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ABSTRACT: Primary effusion lymphoma (PEL) cells are predominantly infected by the latent form of Kaposi's sarcoma-associated herpesvirus (KSHV), with virus reactivation occurring in a small percentage of cells. Latency enables KSHV to persist in the host cell and promotes tumorigenesis through viral gene expression, thus presenting a major barrier to the elimination of KSHV and the treatment of PEL. Therefore, it is important to identify cellular genes that are essential for PEL cell survival or the maintenance of KSHV latency. Here we report that cyclin-dependent kinase 1 (Cdk1) inhibition can induce both apoptosis and KSHV reactivation in a population of PEL cells. Caspases, but not p53, are required for PEL cell apoptosis induced by Cdk1 inhibition. p38 kinase is activated by Cdk1 inhibition and mediates KSHV reactivation. Interestingly, upon Cdk1 inhibition, KSHV is reactivated predominantly in the nonapoptotic subpopulation of PEL cells. We provide evidence that this is due to mutual inhibition between apoptosis and KSHV reactivation. In addition, we found that KSHV reactivation activates protein kinase B (AKT/PKB), which promotes cell survival and facilitates KSHV reactivation. Our study thus establishes a key role for Cdk1 in PEL cell survival and the maintenance of KSHV latency and reveals a multifaceted relationship between KSHV reactivation and PEL cell apoptosis.
Journal of Virology 04/2012; 86(12):6668-76. · 5.40 Impact Factor
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Shaoying Lee,
Lukasz Salwinski,
Chaoying Zhang,
Derrick Chu,
Claire Sampankanpanich,
Nichole A Reyes,
Abbey Vangeloff,
Fangfang Xing, Xudong Li,
Ting-Ting Wu,
Sudhir Sahasrabudhe,
Hongyu Deng,
Douglas J Lacount,
Ren Sun
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ABSTRACT: Genome-wide yeast two-hybrid (Y2H) screens were conducted to elucidate the molecular functions of open reading frames (ORFs) encoded by murine γ-herpesvirus 68 (MHV-68). A library of 84 MHV-68 genes and gene fragments was generated in a Gateway entry plasmid and transferred to Y2H vectors. All possible pair-wise interactions between viral proteins were tested in the Y2H assay, resulting in the identification of 23 intra-viral protein-protein interactions (PPIs). Seventy percent of the interactions between viral proteins were confirmed by co-immunoprecipitation experiments. To systematically investigate virus-cellular protein interactions, the MHV-68 Y2H constructs were screened against a cellular cDNA library, yielding 243 viral-cellular PPIs involving 197 distinct cellar proteins. Network analyses indicated that cellular proteins targeted by MHV-68 had more partners in the cellular PPI network and were located closer to each other than expected by chance. Taking advantage of this observation, we scored the cellular proteins based on their network distances from other MHV-68-interacting proteins and segregated them into high (Y2H-HP) and low priority/not-scored (Y2H-LP/NS) groups. Significantly more genes from Y2H-HP altered MHV-68 replication when their expression was inhibited with siRNAs (53% of genes from Y2H-HP, 21% of genes from Y2H-LP/NS, and 16% of genes randomly chosen from the human PPI network; p<0.05). Enriched Gene Ontology (GO) terms in the Y2H-HP group included regulation of apoptosis, protein kinase cascade, post-translational protein modification, transcription from RNA polymerase II promoter, and IκB kinase/NFκB cascade. Functional validation assays indicated that PCBP1, which interacted with MHV-68 ORF34, may be involved in regulating late virus gene expression in a manner consistent with the effects of its viral interacting partner. Our study integrated Y2H screening with multiple functional validation approaches to create γ-herpes viral-viral and viral-cellular protein interaction networks.
PLoS Pathogens 10/2011; 7(10):e1002297. · 9.13 Impact Factor
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ABSTRACT: The ability to control cellular functions can bring about many developments in basic biological research and its applications. The presence of multiple signals, internal as well as externally imposed, introduces several challenges for controlling cellular functions. Additionally the lack of clear understanding of the cellular signaling network limits our ability to infer the responses to a number of signals. This work investigates the control of Kaposi's sarcoma-associated herpesvirus reactivation upon treatment with a combination of multiple signals. We utilize mathematical model-based as well as experiment-based approaches to achieve the desired goals of maximizing virus reactivation. The results show that appropriately selected control signals can induce virus lytic gene expression about ten folds higher than a single drug; these results were validated by comparing the results of the two approaches, and experimentally using multiple assays. Additionally, we have quantitatively analyzed potential interactions between the used combinations of drugs. Some of these interactions were consistent with existing literature, and new interactions emerged and warrant further studies. The work presents a general method that can be used to quantitatively and systematically study multi-signal induced responses. It enables optimization of combinations to achieve desired responses. It also allows identifying critical nodes mediating the multi-signal induced responses. The concept and the approach used in this work will be directly applicable to other diseases such as AIDS and cancer.
