHV displays two distinct and alternative phases of the life cycle: latent and lytic replication (Boshoff and Chang 2001). Latent phase makes KSHV evade the host immune surveillance and establish a persistent infection to promote tumor development (Lee et al. 2010; Ye et al. 2011), while lytic replication facilitates the spread of the virus and causes the associated diseases (Mercader et al. 2000; Ye et al. 2011). The KSHV genome encodes more than 90 open reading frames (ORFs) and at least 25 mature microRNAs (Ziegelbauer 2011). The lytic genes are classified into three major groups based on expression kinetics, as follows: immediate early (IE) genes, early genes, and late genes. The IE genes are expressed immediately after primary infection or upon reactivation from latency. The IE genes encode for proteins with regulatory functions in activating the cascade of downstream genes and initiating viral transcription. After the IE genes, but before viral DNA synthesis, early genes are made, including K1 (Bowser et al. 2006), viral interferon factor-1 (Chen et al. 2000), ORF57 (Han and Swaminathan 2006), polyadenylated nuclear (PAN) RNA (Song et al. 2002), and viral interleukin-6 (vIL-6) (Deng et al. 2002). Late genes, such as K8.1 and ORF65, usually do not appear until 30 h after induction (Sarid et al. 1998; Sun et al. 1999).
Folia Microbiol
ORF50, which encodes replication and transcription activator (RTA), is the best-characterized IE gene of KSHV, and is necessary and sufficient to drive KSHV lytic replication and the production of viral progeny (Greene et al. 2007). As the first expressed lytic gene, RTA is followed by early genes and late genes. After the expression of viral lytic genes, viral DNA replication, capsid packaging, and virion release completes the viral lytic replication cycle (Ye et al. 2011). RTA can autoactivate its own promoter and transactivate downstream lytic genes (Deng et al. 2000). Moreover, as a transcriptional activator, RTA not only activates promoters by binding DNA directly, but also interacts with cellular proteins, such as octamer-1 (Carroll et al. 2007), KSHV RTA binding protein (Wang et al. 2001b), and RBP-Jκ (Liang et al. 2002). Therefore, many studies indicate that RTA plays a central role in the physiologic mechanisms controlling reactivation of KSHV from latency. To further investigate the biochemical and biological functions, the rabbit polyclonal antibody against KSHV lytic gene encoding protein RTA were generated and evaluated in this study. These results suggest that the prepared antibody will be useful for studying the role of RTA on KSHV infection and pathogenesis.
Materials and methods Cells culture, transfection, and animals EA.hy926, BJAB, and PEL cell lines, including BCBL-1 and JSC-1 cells, were cultured in RPMI-1640 containing 10 % heat-inactivated fetal bovine serum (FBS), 2 mmol/L L-glutamine, 100 U/mL of penicillin, and 100 μg/mL of streptomycin at 37 °C in a humidified, 5 % CO2 atmosphere. The human embryonic kidney fibroblast cell line, 293 T, was maintained in Dulbecco's modified Eagle's medium supplemented with 10 % FBS. Transfections were conducted with LipofectamineTM 2000 (Invitrogen, NY, USA) according to the manufacturer’s instructions. Male New Zealand white rabbits (3 months old, weighing 3 kg) were purchased from BaiQi Biotechnology (Suzhou, China). The protocol was approved by the Animal Care and Ethical Committee of Nanjing Medical University. All procedures were in accordance with the guidelines of the Institute for Laboratory Animal Research of the Nanjing Medical University. Plasmid construction The expression vector, p3FlagBsd, which fused with a triple FLAG epitope on the N-terminal of pcDNA3.1, was kindly provided by Dr. Shou-Jiang Gao (University of Southern California, Los Angeles, CA, USA) (Chen 2006). The fragment
of KSHV RTA (NCBI Reference Sequence: YP_001129401) was amplified from total BCBL-1 cDNA by polymerase chain reaction (PCR) with the following primers, which contained the Nhe I and Xho I restriction sites (underlined): 5′-CTA GCT AGC TAG GCC ACC ATG GCG CAA GAT GAC AAG GGT A-3′ (sense); and 5′-CCG CTC GAG TAG GGT TTC TTA CGC CGG CAT-3′ (anti-sense). The amplified PCR fragment was inserted into p3FlagBsd and sequenced using a commercial service (Invitrogen Biotechnology Company, Shanghai, China). Finally, following transfection 293 T and EA.hy926 cells with pRTA-Flag, RTA-Flag fusion protein was detected by Western blotting. Polypeptides composition and polyclonal antibodies production SUMOplot software was used to analyze the sequences