Modulation of Kaposi's sarcoma-associated herpesvirus infection and replication by MEK/ERK, JNK, and p38 multiple mitogen-activated protein kinase pathways during primary infection

Kaposi's sarcoma-associated herpesvirus (KSHV) is etiologically associated with Kaposi's sarcoma, a dominant AIDS-related tumor of endothelial cells, and several other lymphoproliferative malignancies. While activation of the phosphatidylinositol 3-kinase-protein kinase C-MEK-ERK pathway is essential for KSHV infection, we have recently shown that KSHV also activates JNK and p38 mitogen-activated protein kinase (MAPK) pathways during primary infection (J. Xie, H. Y. Pan, S. Yoo, and S.-J. Gao, J. Virol. 79:15027-15037, 2005). Here, we found that activation of both JNK and p38 pathways was also essential for KSHV infection. Inhibitors of all three MAPK pathways reduced KSHV infectivity in both human umbilical vein endothelial cells (HUVEC) and 293 cells. These inhibitory effects were dose dependent and occurred at the virus entry stage of infection. Consistently, inhibition of all three MAPK pathways with dominant-negative constructs reduced KSHV infectivity whereas activation of the ERK pathway but not the JNK and p38 pathways enhanced KSHV infectivity. Importantly, inhibition of all three MAPK pathways also reduced the yield of infectious virions during KSHV productive infection of HUVEC. While the reduction of infectious virions was in part due to the reduced infectivity, it was also the result of direct modulation of KSHV lytic replication by the MAPK pathways. Accordingly, KSHV upregulated the expression of RTA (Orf50), a master transactivator of KSHV lytic replication, and activated its promoter during primary infection. Furthermore, KSHV activation of RTA promoter during primary infection was modulated by all three MAPK pathways, predominantly through their downstream target AP-1. Together, these results indicate that, by modulating multiple MAPK pathways, KSHV manipulates the host cells to facilitate its entry into the cells and postentry productive lytic replication during primary infection.     

Activation of mitogen-activated protein kinase and NF-kappaB pathways by a Kaposi's sarcoma-associated herpesvirus K15 membrane protein

The K15 gene of Kaposi's sarcoma-associated herpesvirus (also known as human herpesvirus 8) consists of eight alternatively spliced exons and has been predicted to encode membrane proteins with a variable number of transmembrane regions and a common C-terminal cytoplasmic domain with putative binding sites for SH2 and SH3 domains, as well as for tumor necrosis factor receptor-associated factors. These features are reminiscent of the latent membrane proteins LMP-1 and LMP2A of Epstein-Barr virus and, more distantly, of the STP, Tip, and Tio proteins of the related gamma(2)-herpesviruses herpesvirus saimiri and herpesvirus ateles. These viral membrane proteins can activate a number of intracellular signaling pathways. We have therefore examined the abilities of different K15-encoded proteins to initiate intracellular signaling. We found that a 45-kDa K15 protein derived from all eight K15 exons and containing 12 predicted transmembrane domains in addition to the cytoplasmic domain activated the Ras/mitogen-activated protein kinase (MAPK) and NF-kappaB pathways, as well as (more weakly) the c-Jun N-terminal kinase/SAPK pathway. Activation of the MAPK and NF-kappaB pathways required phosphorylation of tyrosine residue 481 within a putative SH2-binding site (YEEVL). This motif was phosphorylated by the tyrosine kinases Src, Lck, Yes, Hck, and Fyn. The region containing the YEEVL motif interacted with tumor necrosis factor receptor-associated factor 2 (TRAF-2), and a dominant negative TRAF-2 mutant inhibited the K15-mediated activation of the Ras/MAPK pathway, suggesting the involvement of TRAF-2 in the initiation of these signaling routes. In contrast, several smaller K15 protein isoforms activated these pathways only weakly. All of the K15 isoforms tested were, however, localized in lipid rafts, suggesting that incorporation into lipid rafts is not sufficient to initiate signaling. Additional regions of K15, located presumably in exons 2 to 5, may therefore contribute to the activation of these pathways. These findings illustrate that the 45-kDa K15 protein engages pathways similar to LMP1, LMP2A, STP, Tip, and Tio but combines functional features that are separated between LMP1 and LMP2A or STP and Tip.     

