- Open Access
Molecular interplay between T-Antigen and splicing factor, arginine/serine-rich 1 (SRSF1) controls JC virus gene expression in glial cells
© Craigie et al. 2015
- Received: 10 August 2015
- Accepted: 15 November 2015
- Published: 24 November 2015
Human polyomavirus JCV is the etiologic agent of progressive multifocal leukoencephalopathy (PML), a fatal demyelinating disease characterized by lytic infection of glial cells in the central nervous system. PML is seen primarily in immunosuppressed patients and is mainly classified as an AIDS-defining disease. In addition to structural capsid proteins, JCV encodes multiple regulatory proteins, including T-antigen and agnoprotein, which are required for functional lytic infection. Previous studies have suggested that molecular interaction between viral proteins and host factors play an important role in reactivation of JCV and progression of the viral life cycle in glial cells. Recently, serine/arginine rich splicing factor 1 (SRSF1), a cellular alternative splicing factor, was identified as a strong negative regulator of JCV in glial cells. SRSF1 inhibits JCV gene expression and viral replication by directly interacting with viral promoter sequences. Here, we have investigated possible impact of JCV regulatory proteins, T-antigen and agnoprotein, on SRSF1-mediated suppression of JCV gene expression in glial cells.
Reporter gene analysis has suggested that T-antigen rescues viral transcriptional suppression mediated by SRSF1. Further analyses have revealed that T-antigen promotes viral gene expression by suppressing SRSF1 gene transcription in glial cells. A subsequent ChIP analysis revealed that T-antigen associates with the promoter region of SRSF1 to induce the transcriptional suppression.
These findings have revealed a molecular interplay between cellular SRSF1 and viral T-antigen in controlling JCV gene expression, and may suggest a novel mechanism of JCV reactivation in patients who are at risk of developing PML.
- Progressive Multifocal Leukoencephalopathy
- Progressive Multifocal Leukoencephalopathy
- T98G Cell
- HCMV Infection
- Viral Life Cycle
JC virus (JCV) is a human polyomavirus and the etiological agent of progressive multifocal leukoencephalopathy (PML), a fatal demyelinating disease of the white matter [1–3]. PML is primarily found in AIDS-patients, with between 3 % and 5 % of patients developing PML [4, 5]. However, PML has recently been diagnosed in patients undergoing immunomodulatory therapy with monoclonal antibodies against immune cells, such as Natalizumab, Rituximab, and Efalizumab, which indicates an immune component to the reactivation of the virus from latent reservoirs [6–9]. JCV is a non-enveloped polyomavirus with a genome comprised of 5 kb of double-stranded circular DNA with a bidirectional non-coding control region separating the early and late coding regions . The early coding region of JCV encodes regulatory proteins, T-antigen, small t-antigen, and T’ splice variants expressed upon alternative splicing of the primary viral transcript . The late coding region of JCV encodes the viral capsid proteins, VP1, VP2, and VP3, which are required for the formation of the viral capsid, as well as the small regulatory protein agnoprotein.
Previous studies have demonstrated that JCV undergoes cell-type specific activation, primarily in glial cells, which is proposed to be regulated at the transcriptional level . Cellular proteins, such as SRSF1, play a major role in controlling JCV infection in glial cells . We have recently identified alternative splicing factor SRSF1 as a negative regulator of JCV gene expression and replication in glial cells . SRSF1 functions by targeting the JCV promoter and strongly inhibits JCV early and late gene transcription. Likewise, the down regulation of SRSF1 in astrocytes increases levels of viral gene expression and replication . SRSF1 is a strong negative regulator of JCV gene expression as it suppresses both early and late gene transcription in glial cells [10, 13, 14]. Moreover, SRSF1 interaction with the CR3 region of JCV promoter sequences has been shown to be required for the ability of SRSF1 to regulate JCV gene expression and replication . In order to proceed to a productive replication cycle, JC virus has to initiate the transcription of viral early and late genes by releasing the transcriptional silencing caused by host factors, such as SRSF1.
