Xiaoping Sun

X-Box-Binding Protein 1 Activates Lytic Epstein-Barr Virus Gene Expression in Combination with Protein Kinase D

ABSTRACT Epstein-Barr virus (EBV) establishes a latent form of infection in memory B cells, while antibody-secreting plasma cells often harbor the lytic form of infection. The switch between latent and lytic EBV infection is mediated by the two viral immediate-early proteins BZLF1 (Z) and BRLF1 (R), which are not expressed in latently infected B cells. Here we demonstrate that a cellular transcription factor that plays an essential role in plasma cell differentiation, X-box-binding protein 1 (XBP-1), also activates the transcription of the two EBV immediate-early gene promoters. In reporter gene assays, XBP-1 alone was sufficient to activate the R promoter, whereas the combination of XBP-1 and protein kinase D (PKD) was required for efficient activation of the Z promoter. Most importantly, the expression of XBP-1 and activated PKD was sufficient to induce lytic viral gene expression in EBV-positive nasopharyngeal carcinoma cells and lymphoblastoid cells, while an XBP-1 small interfering RNA inhibited constitutive lytic EBV gene expression in lymphoblastoid cells. These results suggest that the plasma cell differentiation factor XBP-1, in combination with activated PKD, can mediate the reactivation of EBV, thereby allowing the viral life cycle to be intimately linked to plasma cell differentiation.

Hsp90 inhibitors block outgrowth of EBV-infected malignant cells in vitro and in vivo through an EBNA1-dependent mechanism

EBV causes infectious mononucleosis and is associated with certain malignancies. EBV nuclear antigen 1 (EBNA1) mediates EBV genome replication, partition, and transcription, and is essential for persistence of the viral genome in host cells. Here we demonstrate that Hsp90 inhibitors decrease EBNA1 expression and translation, and that this effect requires the Gly-Ala repeat domain of EBNA1. Hsp90 inhibitors induce the death of established, EBV-transformed lymphoblastoid cell lines at doses nontoxic to normal cells, and this effect is substantially reversed when lymphoblastoid cell lines are stably infected with a retrovirus expressing a functional EBNA1 mutant lacking the Gly-Ala repeats. Hsp90 inhibitors prevent EBV transformation of primary B cells, and strongly inhibit the growth of EBV-induced lymphoproliferative disease in SCID mice. These results suggest that Hsp90 inhibitors may be particularly effective for treating EBV-induced diseases requiring the continued presence of the viral genome.

Hsp90 inhibitor 17-DMAG decreases expression of conserved herpesvirus protein kinases and reduces virus production in Epstein-Barr virus-infected cells

ABSTRACT All eight human herpesviruses have a conserved herpesvirus protein kinase (CHPK) that is important for the lytic phase of the viral life cycle. In this study, we show that heat shock protein 90 (Hsp90) interacts directly with each of the eight CHPKs, and we demonstrate that an Hsp90 inhibitor drug, 17-dimethylaminoethylamino-17-demethoxygeldanamycin (17-DMAG), decreases expression of all eight CHPKs in transfected HeLa cells. 17-DMAG also decreases expression the of the endogenous Epstein-Barr virus protein kinase (EBV PK, encoded by the BGLF4 gene) in lytically infected EBV-positive cells and inhibits phosphorylation of several different known EBV PK target proteins. Furthermore, 17-DMAG treatment abrogates expression of the human cytomegalovirus (HCMV) kinase UL97 in HCMV-infected human fibroblasts. Importantly, 17-DMAG treatment decreased the EBV titer approximately 100-fold in lytically infected AGS-Akata cells without causing significant cellular toxicity during the same time frame. Increased EBV PK expression in 17-DMAG-treated AGS-Akata cells did not restore EBV titers, suggesting that 17-DMAG simultaneously targets multiple viral and/or cellular proteins required for efficient viral replication. These results suggest that Hsp90 inhibitors, including 17-DMAG, may be a promising group of drugs that could have profound antiviral effects on herpesviruses.

Fitness of a Turnip Crinkle Virus Satellite RNA Correlates with a Sequence-Nonspecific Hairpin and Flanking Sequences That Enhance Replication and Repress the Accumulation of Virions

ABSTRACT satC, a satellite RNA associated with Turnip crinkle virus (TCV), enhances the ability of the virus to colonize plants by interfering with stable virion accumulation (F. Zhang and A. E. Simon, unpublished data). Previous results suggested that the motif1-hairpin (M1H), a replication enhancer on minus strands, forms a plus-strand hairpin flanked by CA-rich sequence that may be involved in enhancing systemic infection (G. Zhang and A. E. Simon, J. Mol. Biol. 326:35-48, 2003). In this study, sequence and structural requirements of the M1H were further assayed by replacing the 28-base M1H with 10 random bases and then subjecting the pool of satellite RNA to functional selection in plants. Unlike previous results with 28-base replacement sequences (G. Zhang and A. E. Simon, J. Mol. Biol. 326:35-48, 2003), only a few of the 10-base SELEX (systematic evolution of ligands by exponential enrichment) assay winners contained short motifs in their minus-sense orientation that were similar to TCV replication elements. However, all second- and third-round winning replacement sequences folded into hairpins flanked by CA-rich sequence predicted to be more stable on plus strands than minus strands. Plus strands of several of the most fit satellite RNAs contained insertions of CA-rich sequence at the base of their hairpins whose presence correlated with enhanced replication and reduced detection of virions. Deletion of the M1H resulted in no detectable virions despite very low satellite accumulation. These results support the hypothesis that a sequence-nonspecific plus-strand hairpin brings together flanking CA-rich sequences in the M1H region that confers fitness to satC by reducing the accumulation of stable virions.