Upregulation of the TLR3 pathway by Kaposi's sarcoma-associated herpesvirus during primary infection

Kaposi's sarcoma-associated herpesvirus (KSHV) is associated with several different human malignancies, including Kaposi's sarcoma, primary effusion lymphoma, and multicentric Castleman's disease. KSHV establishes lifelong latency in the host and modulates the host immune response. Innate immunity is critical for controlling de novo viral infection. Toll-like receptors (TLRs) are key components of the innate immune system, and they serve as pathogen recognition receptors that stimulate the host antiviral response. In particular, TLR3 has been implicated in RNA virus recognition. Currently, there is no information regarding how KSHV infection modulates any TLR pathway. We report the first evidence that KSHV upregulates TLR3 expression in human monocytes during primary infection. This is also the first demonstration of a human DNA tumor virus upregulating TLR3, a TLR that thus far has been associated with the recognition of RNA viruses. We found that KSHV upregulates the TLR3 pathway and induces TLR3-specific cytokines and chemokines, including beta 1 interferon (IFN-beta1) and CXCL10 (IP-10). Small interfering RNAs directed against TLR3 greatly reduced the ability of KSHV to upregulate IFN-beta1 and CXCL10 upon infection.     

Control of Kaposi's sarcoma-associated herpesvirus reactivation induced by multiple signals

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.     

Role of protein kinase C delta in reactivation of Kaposi's sarcoma-associated herpesvirus

TPA (12-O-tetradecanoylphorbol-13-acetate), a well-known activator of protein kinase C (PKC), can experimentally induce reactivation of Kaposi's sarcoma-associated herpesvirus (KSHV) in certain latently infected cells. We selectively blocked the activity of PKC isoforms by using GF 109203X or rottlerin and demonstrated that this inhibition largely decreased lytic KSHV reactivation by TPA. Translocation of the PKCdelta isoform was evident shortly after TPA stimulation. Overexpression of the dominant-negative PKCdelta mutant supported an essential role for the PKCdelta isoform in virus reactivation, yet overexpression of PKCdelta alone was not sufficient to induce lytic reactivation of KSHV, suggesting that additional signaling molecules participate in this pathway.     

Structural analysis of the Kaposi's sarcoma-associated herpesvirus K1 protein

The K1 protein of Kaposi's sarcoma-associated herpesvirus (KSHV) efficiently transduces extracellular signals to elicit cellular activation events through its cytoplasmic immunoreceptor tyrosine-based activation motif (ITAM). In addition, the extracellular domain of K1 demonstrates regional homology with the immunoglobulin (Ig) family and contains conserved regions (C1 and C2) and variable regions (V1 and V2). To generate mouse monoclonal antibodies directed against the KSHV K1 protein, BALB/c mice were primed and given boosters with K1 protein purified from mammalian cells. Twenty-eight hybridomas were tested for reactivity with K1 protein by enzyme-linked immunosorbent assay, immunofluorescence, flow cytometry, immunohistochemistry, and immunoblotting. Deletion mutants of the K1 extracellular domain were used to map the epitope of each antibody. All antibodies were directed to the Ig, C1, and C2 regions of K1. Furthermore, antibody recognition of a short sequence (amino acids 92 to 125) of the C2 region overlapping with the Ig region of K1 efficiently induced intracellular free calcium mobilization; antibody recognition of the other regions of K1 did not. The efficient signal transduction of K1 induced by antibody stimulation required both the ITAM sequence of the cytoplasmic domain and the normal structure of the extracellular domain. Finally, immunological assays showed that K1 was expressed during the early lytic cycle of viral replication in primary effusion lymphoma cells. K1 was readily detected in multicentric Castleman's disease tissues, whereas it was not detected in Kaposi's sarcoma lesions, suggesting that K1 is preferentially expressed in lymphoid cells. Thus, these results indicate that the conserved regions, particularly the Ig and C2 regions, of the K1 extracellular domain are exposed on the outer surface and play an important role in K1 structure and signal transduction, whereas the variable regions of K1 appear to be away from the surface.     