While cellular factors hold a negative pressure on viral gene expression in infected cells, viruses have evolved and developed strategies to bypass host defense mechanisms by utilizing mainly their regulatory proteins [15–22]. Here we investigated possible role of JCV regulatory proteins, T-antigen and agnoprotein, in regulation of viral transcriptional suppression mediated by SRSF1. Our results suggest that T-antigen but not agnoprotein is able to rescue and initiate viral transcription suppressed by SRSF1. These results have revealed a novel interaction between T-antigen and SRSF1 in controlling JCV gene transcription and replication, which may suggest a unique mechanism of JCV reactivation in patients who are at risk of developing PML.
Effect of JCV regulatory proteins, T-antigen and agnoprotein, on SRSF1-mediated suppression of JCV transcription
T-Antigen inhibits SRSF1 expression in glial cells
T-Antigen targets SRSF1 promoter and inhibits its transcription
Viral life cycles within the body are a fight between the ability of the host to recognize and quarantine or destroy the viruses and the virus’s ability to avoid this detection or avoid the mechanisms used to alter viral function. Therefore, the interplay between viral regulatory proteins and the host defense factors is a major regulator of the viral life cycle. While cellular factors holds a negative pressure on viral gene expression in infected cells, viruses have evolved and developed strategies to bypass host defense mechanisms by utilizing mainly their regulatory proteins [15–22]. One example by which a viral protein interacts with a host anti-viral protein is within the infection process of human cytomegalovirus (HCMV). When HCMV is detected by cells, an interferon gamma response is induced, resulting in the expression of multiple antiviral proteins, including the iron-sulfur cluster-binding antiviral protein viperin [15–17]. During the infection cycle, pre-expression of viperin results in inhibition of HCMV infection, however, the virus also is able to induce viperin expression independently of the IFN-dependent pathway [17, 18]. Although viperin contains antiviral activity, HCMV-induced viperin functions to disrupt cellular metabolism through localization to the mitochondria in infected cells, which results in enhancement of the HCMV infection process [18–22]. HCMV encodes a viral protein, viral mitochondrial inhibitor of apoptosis (vMIA), which interacts with viperin during HCMV infection, thus facilitating viral replication .
Similar to HCMV, JCV encodes viral regulatory proteins which are required for the progression of the lytic viral life cycle. We recently identified that cellular SRSF1 can overcome JCV reactivation by suppressing the expression of the viral genes [10, 13, 14]. Here, we investigated the molecular interplay between SRSF1 and JCV regulatory proteins T-antigen and agnoprotein in regulation of the viral transcription. Our results demonstrate that T-antigen is able to rescue the SRSF1-mediated transcriptional suppression seen in both early and late coding regions of the JCV genome. Likewise, it was found that T-antigen alone significantly increased viral transcription, however, a second viral regulatory protein, agnoprotein, had no effect on JCV transcription. To determine the mechanism behind T-antigen rescue of transcriptional suppression mediated by SRSF1, the impact of T-antigen expression on SRSF1 expression was analyzed. T-antigen expression decreased the expression of SRSF1, thus allowing for increased viral transcription. This step is hypothesized to be highly important within the JCV infection process, as T-antigen acts as a transcription factor for viral transcription and is able to autoregulate the viral promoter to drive the required viral processes for a productive infection cycle: including DNA replication and transcription of the late region allowing for agnoprotein and the capsid proteins to be expressed [23, 24]. The observed suppression of SRSF1 levels within cells expressing T-antigen was then demonstrated to be due to the T-antigen interaction with the SRSF1 promoter region leading to transcriptional inhibition.
In summary, our results demonstrate a novel role of T-antigen in SRSF1-mediated suppression of JCV gene expression and provide basis for the further investigation of the role of the molecular interplay between cellular SRSF1 and viral T-antigen in JCV reactivation in glial cells leading to initiation of productive viral life cycle in patients who are at risk of developing PML.
Cell lines and culture
The human glioblastoma multiforme cell line, T98G, was obtained from American Type Culture Collection (ATCC) and grown in Dulbecco’s Modified Eagle’s Medium (DMEM) containing 5 % heat-inactivated fetal bovine serum (FBS) and penicillin/streptomycin (100 ug/ml). T98G cells were maintained at 37 °C in a humidified environment with 5 % CO2. Primary human fetal astrocytes were cultured from the fetal human brain of a donor and provided by the Comprehensive NeuroAIDS Core facility at Temple University and cultured in Dulbecco’s Modified Eagle’s Medium/Nutrient Mixture F-12 (DMEM/F-12) containing 10 % heat-inactivated fetal bovine serum (FBS) penicillin/streptomycin (100 μg/ml), GlutaMax (100 μg/mL) and insulin (100 μg/mL). Cultured PHFA cells were maintained at 37 °C in a humidified atmosphere with 5 % CO2.