of RTA on the basis of a series of parameters, including antigen hydrophilicity, N-mer, beta-turn, flexibility, and accessibility (Xue et al. 2006). Combined with the hydrophilic prediction by ProtScale, three RTA polypeptides coupled with keyhole limpet hemocyanin (KLH) were separately designed. Then, the designed polypeptides were synthesized and purified by reverse-phase high-performance liquid chromatography using a commercial service (BaiQi Biotechnology, Suzhou, China). Anti-recombinant RTA rabbit serum was then obtained from six adult male New Zealand white rabbits immunized with 400 μg/animal of purified recombinant KLH-conjugated polypeptides emulsified in complete Freund’s adjuvant (Sigma-Aldrich, MO, USA) by subcutaneous injection on the dorsum (each polypeptide was used to immunize two rabbits, e.g., RB1A and RB1B were immunized with polypeptide no. 1). Each rabbit was booster-immunized with 200 μg of RTA-KLH polypeptides emulsified in incomplete Freund’s adjuvant by subcutaneous injection every 2 weeks. Blood was obtained from ear marginal veins of each rabbit to prepare non-immune serum before immunization. After four booster immunizations, about 80–100 mL blood was harvested again by carotid artery, and then 30–40 mL antiserum was taken apart for final collection. The immunization efficiency was subsequently analyzed by enzyme-linked immunosorbent assays (ELISAs) and Western blottings, and serum samples were stored at −80 °C. Protein purification Anti-RTA antisera was purified with caprylic acid-ammonium sulfate precipitation, according to previously published protocols (Ruan et al. 2005; Yang et al. 2012). One volume of antiserum was diluted with four volumes of 0.06 mol/L acetic acid buffer (17.4 mol/L glacial acetic acid was diluted to 0.06 mol/L with distilled water) and adjusted to pH 4.5 with 5 mol/L NaOH. Caprylic acid was added drop-by-drop at
Folia Microbiol
room temperature to a final concentration of 25 μL caprylic acid per milliliter sample. After vigorous mixing for 30 min, the sample was centrifuged (10,000×g for 10 min at 4 °C) to remove floaters and the precipitate. The supernatant was filtered through filter paper three times. The solution was then adjusted to pH 7.4 and kept in an ice bath for 10 min. Solid ammonium sulfate (0.277 g per mL) was added to the solution. After stirring for 30 min at 4 °C, the solution was centrifuged (10,000×g for 10 min). The supernatant was discarded. The precipitate was dissolved in phosphate-buffered saline (PBS) and dialyzed against PBS until no NH4+ was present. Finally, the purity of the antibody was monitored by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE).
ELISA Indirect ELISA was performed to determine the titer of anti-RTA. Briefly, the purified RTA protein was diluted to 1 mg/L in 50 mmol/L carbonate-bicarbonate buffer (pH 9.6) and coated on plates at 100 μL per well in 96-well immunoplates at 4 °C overnight. The wells were washed three times with PBS buffer. The coated wells were blocked with 100 μL of 10 % fetal calf serum for 2 h at 37 °C, then incubated with 100 μL of anti-RTA with serial 1:2 dilutions (normal serum was the negative control). After incubation for 1 h at 37 °C, the wells were washed and incubated with 100 μL of horseradish peroxidase (HRP)-conjugated goat antirabbit IgG (dilution, 1:5,000; Santa Cruz Biotechnology, Inc., CA, USA) for 1 h at 37 °C. The wells were then washed five times with PBS buffer and incubated with 100 μL of enzyme substrates (tetramethyl benzidine) for 30 min at 37 °C. Color development was then stopped, and the absorbance was measured at 450 nm using a microplate reader. The relational reagents of ELISA assay were purchased from TIANGEN Biotechnology. Fig. 1 Amplification of KSHV RTA and construction of recombination plasmid pRTAFlag. a Amplification of the fragment of RTA by PCR. b PCR amplification of recombinant plasmid pRTA-Flag