PLoS ONE 01/2011; 6(6):e20998. · 4.09 Impact Factor
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ABSTRACT: Kaposi's sarcoma (KS) and primary effusion lymphoma (PEL) cells are predominantly infected with latent Kaposi's sarcoma-associated herpesvirus (KSHV), presenting a barrier to the destruction of tumor cells. Latent KSHV can be reactivated to undergo lytic replication. Here we report that in PEL cells, oxidative stress induced by upregulated reactive oxygen species (ROS) can lead to KSHV reactivation or cell death. ROS are upregulated by NF-κB inhibition and are required for subsequent KSHV reactivation. Disruption of the intracellular redox balance through depletion of the antioxidant glutathione or inhibition of the antioxidant enzyme catalase also induces KSHV reactivation, suggesting that hydrogen peroxide induces reactivation. In addition, p38 signaling is required for KSHV reactivation induced by ROS. Furthermore, treatment of PEL cells with a higher concentration of the NF-κB inhibitor than that used for inducing KSHV reactivation further upregulates ROS and induces massive cell death. ROS, but not p38 signaling, are required for PEL cell death induced by NF-κB inhibition as well as by glutathione depletion. Importantly, anticancer drugs, such as cisplatin and arsenic trioxide, also induce KSHV reactivation and PEL cell death in a ROS-dependent manner. Our study thus establishes a critical role for ROS and oxidative stress in the regulation of KSHV reactivation and PEL cell death. Disrupting the cellular redox balance may be a potential strategy for treating KSHV-associated lymphoma.
Journal of Virology 11/2010; 85(2):715-24. · 5.40 Impact Factor
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ABSTRACT: Myc is deregulated by Kaposi's sarcoma-associated herpesvirus (KSHV) latent proteins, but its role in KSHV latency is not clear. We found that Myc knockdown with RNA interference (RNAi) induced KSHV reactivation and increased the protein and mRNA levels of RTA, a key viral regulator of KSHV reactivation. Myc knockdown increased, whereas Myc overexpression inhibited, RTA promoter activity. KSHV reactivation and the activation of the RTA promoter induced by Myc depletion were inhibited by c-Jun N-terminal kinase (JNK) and p38 inhibitors but not by a MEK1 inhibitor. Myc knockdown inhibited primary effusion lymphoma (PEL) cell proliferation through inducing apoptosis and G(1) cell cycle arrest. Thus, Myc may be a key cellular node coupling cellular transformation and KSHV latency.
Journal of Virology 09/2010; 84(17):8945-8. · 5.40 Impact Factor
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ABSTRACT: How cellular factors regulate gammaherpesvirus lytic replication is not well understood. Here, through functional screening of a cellular kinase expression library, we identified mitogen-activated protein kinase kinase kinase 8 (MAP3K8/Tpl2) as a positive regulator of murine gammaherpesvirus 68 (MHV-68 or gammaHV-68) lytic gene expression and replication. Tpl2 enhances MHV-68 lytic replication by upregulating lytic gene expression and promoter activities of viral lytic genes, including RTA and open reading frame 57 (ORF57). By screening a cellular transcription factor library, we identified the Fos AP-1 transcription factor as a downstream factor that is both necessary and sufficient for mediating the enhancement of MHV-68 lytic replication by Tpl2. In addition, Tpl2 stimulates the promoter activities of key viral lytic genes, including RTA and ORF57, in an AP-1-dependent manner. We identified an AP-1-responsive element on the MHV-68 RTA promoter as the cis element mediating the upregulation of RTA promoter activity by Tpl2. MHV-68 lytic infection upregulates Fos expression, AP-1 activity, and RTA promoter activity in a Tpl2-dependent manner. We constructed a mutant MHV-68 virus that abolished this AP-1-responsive element. This mutant virus exhibited attenuated lytic replication kinetics, indicative of a critical role of this AP-1-responsive element during lytic replication. Moreover, Tpl2 knockdown inhibited the lytic replication of wild-type MHV-68 (MHV-68-WT) but not that of the MHV-68 mutant virus, indicating that endogenous Tpl2 promotes efficient virus lytic replication through AP-1-dependent upregulation of RTA expression. In summary, through tandem functional screens, we identified the Tpl2/AP-1 signaling transduction pathway as a positive regulator of MHV-68 lytic replication.