The T-antigen gene was cloned into the eukaryotic expression vector pcDNA3.1 (+) at the EcoRI restriction enzyme site and designated as pcDNA3.1-T-antigen previously . pCGT7-SF2/ASF (SRSF1) expression plasmid was kindly provided by Javier F. Cáceres (Medical Research Council Human Genetics Unit, Western General Hospital, Edinburgh EH4 2XU, Scotland, United Kingdom) and was described previously . The pCGT7-agno plasmid was described previously . Briefly, agnoprotein coding sequence was cloned into the eukaryotic expression plasmid pCGT7 at Xba1/BamH1 restriction enzyme sites and designated as pCGT7-agno. The luciferase reporter construct pLuc-JCV-Early and pLuc-JCV-Late were made by blunt end cloning of the full-length Mad-1 NCCR into the SmaI site immediately upstream of the luciferase gene in the plasmid pGL3 (Promega, Madison WI) and described previously . The luciferase reporter plasmid pLuc-SRSF1 was made by cloning the −1000 to +49 promoter region of SRSF1 gene into pGL3 vector at BamH1 site.
Luciferase reporter assay
T98G cells were plated in 6-well tissue culture dishes and transiently transfected with pLuc-JCV-Early, pLuc-JCV-Late, or pLuc-SRSF1-1000 bp reporter plasmids in the presence or absence of expression plasmids for T-antigen and agnoprotein or pCDNA3.1 as control. At 48 h post-transfection, cells were extracted and lysed using reporter lysis buffer for the luciferase reporter system provided by the manufacturer Promega. After cell lysis, luciferase activity of samples was determined through the use of luciferase assay reagent (LAR). The luciferase activities were then corrected for protein concentrations and normalized to the basal levels of transcription, allowing for determination of the fold changes.
Chromatin Immunoprecipitation Assay (ChIP)
T98G cells were transfected with pCDNA3.1-T-antigen. At 48 h post-transfection, proteins were cross-linked to DNA using formaldehyde at a final concentration of 1 %. After cross-linking, cells were lysed and subject to sonication to fragment the chromatin. After sonication, cells were incubated overnight with Protein G beads and anti-T antigen antibody (pAb-416, Calbiochem) for immunoprecipiation. After immunoprecipiation, the beads were washed and bound proteins were eluted using ChIP elution buffer (1 % SDS, 100 mM NaHCO3). After elution, cross-linking was reversed by using 5 M NaCl followed by Proteinase and RNase treatment. The sample was then purified for DNA using phenol/chloroform extraction followed by ethanol precipitation. Obtained DNA fragments were analyzed by PCR using primers; SRSF1-Promoter-Forward (−1000 to +47): 5’-ACCTTCCAAAGCTTTCCAGATTTCAG-3’ and SRSF1-Promoter-Reverse (+47 to +27): 5’-ACCTTCCACTCGAGGAAGGAAACAGC-3’. The PCR conditions were as follows: denaturing at 95° for 30 s, annealing at 58 °C for 40 s, and extension at 72 °C for 65 s. The PCR products were then resolved on a 1 % agarose gel.
Whole cell protein extracts were washed with PBS and lysed with TNN lysis buffer with protease inhibitors. Purified protein extracts were then heated to 95 °C for 5 min and resolved through sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). After resolution, the gel was transferred to a nitrocellulose membrane (Whatman, Germany) for three hours at 250 mÅ at 4 °C in transfer buffer (25 mM Tris pH 7.4, 200 mM glycine, 20 % methanol). After transfer, membranes were blocked for thirty minutes at room temperature with 5 % non-fat dry milk in 1X phosphate-buffered saline containing 0.1 % Tween-20 (PBST). After blocking, membranes were washed and incubated with primary antibodies at a 1:1000 dilution overnight at 4 °C. After primary antibody incubation, membranes were washed three times in PBST and incubated with secondary antibodies at a 1:5000 dilution at room temperature. Following secondary antibody incubation, membranes were visualized with the Odyssey CLx Imaging System (LI-COR). Primary antibodies used were anti-T-antigen (pAb-416, Calbiochem), anti-SRSF1 (ab12910, Abcam), anti-agnoprotein (Pab7903, house raised), anti-β-Tubulin (LI-COR), and anti-GAPDH (Cell Signaling Technology). Secondary antibodies used were IRDye 800CW goat anti-mouse (LI-COR) and IRDye 680RD goat anti-rabbit (LI-COR).