Western blotting The 293 T and EA.hy926 cell lysates, which were transfected with pRTA-Flag for 48 h, or BJAB, BCBL-1, and JSC-1 cell lysates were separated by 10 % SDS-PAGE, transferred to an Immobilon P (polyvinylidene difluoride) membrane, and blocked with 5 % powdered milk in TBST (50 mmol/L Tris, pH 7.5, 150 mmol/L NaCl, 0.01 % Tween 20). The membrane was then incubated with primary antibodies diluted in 2.5 % powdered milk in TBST, washed extensively, and incubated with HRP-conjugated species-specific secondary antibodies (Santa Cruz Biotechnology, Inc). The primary antibodies used in Western blotting were the prepared antibodies, anti-Flag M2 rabbit monoclonal antibody (MAb) from Cell Signaling Technology (Beverly, MA, USA), anti-vIL-6 rabbit polyclonal antibody (PAb) from Advanced Biotechnologies, Inc. (Columbia, MD, USA), and anti-GAPDH mouse MAb from Santa Cruz Biotechnology, Inc. Proteins were visualized with enhanced chemiluminescence reagents (Cell Signaling Technology), according to the manufacturer’s instructions. The data shown were repeated at least three times to confirm the results. Immunofluorescence assay (IFA) BJAB cells and BCBL-1 cells treated with or without 12-Otetradecanoylphorbol-13-acetate (TPA; 20 ng/mL, Sigma, USA) for 48 h (107/mL) were washed and smeared on chamber slides. Slides were incubated with anti-RTA antibody from RB3A rabbit immunized with polypeptide no. 3 or antiKSHV ORF59 (clone 11D1; IgG2b) mouse MAb from Advanced Biotechnologies, Inc. (Columbia, MD, USA) at 1:100 dilution. Alexa Fluor 647 (Invitrogen)-conjugated goat antirabbit antibody (1:200 dilution) was used as a secondary antibody for detection of specific signal. The cells were counterstained with 4’, 6’-diamidino-2-phenylindole (DAPI). Images were observed and recorded with a Zeiss Axiovert 200 M epifluorescence microscope (Carl Zeiss, Inc.).
Folia Microbiol
Fig. 2 Detection of recombination RTA protein in 293 T and EA.hy926 cells with the anti-Flag antibody. The expression of the recombinant RTA protein in 293 T (a) or EA.hy926 (b) cells transfected with pRTA-Flag or empty vector was detected with the anti-Flag antibody in Western blotting
Results Amplification of KSHV RTA and construction of recombination plasmid pRTA-Flag The RTA fragment was cloned by PCR amplification and confirmed with 1.0 % agarose gel (w/v) electrophoresis by detection of the expected 3,538 bp fragment (Fig. 1a). A fragment consistent with RTA was inserted between the Nhe I and Xho I sites of the expression vector, p3FlagBsd. After verification by DNA sequencing, recombinant plasmid pRTA-Flag showed an identical sequence to that previously registered in GenBank (accession number YP_001129401; data not shown; Fig. 1b). Expression of recombination RTA protein in 293 T and EA.hy926 cells Western blotting was performed to detect the expression of full-length RTA protein with anti-Flag as the primary antibody. 293 T and EA.hy926 cells were transfected with pRTA-Flag for 48 h, and the lysates of cells were detected by Western blotting. A single band at about 110 kDa was detected with anti-Flag, which stood for RTA-Flag fusion protein (Fig. 2a and b). Preparation, purification, and identification of RTA-KLH fusion protein On the basis of a series of parameters, we separately designed three antigenic polypeptides of KSHV RTA (Table 1), and then RTA protein was prepared and purified according to a routine protocol. Polyclonal antibodies against RTA were generated in six adult male New Zealand white rabbits by immunization with purified recombinant RTA-KLH polypeptides Table 1
(anti-RTA). Next, according to the normal protocol, the antiRTA IgG was purified with caprylic acid–ammonium sulfate precipitation and the purity of the prepared antibodies was monitored by SDS-PAGE to observe the expected RTA immunoglobulin heavy and light chains without irrelevant bands (data not shown). Detection of the RTA antisera titer by ELISA The polyclonal antibodies against full-length of RTA were generated by immunizing rabbits with RTA-KLH (anti-RTAKLH antisera). With indirect ELISA, the anti-RTA antisera were assayed at different dilutions ranged from 1:1,000–1:8, 000 for reactivity with RTA or the control proteins (Table 2). The titers of anti-RTA antisera were determined to be 1:11,000. Detection of full-length RTA protein by prepared antibodies To determine whether the anti-RTA antibodies detect fulllength RTA, the whole cell lysates of 293 T cells transfected with pRTA-Flag were subjected to SDS-PAGE. Only the prepared antibodies from RB3A and RB3B immunized with polypeptide no. 3 specifically recognized the full-length RTA expressed in transfected 293 T cells (Fig. 3a–f). PEL cells, including BCBL-1 (KSHV+ EBV−) and JSC-1 (KSHV+ EBV+) cells were used to determine whether the prepared antibodies were suitable for RTA detection, while the human B cell lymphoma BJAB cells (KSHV− EBV−) were acted as the negative control. The anti-RTA antibody from RB3A was preferred for realistic applications because of the superior performance, as shown in Fig. 3. Western blotting revealed that the anti-RTA antibody from RB3A clearly detected full-length RTA protein in JSC-1 and BCBL-1 cells
Epitope candidates designed for RTA (no. 1–3)
Sequence no.