TAK1-TAB1 fusion protein: a novel constitutively active mitogen-activated protein kinase kinase kinase that stimulates AP-1 and NF-kappaB signaling pathways

TAK1 mitogen-activated protein kinase kinase kinase (MAP3K) is activated by its specific activator, TAK1-binding protein 1 (TAB1). A constitutively active TAK1 mutant has not yet been generated due to the indispensable requirement of TAB1 for TAK1 kinase activity. In this study, we generated a novel constitutively active TAK1 by fusing its kinase domain to the minimal TAK1-activation domain of TAB1. Co-immunoprecipitation assay demonstrated that these domains interacted intra-molecularly. The TAK1-TAB1 fusion protein showed a significant MAP3K activity in vitro and activated c-Jun N-terminal kinase/p38 MAPKs and IkappaB kinase in vivo, which was followed by increased production of interleukin-6. These results indicate that the fusion protein is useful for characterizing the physiological roles of the TAK1-TAB1 complex.     

Kaposi's sarcoma-associated herpesvirus infection promotes invasion of primary human umbilical vein endothelial cells by inducing matrix metalloproteinases

Matrix metalloproteinases (MMPs) play important roles in cancer invasion, angiogenesis, and inflammatory infiltration. Kaposi's sarcoma is a highly disseminated angiogenic tumor of proliferative endothelial cells linked to infection by Kaposi's sarcoma-associated herpesvirus (KSHV). In this study, we showed that KSHV infection increased the invasiveness of primary human umbilical vein endothelial cells (HUVEC) in a Matrigel-based cell invasion assay. KSHV-induced cell invasion was abolished by an inhibitor of MMPs, BB-94, and occurred in both autocrine- and paracrine-dependent fashions. Analysis by zymography and Western blotting showed that KSHV-infected HUVEC cultures had increased secretion of MMP-1, -2, and -9. KSHV increased the secretion of MMP-2 within 1 h following infection without upregulating its mRNA expression level. In contrast, the secretion of MMP-1 and -9 was not increased until 6 h after KSHV infection and was correlated with the upregulation of their mRNA expression levels. Promoter analysis by reporter assays and electrophoretic mobility shift assays identified an AP-1 cis-element as the dominant KSHV-responsive site in the MMP-1 promoter. Together, these results suggest that KSHV infection modulates the production of multiple MMPs to increase cell invasiveness and thus contributes to the pathogenesis of KSHV-induced malignancies.     

Ribosomal protein S6 interacts with the latency-associated nuclear antigen of Kaposi's sarcoma-associated herpesvirus

The latency-associated nuclear antigen (LANA) is central to the maintenance of Kaposi's sarcoma-associated herpesvirus (KSHV) and to the survival of KSHV-carrying tumor cells. In an effort to identify interaction partners of LANA, we purified authentic high-molecular-weight complexes of LANA by conventional chromatography followed by immunoprecipitation from the BC-3 cell line. This is the first analysis of LANA-interacting partners that is not based on forced ectopic expression of LANA. Subsequent tandem mass spectrometry (MS/MS) analysis identified many of the known LANA-interacting proteins. We confirmed LANA's interactions with histones. Three classes of proteins survived our stringent four-step purification procedure (size, heparin, anion, and immunoaffinity chromatography): two heat shock proteins (Hsp70 and Hsp96 precursor), signal recognition particle 72 (SRP72), and 10 different ribosomal proteins. These proteins are likely involved in structural interactions within LANA high-molecular-weight complexes. Here, we show that ribosomal protein S6 (RPS6) interacts with LANA. This interaction is mediated by the N-terminal domain of LANA and does not require DNA or RNA. Depletion of RPS6 from primary effusion lymphoma (PEL) cells dramatically decreases the half-life of full-length LANA. The fact that RPS6 has a well-established nuclear function beyond its role in ribosome assembly suggests that RPS6 (and by extension other ribosomal proteins) contributes to the extraordinary stability of LANA.     

The cyclin encoded by Kaposi's sarcoma-associated herpesvirus stimulates cdk6 to phosphorylate the retinoblastoma protein and histone H1.