The authors thank the past and present members of the Department of Neuroscience/Center for Neurovirology for sharing their ideas and reagents, particularly Drs. Kamel Khalili and Jennifer Gordon. Research reported in this publication was supported by the National Institute of Allergy and Infectious Diseases of the National Institutes of Health under Award number R01AI101192. This study utilized services offered by core facilities of the Comprehensive NeuroAIDS Center (CNAC NIMH Grant Number P30MH092177) at Temple University Lewis Katz School of Medicine. The funding organizations played no role in the design of the study, in the collection, analysis, and interpretation of the data and in the decision to submit the manuscript for publication.
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
- Wollebo HS, Woldemichaele B, Khalili K, Safak M, White MK. Epigenetic regulation of polyomavirus JC. Virol J. 2013;23:264–73.View ArticleGoogle Scholar
- Berger JR, Khalili K. The pathogenesis of progressive multifocal leukoencephalopathy. Discov Med. 2011;12:495–503.PubMedGoogle Scholar
- Weber T. Progressive multifocal leukoencephalopathy. Neurol Clin. 2008;26:833–54.View ArticlePubMedGoogle Scholar
- Holman RC, Janssen RS, Buehler JW, Zelasky MT, Hooper WC. Epidemiology of progressive multifocal leukoencephalopathy in the United States: analysis of national mortality and AIDS surveillance data. Neurol. 1991;41:1733–6.View ArticleGoogle Scholar
- San-Andres FJ, Rubio R, Castilla J, Pulido F, Palao G, de Pedro I, et al. Incidence of acquired immunodeficiency syndrome-associated opportunistic diseases and the effect of treatment on a cohort of 1115 patients infected with human immunodeficiency virus. Clin Infect Dis. 2003;36:1177–85.View ArticlePubMedGoogle Scholar
- Sandborn WJ, Colombel JF, Enns R, Feagan BG, Hanauer SB. Natalizumab induction and maintenance therapy for Crohn’s disease: International efficacy of Natalizumab as active Crohn’s therapy, evaluation of natalizumab as continuous therapy trial group. N Engl J Med. 2005;353:1912–25.View ArticlePubMedGoogle Scholar
- Langer-Gould A, Atlas SW, Green AJ, Bollen AW, Pelletier D. Progressive multifocal leukoencephalopathy in a patient treated with natalizumab. N Engl J Med. 2005;353:375–81.View ArticlePubMedGoogle Scholar
- Kleinschimdt-DeMasters BK, Tyler KL. Progressive multifocal leukoencephalopathy complicating treatment with natalizumab and interferon beta-1a for multiple sclerosis. N Engl J Med. 2005;353:369–74.View ArticleGoogle Scholar
- Carson KR, Focosi D, Major EO, Petrini M, Richey EA, West DP, et al. Monoclonal antibody-associated progressive multifocal leukoencephalopathy in patients treated with rituximab, natalizumab, and efalizumab: A review from the research on adverse drug events and reports project. Lancet Oncol. 2009;10:816–24.Google Scholar
- Sariyer IK, Khalili K. Regulation of human neurotropic JC virus replication by alternative splicing factor SF2/ASF in glial cells. PLoS ONE. 2011;6, e14630.PubMed CentralView ArticlePubMedGoogle Scholar
- Trowbridge PW, Frisque RJ. Identification of three new JC virus proteins generated by alternative splicing of the early viral mRNA. Neurovirol. 1995;1:195–206.View ArticleGoogle Scholar
- White MK, Safak M, Khalili K. Regulation of gene expression in primate polyomaviruses. J Virol. 2009;83:10846–56.PubMed CentralView ArticlePubMedGoogle Scholar