Epitope
Len
Start
End
Rabbit no.
1 2 3
NH2-AQDDKGKKLRRSC-CONH2 NH2-KKRKALTVPEADT-CONH2 NH2-ATPANEVQESGTLYQLHQWRNYFRD-CONH2
13 13 25
2 527 667
14 539 691
RB1A-B RB2A-B RB3A-B
Folia Microbiol Table 2 Detection of antibody titer in serum by indirect ELISA No.
Antigen
Blank
Negative 1:1,000
Negative 1:4,000
Positive 1:1,000
Positive 1:4,000
Positive 1:8,000
RB1A RB1B RB2A RB2B RB3A RB3B
1 1 2 2 3 3
0.079 0.080 0.082 0.090 0.083 0.093
0.085 0.109 0.132 0.166 0.159 0.168
0.080 0.088 0.091 0.103 0.094 0.110
2.561 3.528 3.741 3.892 3.485 3.560
1.288 1.564 1.965 2.141 1.613 1.823
0.612 0.996 1.124 0.870 1.092 1.057
Blank normal serum collected from each rabbit before immunization; Negative immunized serum collected from each rabbit when A450 value1.0 with 1:4,000 dilution.
after treatment with TPA for 48 h, but not in BJAB cells (Fig. 4). Furthermore, the expression of KSHV RTA was found since 6 h after TPA treatment in BCBL-1 cells and gradually increased with time going, but not in BJAB cells (Fig. 5). Simultaneously, the commercially anti-KSHV vIL-6 rabbit polyclonal antibody was used as the positive control (Figs. 4 and 5). To determine the most appropriate application concentration, different dilution rate of anti-RTA antibody from RB3Awas used in Western blottings. As shown in Figs. 4 and 5, the prepared antibody could react specifically with RTA at dilution of 1:2,000 and 1:1,000. Moreover, IFA was performed to evaluate whether the antiRTA antibody could recognize the native protein in the KSHV-infected PEL cells. We found that native RTA was obviously detected by anti-RTA antibody from RB3A in BCBL-1 cells treated without or with TPA for 48 h, but not
in KSHV-negative BJAB cells (Fig. 6a). The commercially anti-KSHV ORF59 mouse monoclonal antibody was also used as the positive control in IFA (Fig. 6b).