Triptolide inhibits transcription of hTERT through down-regulation of transcription factor specificity protein 1 in primary effusion lymphoma cells

Primary effusion lymphoma (PEL) is a rare and aggressive non-Hodgkins lymphoma. Human telomerase reverse transcriptase (hTERT), a key component responsible for the regulation of telomerase activity, plays important roles in cellular immortalization and cancer development. Triptolide purified from Tripterygium extracts displays a broad-spectrum bioactivity profile, including immunosuppressive, anti-inflammatory, and anti-tumor. In this study, it is investigated whether triptolide reduces hTERT expression and suppresses its activity in PEL cells. The mRNA and protein levels of hTERT were examined by real time-PCR and Western blotting, respectively. The activity of hTERT promoter was determined by Dual luciferase reporter assay. Our results demonstrated that triptolide decreased expression of hTERT at both mRNA and protein levels. Further gene sequence analysis indicated that the activity of hTERT promoter was suppressed by triptolide. Triptolide also reduced the half-time of hTERT. Additionally, triptolide inhibited the expression of transcription factor specificity protein 1(Sp1) in PEL cells. Furthermore, knock-down of Sp1 by using specific shRNAs resulted in down-regulation of hTERT transcription and protein expression levels. Inhibition of Sp1 by specific shRNAs enhanced triptolide-induced cell growth inhibition and apoptosis. Collectively, our results demonstrate that the inhibitory effect of triptolide on hTERT transcription is possibly mediated by inhibition of transcription factor Sp1 in PEL cells.

Short Internal Sequences Involved in Replication and Virion Accumulation in a Subviral RNA of Turnip Crinkle Virus

ABSTRACT cis-acting sequences and structural elements in untranslated regions of viral genomes allow viral RNA-dependent RNA polymerases to correctly initiate and transcribe asymmetric levels of plus and minus strands during replication of plus-sense RNA viruses. Such elements include promoters, enhancers, and transcriptional repressors that may require interactions with distal RNA sequences for function. We previously determined that a non-sequence-specific hairpin (M1H) in the interior of a subviral RNA (satC) associated with Turnip crinkle virus is required for fitness and that its function might be to bridge flanking sequences (X. Sun and A. E. Simon, J. Virol. 77:7880-7889, 2003). To establish the importance of the flanking sequences in replication and satC-specific virion repression, segments on both sides of M1H were randomized and subjected to in vivo functional selection (in vivo SELEX). Analyses of winning (functional) sequences revealed three different conserved elements within the segments that could be specifically assigned roles in replication, virion repression, or both. One of these elements was also implicated in the molecular switch that releases the 3′ end from its interaction with the repressor hairpin H5, which is possibly involved in controlling the level of minus-strand synthesis.

Triptolide inhibits proliferation of Epstein-Barr virus-positive B lymphocytes by down-regulating expression of a viral protein LMP1.

Epstein-Barr virus (EBV) infects various types of cells and mainly establishes latent infection in B lymphocytes. The viral latent membrane protein 1 (LMP1) plays important roles in transformation and proliferation of B lymphocytes infected with EBV. Triptolide is a compound of Tripterygium extracts, showing anti-inflammatory, immunosuppressive, and anti-cancer activities. In this study, it is determined whether triptolide inhibits proliferation of Epstein-Barr virus-positive B lymphocytes. The CCK-8 assays were performed to examine cell viabilities of EBV-positive B95-8 and P3HR-1 cells treated by triptolide. The mRNA and protein levels of LMP1 were examined by real time-PCR and Western blotting, respectively. The activities of two LMP1 promoters (ED-L1 and TR-L1) were determined by Dual luciferase reportor assay. The results showed that triptolide inhibited the cell viability of EBV-positive B lymphocytes, and the over-expression of LMP1 attenuated this inhibitory effect. Triptolide decreased the LMP1 expression and transcriptional levels in EBV-positive B cells. The activity of LMP1 promoter ED-L1 in type III latent infection was strongly suppressed by triptolide treatment. In addition, triptolide strongly reduced growth of B95-8 induced B lymphoma in BALB/c nude mice. These results suggest that triptolide decreases proliferation of EBV-induced B lymphocytes possibly by a mechanism related to down-regulation of the LMP1 expression.

Triptolide decreases expression of latency-associated nuclear antigen 1 and reduces viral titers in Kaposi's sarcoma-associated and herpesvirus-related primary effusion lymphoma cells.