Journal of Virology 11/2009; 84(4):1881-90. · 5.40 Impact Factor
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Li Peng,
Ting-Ting Wu,
Jason H Tchieu,
Jun Feng,
Helen J Brown,
Jiaying Feng, Xudong Li,
Jing Qi,
Hongyu Deng,
Igor Vivanco,
Ingo K Mellinghoff,
Christina Jamieson,
Ren Sun
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ABSTRACT: Cellular signalling pathways are critical in regulating the balance between latency and lytic replication of herpesviruses. Here, we investigated the effect of the phosphatidylinositol 3-kinase (PI3K)-Akt pathway on replication of two gamma-2 herpesviruses, murine gammaherpesvirus-68 (MHV-68) and human herpesvirus-8/Kaposi's sarcoma-associated herpesvirus (HHV-8/KSHV). We found that de novo infection of MHV-68 induced PI3K-dependent Akt activation and the lytic replication of MHV-68 was enhanced by inhibiting the PI3K-Akt pathway with both chemical inhibitors and RNA interference technology. Inhibiting the activity of Akt using Akt inhibitor VIII also facilitated the reactivation of KSHV from latency. Both lytic replication and latency depend on the activity of viral transactivator RTA and we further show that the activity of RTA is increased by reducing Akt1 expression. The data suggest that the PI3K-Akt pathway suppresses the activity of RTA and thereby contributes to the maintenance of viral latency and promotes tumorigenesis.
Journal of General Virology 10/2009; 91(Pt 2):463-9. · 3.36 Impact Factor
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ABSTRACT: Gammaherpesviruses, including the human pathogens Epstein-Barr virus and Kaposi's sarcoma-associated herpesvirus, are causative agents of lymphomas and other malignancies. The structural characterization of these viruses has been limited due to difficulties in obtaining adequate amount of virion particles. Here we report the first three-dimensional structural characterization of a whole gammaherpesvirus virion by an emerging integrated approach of cryo-electron tomography combined with single-particle cryo-electron microscopy, using murine gammaherpesvirus-68 (MHV-68) as a model system. We found that the MHV-68 virion consists of distinctive envelope and tegument compartments, and a highly conserved nucleocapsid. Two layers of tegument are identified: an inner tegument layer tethered to the underlying capsid and an outer, flexible tegument layer conforming to the overlying, pleomorphic envelope, consistent with the sequential viral tegumentation process inside host cells. Surprisingly, comparison of the MHV-68 virion and capsid reconstructions shows that the interactions between the capsid and inner tegument proteins are completely different from those observed in alpha and betaherpesviruses. These observations support the notion that the inner layer tegument across different subfamilies of herpesviruses has evolved significantly to confer specific characteristics related to viral-host interactions, in contrast to a highly conserved capsid for genome encapsidation and protection.
Journal of Structural Biology 04/2008; 161(3):428-38. · 3.41 Impact Factor
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ABSTRACT: RTA (replication and transcription activator; also referred to as ORF50, Lyta, and ART), an immediate-early gene product of Kaposi's sarcoma-associated herpesvirus (KSHV)/human herpesvirus 8, disrupts latency and drives lytic replication. RTA activates the expression of polyadenylated nuclear (PAN) RNA (also known as T1.1 or nut-1) of KSHV. This novel noncoding PAN RNA is the most abundant lytic transcript of KSHV; therefore, studying PAN RNA expression serves as a model system for understanding how RTA transactivates target genes during lytic replication. The RTA-responsive element of the PAN promoter (pPAN RRE) was previously identified, and our data suggested direct binding of full-length RTA to the pPAN RRE. Here, we present a detailed analysis of specific interactions between RTA and the PAN promoter. We expressed and purified the DNA-binding domain of RTA (Rdbd) to near homogeneity and measured its affinity for the pPAN RRE. In electrophoretic mobility shift assays (EMSAs), the dissociation constant (K(d)) of Rdbd on the pPAN RRE was determined to be approximately 8 x 10(-9) M, suggesting a strong interaction between RTA and DNA. The specificity of RTA binding to the PAN promoter was confirmed with supershift assays. The Rdbd binding sequences on the PAN promoter were mapped within a 16-bp region of the pPAN RRE by methylation interference assays. However, the minimal DNA sequence for Rdbd binding requires an additional 7 bp on both sides of the area mapped by interference assays, suggesting that non-sequence-specific as well as sequence-specific interactions between RTA and DNA contribute to high-affinity binding. To better understand the molecular interactions between RTA and the PAN promoter, an extensive mutagenesis study on the pPAN RRE was carried out by using EMSAs and reporter assays. These analyses revealed base pairs critical for both Rdbd binding in vitro and RTA transactivation in vivo of the PAN promoter. The results from methylation interference, deletion analysis, and mutagenesis using EMSAs and reporter assays were closely correlated and support the hypothesis that RTA activates PAN RNA expression through direct binding to DNA.
Journal of Virology 06/2002; 76(10):5000-13. · 5.40 Impact Factor