Fig. 3 Detection of full-length RTA protein in 293 T cells with the prepared antibodies. a, b Recombinant KSHV RTA protein was detected with anti-RTA antibodies from RB1A and RB1B (rabbits immunized with polypeptide no. 1); c, d Recombinant KSHV RTA protein was
detected with anti-RTA antibodies from RB2A and RB2B (rabbits immunized with polypeptide no. 2); e, f Recombinant KSHV RTA protein was detected with anti-RTA antibodies from RB3A and RB3B (rabbits immunized with polypeptide no. 3)
Discussion KSHV infection undergoes latent and lytic phases, and viral lytic replication plays a critical role in the process of KSHV pathogenesis. The switch from latent to lytic gene expression is governed by an immediate–early gene product (RTA), which is encoded by ORF50. The RTA protein consists of 691 amino acids and is predicted to have a molecular mass of 73.7 kDa; however, the expressed protein, when analyzed by Western blotting, appears to be approximately 110 kDa, which may due to post-translation modification,
Folia Microbiol
Fig. 4 Detection of RTA in PEL cell lines with prepared antibody. BJAB cells and PEL cell lines, including BCBL-1 and JSC-1 cells, were collected at 48 h after treatment with or without TPA (20 ng/mL), and total proteins were analyzed by Western blotting with anti-RTA antibody from RB3A rabbit immunized with polypeptide no. 3 (1:2,000 or 1:1,000 dilution) or anti-KSHV vIL-6 rabbit polyclonal antibody. M: protein ladder (#SM0671, Fermentas)
phophorylation, or other mechanisms (Lukac et al. 1999). The RTA protein contains an N-terminal DNA-binding domain, a central dimerization domain, a C-terminal acidic activation domain, and two nuclear localization signals (Chen et al. 2000; Lukac et al. 1999). The DNA-binding domain of RTA is located at the amino terminus (aa 1–530), while the activation domain is located at the carboxyl terminus of the protein (aa 486–691) (Lukac et al. 1999). The activation domain also contains four repeated units of a highly hydrophobic domain, known as activation domains 1-4 (AD1-AD4) (Lukac et al. 1999; Wang et al. 2001a), with sequence homology to other transcriptional factors. These features suggest that RTA is a member of transcriptional factors. RTA is necessary and sufficient to trigger the KSHV lytic cascade and the production of viral progeny. RTA has been shown to autoregulate its own expression and transactivate a multitude of downstream viral genes, including PAN (Song et al. 2002), ORF57 (Han and Swaminathan 2006), ORF21 (Zhang et al. 1998), ORF59 (Liu et al. 2008), ORF73 (Staudt and Dittmer 2006), ORF74 (Jeong et al. 2001), K1 (Bowser et al. 2006), K2 (Deng et al. 2002), K5 (St Swierzko et al.
Fig. 5 Detection of the dynamic expression of native RTA in BCBL-1 cells with prepared antibody. BCBL-1 cells were collected at 6, 24, or 48 h after treatment with or without TPA (20 ng/mL), while BJAB cells were also collected at 6 h after treatment with or without TPA. Total proteins were analyzed by Western blotting with anti-RTA antibody from RB3A rabbit immunized with polypeptide no. 3 (1:2,000 or 1:1,000 dilution) or anti-KSHV vIL-6 rabbit polyclonal antibody. M: protein ladder (#SM0671, Fermentas)
2000), K8/K-bZIP (Byun et al. 2002), K9 (Ueda et al. 2002), K12 (Chang et al. 2002), and K15 (Wong and Damania 2006). The mechanism of RTA transactivation is either by direct recognition of RTA response elements in the promoters of the targets genes or interaction with cellular and/or viral co-factors bound to the promoters (Chang et al. 2005; Song et al. 2003). Although RTA is a significant lytic protein of KSHV, previous studies usually used Tag-antibody to detect the recombination protein or homemade antibody to detect the native protein without the detailed information (Chen et al. 2000; Lin et al. 2011). For instance, H. Katano et al. developed a rabbit anti-RTA protein polyclonal antibody to react with the RTA protein in HHV-8–infected cells lines, while the expression vector produced almost the full-length of the RTA protein (631 amino acids) as a fusion protein (Katano et al. 2001), and Gary Hayward et al. also generated the rabbit antiserum against a synthetic peptide representing the KSHV RTA segment between amino acids 527 and 539 (Wang et al. 2003). In comparison with these studies, we prepared and purified rabbit polyclonal antibody against the partial activation domain of RTA (aa 667–691), which may get more pure protein for antibody production, make less obstruction on binding to DNA comparing with other viral or cellular factors, and finally detect the native viral protein successfully (Qin et al. 2010). In the current study, we generated anti-RTA antibodies (RA1A, RA1B, RA2A, RA2B, RA3A, and RA3B) from six