Kaposis sarcoma-associated herpesvirus (KSHV) can establish a life-long persistence in the host after primary infection and is associated with certain malignancies, which are resistant to conventional chemotherapeutic agents with a poor prognosis. Latency-associated nuclear antigen 1 (LANA1) encoded by KSHV is essential for segregation, replication and maintenance of viral genome. In addition, LANA1 upregulates the transcriptional activity of signal transducer and activator of transcription 3 (STAT3), which plays an important role in promoting survival of KSHV-associated primary effusion lymphoma (PEL) cells. Furthermore, LANA1 mediates transcriptional modulation of KSHV and host genome in host cells. In the present study, the antitumor effect of triptolide was assessed. CCK-8 assays were performed to demonstrate that the proliferations of PEL cells were efficiently inhibited by triptolide in a dose- and time-dependent manner. Flow cytometric results indicated that triptolide induced cell cycle arrest and apoptosis. Western blot results suggested that triptolide downregulated LANA1 expression and reduced half-life of LANA1 in the KSHV-infected malignant cells. Viral titer experiments indicated that triptolide treatment impaired the number of viral DNA copies and the production of virions in BCBL-1 cells. Triptolide also suppressed STAT3 activity and inhibited secretion of IL-6 in PEL cells. In a mouse xenograft model of primary effusion lymphoma by BCBL-1 cells, triptolide treatment significantly inhibited ascites formation and diffused organ infiltration. These results indicate that triptolide impairs the expression of LANA1 and shows antitumor activity against PEL in vitro and in vivo. Triptolide may be a potential agent for treatment of PEL.

The Hsp70 inhibitor 2-phenylethynesulfonamide inhibits replication and carcinogenicity of Epstein–Barr virus by inhibiting the molecular chaperone function of Hsp70

Epstein–Barr virus (EBV) can infect cells in latent and lytic period and cause serious disease. Epstein–Barr virus nuclear antigen 1 (EBNA1) is essential for the maintenance of the EBV DNA episome, replication and transcription. 2-phenylethynesulfonamide (PES) is a small molecular inhibitor of Heat shock protein 70 (Hsp70), which can interact with Hsp70 and disrupts its association with co-chaperones and substrate proteins of Hsp70. In our study, we found that PES could decrease the expression of EBNA1, which is independent of effects on EBNA1 transcription or proteasomal degradation pathway. The central glycine–alanine repeats domain was not required for inhibition of EBNA1 expression by PES. Also, PES could reduce the amount of intracellular EBV genomic DNA. PES inhibited proliferation and migration but induced cell cycle arrest and apoptosis of EBV positive cells. In addition, silencing of Hsp70 decreased expression of EBNA1 and the amounts of intracellular EBV genomic DNA, and PES increased this effect on a dose-dependent manner. On the contrast, over-expression of Hsp70 enhanced the expression of EBNA1 and the amounts of intracellular EBV genomic DNA, but PES inhibited this effect on a dose-dependent manner. Furthermore, Hsp70 interacted with EBNA1 but PES interfered this interaction. Our results indicate that PES suppresses replication and carcinogenicity of Epstein–Barr virus via inhibiting the molecular chaperone function of Hsp70.

Baicalein inhibits growth of Epstein-Barr virus-positive nasopharyngeal carcinoma by repressing the activity of EBNA1 Q-promoter

Epstein-Barr virus (EBV) can establish a life-long latent infection in the host and is associated with various human malignancies, including nasopharyngeal carcinoma (NPC), the most common cancer originated from nasopharynx. EBV nuclear antigen 1 (EBNA1) is the only viral protein absolutely demanded for segregation, replication, transcription and maintenance of EBV viral genome in host cells. Baicalein, a bioactive flavonoid compound purified from the root of Scutellariae baicaleinsis, displays anti-inflammatory, immunosuppressive, and anti-tumor properties. In this study, the therapeutic effects and functional mechanism of baicalein on EBV-positive human NPC were determined. Cell Counting Kit-8 assays and cell formation colony were performed to investigate that baicalein can suppress proliferation of EBV-infected human NPC cells. Flow cytometric and hoechst 33258 staining results indicated that baicalein induced cell cycle arrest and apoptosis. Western blotting results demonstrated that baicalein down-regulates EBNA1 expression but not reduces the stability and half-life of EBNA1 in EBV-infected NPC cells. Additionally, the mRNA level of EBNA1 was examined by real time-PCR, the activity of EBNA1 Q promoter (Qp) was determined by dual luciferase reporter assay. Considering that transcription factor specificity protein 1 (Sp1) can maintain EBNA1 Qp active. Further analyses also elucidated that baicalein inhibits the expression of Sp1 while knock-down Sp1 by specific shRNAs decreases the expression and transcription levels of EBNA1. Therefore, the results suggested that baicalein may decrease EBNA1 expression level in EBV-positive NPC cells via inhibiting the activity of EBNA1 Q-promoter while over-expression of EBNA1 attenuate the inhibitory effect of baicalein. Finally, it was found that baicalein may strongly reduce growth of tumor in the mouse xenograft model of EBV-positive NPC. These results indicated that baicalein inhibits growth of EBV-positive NPC by repressing the activity of EBNA1 Q-promoter. Baicalein may be used as a therapeutic agent to treat EBV-positive NPC.