- Uleri E, Beltrami S, Gordon J, Dolei A, Sariyer IK. Extinction of tumor antigen expression by SF2/ASF in JCV-transformed cells. Genes and Cancer. 2011;2:728–36.PubMed CentralView ArticlePubMedGoogle Scholar
- Uleri E, Regan P, Dolei A, Sariyer IK. SF2/ASF binding region within JC virus NCCR limits early gene transcription in glial cells. Virol J. 2013;10:147.PubMed CentralView ArticlePubMedGoogle Scholar
- Duschene KS, Broderick JB. The antiviral protein viperin is a radical SAM enzyme. FEBS Lett. 2010;584:1263.PubMed CentralView ArticlePubMedGoogle Scholar
- Shaveta G, Shi J, Chow VT, Song J. Structural characterization reveals that viperin is a radical S-adenosyl-L-methionine (SAM) enzyme. Biochem Biophys Res Commun. 2010;391:1390.View ArticlePubMedGoogle Scholar
- Chin KC, Cresswell P. Viperin, an INF-inducible antiviral protein directly induced by human cytomegalovirus. Proc Natl Acad Sci U S A. 2001;98:15125.PubMed CentralView ArticlePubMedGoogle Scholar
- Seo JY, Yaneva R, Hinson ER, Cresswell P. Human cytomegalovirus directly induces the antiviral protein viperin to enhance infectivity. Science. 2011;332:1093–7.Google Scholar
- Goldmacher VS, Bartle LM, Skaletskaya A, Dionne CA, Kedersha NL, Vater CA, et al. A cytomegalovirus-encoded mitochondria-localized inhibitor of apoptosis structurally unrelated to Bcl-2. Proc Natl Acad Sci U S A. 1999;96:12536.Google Scholar
- Goldmacher VS. vMIA, a viral inhibitor of apoptosis targeting mitochondria. Biochimie. 2002;84:177.View ArticlePubMedGoogle Scholar
- Al-Barazi, H.O., Colberg-Poley, A.M. The human cytomegalovirus UL37 immediate-early regulatory protein is an integral membrane N-glycoprotein which traffics through the endoplasmic reticulum and Golgi apparatus. J Virol. 1996;70:7198–7208.Google Scholar
- Colberg-Poley AM, Patel MB, Erezo DP, Slater JE. Human cytomegalovirus UL37 immediate-early regulatory proteins traffic through the secretory apparatus and to mitochondria. J Gen Virol. 2000;81:1779–89.View ArticlePubMedGoogle Scholar
- Saribas AS, Ozdemir A, Lam C, Safak M. JC virus induced progressive multifocal leukoencephalopathy. Future Virol. 2010;5:313–23.PubMed CentralView ArticlePubMedGoogle Scholar
- Lashgari MS, Tada H, Amini S, Khalili K. Regulation of JCVL promoter function: transactivation of JCVL promoter by JCV and SV40 early proteins. Virol. 1989;170:292–5.View ArticleGoogle Scholar
- Khalili K, Sariyer IK, Safak M. Small tumor antigen of polyomaviruses: role in viral life cycle and cell transformation. J Cell Physiol. 2008;215:309–19.PubMed CentralView ArticlePubMedGoogle Scholar
- Caceres JF, Misteli T, Screaton GR, Spector DL, Krainer AR. Role of the modular domains of SR proteins in subcellular localization and alternative splicing specificity. J Cell Biol. 1997;118:225–38.View ArticleGoogle Scholar
- Otlu O, De Simone F, Lopez-Otalora Y, Khalili K, Sariyer IK. The agnoprotein of Polyomavirus JC is Released by Infected Cells: Evidence for Its Cellular Uptake by Uninfected Neighboring Cells. Virology. 2014;468–470:88–95.View ArticlePubMedGoogle Scholar
- Wollebo HS, Safak M, Del Valle L, Khalili K, White MK. Role for tumor necrosis factor-α in JC virus reactivation and progressive multifocal leukoencephalopathy. J Neuroimmunol. 2011;233(1–2):46–53.PubMed CentralView ArticlePubMedGoogle Scholar