Folia Microbiol Fig. 6 Detection of native RTA in BCBL-1 and BJAB cells with prepared antibody by IFA. BJAB cells and BCBL-1 cells treated with or without TPA (20 ng/mL) for 48 h were examined by IFA with anti-RTA antibody from RB3A rabbit immunized with polypeptide no. 3 (a) or anti-ORF59 mouse monoclonal antibody (b). DAPI (blue) stains nuclei (original magnifications, ×10)
rabbits immunized with three different synthesized antigen peptides coupled with KLH. Western blotting revealed that RA3A and RA3B anti-RTA antibodies from rabbits immunized with no. 3 antigen peptides detected the 110 kDa fulllength RTA protein expressed in 293 T cells. Unfortunately, the remaining antibodies did not recognize the recombination
RTA protein expressed in 293 T cells. The possible explanation for this observation is that the algorithms for structure prediction are poor for immunogenicity. Although the selection of peptides on the basis of the algorithms may be an obvious choice for identification of potential peptides (Ponomarenko and Bourne 2007), the algorithms for structure
Folia Microbiol
prediction were originally designed to determine antigenicity and the proposed sequences may not be immunogenic (Trier et al. 2012). Also, if the peptide template is located internally, the peptide antibody will most likely not be able to interact with the corresponding protein because the peptide is buried in the structure. Another possible explanation is due to the impact of the FLAG-tag on the three-dimensional conformation of recombination protein (Bucher et al. 2002). The binding affinity of most antibodies is influenced by conformational determinants, and antibodies may not bind the same protein in a denatured state (Lipman et al. 2005). The switch from latent to lytic viral replication of KSHV is initiated by an abundance of stimuli that induce the expression of RTA, such as TPA, which cause the demethylation of RTA promoter (Yu et al. 1999). The expression of RTA precedes the expression of all other cycloheximide-resistant IE genes and cycloheximide-sensitive early genes (Sun et al. 1999). Using the prepared antibody, our study confirmed that the expression of RTA strikingly increased in BCBL-1 and JSC-1 cells after TPA treatment for 48 h. In addition, the expression of RTA was markedly raised in BCBL-1 cells from 6 to 24 h following TPA induction. The corresponding results indicated that the produced antibody was able to detect the veritable dynamic expression of this KSHV lytic gene, RTA. In summary, the rabbit polyclonal antibody with specificity for KSHV lytic gene RTA were generated in this study to reveal its expression pattern and biochemical properties, which is functional at least in three different assays: Western blottings, IFA, and ELISA. The other usage of the antibody remains to be identified in future. Because RTA is a critical protein of the KSHV lytic cycle, the prepared antibody may provide a preparative work for the investigation of biological functions of RTA on KSHV infection and pathogenesis. (To facilitate statistics of antibody titer and calculation for practical application, the prepared antibodies were diluted to the final concentration of 1 mg/mL). Acknowledgments We are grateful to Dr. Shou-Jiang Gao (University of Southern California) for generously providing plasmid. This work was supported by grants from the Natural Science Youth Foundation of Jiangsu Province (BK20140908) and Science Development Foundation of Nanjing Medical University (2013NJMU007). Conflict of interest disclosure The authors declare no competing financial interest.
References Boshoff C, Chang Y (2001) Kaposi’s sarcoma-associated herpesvirus: a new DNA tumor virus. Annu Rev Med 52:453–470 Bowser BS, Morris S, Song MJ, Sun R, Damania B (2006) Characterization of Kaposi’s sarcoma-associated herpesvirus (KSHV) K1 promoter activation by Rta. Virology 348:309–327
Bucher MH, Evdokimov AG, Waugh DS (2002) Differential effects of short affinity tags on the crystallization of Pyrococcus furiosus maltodextrin-binding protein. Acta Crystallogr D Biol Crystallogr 58:392–397 Byun H, Gwack Y, Hwang S, Choe J (2002) Kaposi’s sarcoma-associated herpesvirus open reading frame (ORF) 50 transactivates K8 and ORF57 promoters via heterogeneous response elements. Mol Cell 14:185–191 Carroll KD, Khadim F, Spadavecchia S, Palmeri D, Lukac DM (2007) Direct interactions of Kaposi’s sarcoma-associated herpesvirus/ human herpesvirus 8 ORF50/Rta protein with the cellular protein octamer-1 and DNA are critical for specifying transactivation of a delayed–early promoter and stimulating viral reactivation. J Virol 81:8451–8467 Chang H, Dittmer DP, Shin YC, Hong Y, Jung JU (2005) Role of Notch signal transduction in Kaposi’s sarcoma-associated herpesvirus gene expression. J Virol 79:14371–14382 Chang PJ, Shedd D, Gradoville L, Cho MS, Chen LW, Chang J, Miller G (2002) Open reading frame 50 protein of Kaposi’s sarcomaassociated herpesvirus directly activates the viral PAN and K12 genes by binding to related response elements. J Virol 76:3168– 3178 Chang Y, Cesarman E, Pessin MS, Lee F, Culpepper J, Knowles DM, Moore PS (1994) Identification of herpesvirus-like DNA sequences in AIDS-associated Kaposi’s sarcoma. Science 266:1865–1869 Chen J (2006) Serial analysis of binding elements for human transcription factors. Nat Protoc 1:1481–1493 Chen J, Ueda K, Sakakibara S, Okuno T, Yamanishi K (2000) Transcriptional regulation of the Kaposi’s sarcoma-associated herpesvirus viral interferon regulatory factor gene. J Virol 74:8623– 8634 Deng H, Song MJ, Chu JT, Sun R (2002) Transcriptional regulation of the interleukin-6 gene of human herpesvirus 8 (Kaposi’s sarcomaassociated herpesvirus). J Virol 76:8252–8264 Deng H, Young A, Sun R (2000) Auto-activation of the Rta gene of human herpesvirus-8/Kaposi’s sarcoma-associated herpesvirus. J Gen Virol 81:3043–3048 Greene W, Kuhne K, Ye F, Chen J, Zhou F, Lei X, Gao SJ (2007) Molecular biology of KSHV in relation to AIDS-associated oncogenesis. Cancer Treat Res 133:69–127 Han Z, Swaminathan S (2006) Kaposi’s sarcoma-associated herpesvirus lytic gene ORF57 is essential for infectious virion production. J Virol 80:5251–5260 Jeong J, Papin J, Dittmer D (2001) Differential regulation of the overlapping Kaposi’s sarcoma-associated herpesvirus vGCR (orf74) and LANA (orf73) promoters. J Virol 75:1798–1807 Katano H, Sato Y, Itoh H, Sata T (2001) Expression of human herpesvirus 8 (HHV-8)-encoded immediate early protein, open reading frame 50, in HHV-8-associated diseases. J Hum Virol 4:96–102 Lee HR, Brulois K, Wong L, Jung JU (2012) Modulation of immune system by Kaposi’s sarcoma-associated herpesvirus: lessons from viral evasion strategies. Front Microbiol 3:44 Lee HR, Lee S, Chaudhary PM, Gill P, Jung JU (2010) Immune evasion by Kaposi’s sarcoma-associated herpesvirus. Future Microbiol 5: 1349–1365 Liang Y, Chang J, Lynch SJ, Lukac DM, Ganem D (2002) The lytic switch protein of KSHV activates gene expression via functional interaction with RBP-Jkappa (CSL), the target of the Notch signaling pathway. Genes Dev 16:1977–1989 Lin X, Liang D, He Z, Deng Q, Robertson ES, Lan K (2011) miR-K12-75p encoded by Kaposi’s sarcoma-associated herpesvirus stabilizes the latent state by targeting viral ORF50/RTA. PLoS ONE 6:e16224 Lipman NS, Jackson LR, Trudel LJ, Weis-Garcia F (2005) Monoclonal versus polyclonal antibodies: distinguishing characteristics, applications, and information resources. ILAR J 46:258–268
Folia Microbiol Liu Y, Cao Y, Liang D, Gao Y, Xia T, Robertson ES, Lan K (2008) Kaposi’s sarcoma-associated herpesvirus RTA activates the processivity factor ORF59 through interaction with RBP-Jkappa and a cis-acting RTA responsive element. Virology 380:264–275 Lukac DM, Kirshner JR, Ganem D (1999) Transcriptional activation by the product of open reading frame 50 of Kaposi’s sarcomaassociated herpesvirus is required for lytic viral reactivation in B cells. J Virol 73:9348–9361 Mercader M, Taddeo B, Panella JR, Chandran B, Nickoloff BJ, Foreman KE (2000) Induction of HHV-8 lytic cycle replication by inflammatory cytokines produced by HIV-1-infected T cells. Am J Pathol 156: 1961–1971 Ponomarenko JV, Bourne PE (2007) Antibody–protein interactions: benchmark datasets and prediction tools evaluation. BMC Struct Biol 7:64 Qin Y, Liu Z, Zhang T, Wang Y, Li X, Wang J (2010) Generation and application of polyclonal antibody against replication and transcription activator of Kaposi’s sarcoma-associated herpesvirus. Appl Biochem Biotechnol 160:1217–1226 Ruan GP, Ma L, He XW, Meng MJ, Zhu Y, Zhou MQ, Hu ZM, Wang XN (2005) Efficient production, purification, and application of egg yolk antibodies against human HLA-A*0201 heavy chain and light chain (beta2m). Protein Expr Purif 44:45–51 Sarid R, Flore O, Bohenzky RA, Chang Y, Moore PS (1998) Transcription mapping of the Kaposi’s sarcoma-associated herpesvirus (human herpesvirus 8) genome in a body cavity-based lymphoma cell line (BC-1). J Virol 72:1005–1012 Song MJ, Deng H, Sun R (2003) Comparative study of regulation of RTA-responsive genes in Kaposi’s sarcoma-associated herpesvirus/ human herpesvirus 8. J Virol 77:9451–9462 Song MJ, Li X, Brown HJ, Sun R (2002) Characterization of interactions between RTA and the promoter of polyadenylated nuclear RNA in Kaposi’s sarcoma-associated herpesvirus/human herpesvirus 8. J Virol 76:5000–5013 St Swierzko A, Kirikae T, Kirikae F, Hirata M, Cedzynski M, Ziolkowski A, Hirai Y, Kusumoto S, Yokochi T, Nakano M (2000) Biological activities of lipopolysaccharides of Proteus spp. and their interactions with polymyxin B and an 18-kDa cationic antimicrobial protein (CAP18)-derived peptide. J Med Microbiol 49:127–138 Staudt MR, Dittmer DP (2006) Promoter switching allows simultaneous transcription of LANA and K14/vGPCR of Kaposi’s sarcomaassociated herpesvirus. Virology 350:192–205
Sun R, Lin SF, Staskus K, Gradoville L, Grogan E, Haase A, Miller G (1999) Kinetics of Kaposi’s sarcoma-associated herpesvirus gene expression. J Virol 73:2232–2242 Trier NH, Hansen PR, Houen G (2012) Production and characterization of peptide antibodies. Methods 56:136–144 Ueda K, Ishikawa K, Nishimura K, Sakakibara S, Do E, Yamanishi K (2002) Kaposi’s sarcoma-associated herpesvirus (human herpesvirus 8) replication and transcription factor activates the K9 (vIRF) gene through two distinct cis elements by a non-DNA-binding mechanism. J Virol 76:12044–12054 Wang S, Liu S, Wu M, Geng Y, Wood C (2001a) Kaposi’s sarcomaassociated herpesvirus/human herpesvirus-8 ORF50 gene product contains a potent C-terminal activation domain which activates gene expression via a specific target sequence. Arch Virol 146: 1415–1426 Wang S, Liu S, Wu MH, Geng Y, Wood C (2001b) Identification of a cellular protein that interacts and synergizes with the RTA (ORF50) protein of Kaposi’s sarcoma-associated herpesvirus in transcriptional activation. J Virol 75:11961–11973 Wang SE, Wu FY, Yu Y, Hayward GS (2003) CCAAT/enhancer-binding protein-alpha is induced during the early stages of Kaposi’s sarcoma-associated herpesvirus (KSHV) lytic cycle reactivation and together with the KSHV replication and transcription activator (RTA) cooperatively stimulates the viral RTA, MTA, and PAN promoters. J Virol 77(17):9590–9612 Wong EL, Damania B (2006) Transcriptional regulation of the Kaposi’s sarcoma-associated herpesvirus K15 gene. J Virol 80:1385–1392 Xue Y, Zhou F, Fu C, Xu Y, Yao X (2006) SUMOsp: a web server for sumoylation site prediction. Nucleic Acids Res 34:W254–W257 Yang Y, Wang B, Yang D, Lu M, Xu Y (2012) Prokaryotic expression of woodchuck cytotoxic T lymphocyte antigen 4 (wCTLA-4) and preparation of polyclonal antibody to wCTLA-4. Protein Expr Purif 81:181–185 Ye F, Lei X, Gao SJ (2011) Mechanisms of Kaposi’s sarcoma-associated herpesvirus latency and reactivation. Adv Virol 2011 Yu Y, Black JB, Goldsmith CS, Browning PJ, Bhalla K, Offermann MK (1999) Induction of human herpesvirus-8 DNA replication and transcription by butyrate and TPA in BCBL-1 cells. J Gen Virol 80(Pt 1):83–90 Zhang L, Chiu J, Lin JC (1998) Activation of human herpesvirus 8 (HHV-8) thymidine kinase (TK) TATAA-less promoter by HHV-8 ORF50 gene product is SP1 dependent. DNA Cell Biol 17:735–742 Ziegelbauer JM (2011) Functions of Kaposi’s sarcoma-associated herpesvirus microRNAs. Biochim Biophys Acta 1809:623–630