Kaposi's sarcoma-associated herpesvirus (KSHV or human herpesvirus 8) is a novel gammaherpesvirus implicated in the cause of Kaposi's sarcoma and certain malignancies of lymphatic origin. One of the candidate genes possibly involved in promoting tumor development is an open reading frame (ORF) with sequence similarity to human type D cyclin genes. This cyclin-like gene, when expressed in tissue culture cells, promotes phosphorylation and inactivation of the retinoblastoma tumor suppressor protein and thereby may result in deregulation of cell division control. We report here the biochemical characterization of this cyclin (KSHV-cyc) and the kinase activity that it elicits upon expression in tissue culture cells. We demonstrate that the kinase activity associated with KSHV-cyc is sensitive to the cdk inhibitor p27 (KIP) and due to activation of cdk6. However, in contrast to cdk6 activated by cellular type D cyclins, the cdk6 activated by KSHV-cyc is capable of phosphorylating not only the retinoblastoma protein but also histone H1. This finding implies that activation by KSHV-cyc alters the substrate preference of this cdk. This may have important physiological consequences in that the kinase activity triggered by this viral cyclin may abrogate cell cycle checkpoints in addition to those targeted by cellular cyclin D-cdk6 kinase.     

Erythropoietin induction of tissue inhibitors of metalloproteinase-1 expression and secretion is mediated by mitogen-activated protein kinase and phosphatidylinositol 3-kinase pathways.

In the present study, we demonstrate that erythropoietin (Epo) induces the expression and the release of tissue inhibitors of metalloproteinase-1 (TIMP-1) in a time- and dose-dependent manner in Epo-dependent cell line UT-7 cells and in normal human erythroid progenitor cells from cord blood (CD36+) and required de novo protein synthesis. TIMP-1 was not expressed in the absence of Epo. Inhibition of the mitogen-activated protein kinase pathway by the specific inhibitors PD98059 and U0126 and of phosphatidylinositol 3-kinase by LY294002, strongly inhibited Epo-induced TIMP-1 expression and secretion. In the absence of Epo, both latent and active forms of matrix metalloproteinase-9 (MMP-9) were secreted into media. Upon Epo stimulation, MMP-9 and pro-MMP-9 secretion was inhibited in a dose-dependent manner parallel to TIMP-1 induction. The addition of PD98059, U0126, and LY294002 in the presence of Epo restored MMP-9 production in UT-7 and CD36+ cells. Our findings strongly suggest an inversely coordinated regulation of the TIMP-1 gene and MMP-9 production by Epo via mitogen-activated protein kinase and phosphatidylinositol 3-kinase pathways.     

Productive lytic replication of a recombinant Kaposi's sarcoma-associated herpesvirus in efficient primary infection of primary human endothelial cells

Kaposi's sarcoma-associated herpesvirus (KSHV) is linked to the development of Kaposi's sarcoma (KS), a vascular spindle cell tumor primarily consisting of proliferating endothelial cells. Although KSHV has been shown to infect primary human endothelial cells and convert them into spindle shapes, KSHV infection is largely latent, and efforts to establish a highly efficient and sustainable infection system have been unsuccessful. A recombinant KSHV, BAC36, that has high primary-infection efficiency in 293 cells has been obtained (F. C. Zhou, Y. J. Zhang, J. H. Deng, X. P. Wang, H. Y. Pan, E. Hettler, and S. J. Gao, J. Virol. 76:6185-6196, 2002). BAC36 contains a green fluorescent protein cassette which can be used to conveniently monitor viral infection. Here, we describe the establishment of a KSHV lytic-replication-permissive infection cell model using BAC36 virions to infect primary human umbilical vein endothelial cell (HUVEC) cultures. BAC36 infection of HUVEC cultures has as high as 90% primary-infection efficiency and consists of two phases: a permissive phase, in which the cultures undergo active viral lytic replication, producing a large number of virions and concomitantly resulting in large-scale cell death, and a latent phase, in which the surviving cells from the permissive phase switch into latent infection, with a small number of cells undergoing spontaneous viral lytic replication, and proliferate into bundles of spindle cells with KS slit-like spaces. An assay for determining the KSHV titer in a virus preparation has also been developed. The cell model should be useful for examining KSHV infection and replication, as well as for understanding the development of KS.     

Regulation of Nm23-H1 and cell invasiveness by Kaposi's sarcoma-associated herpesvirus

The Kaposi's sarcoma-associated herpesvirus (KSHV) is the causative agent of Kaposi's sarcoma (KS), and the induction of an invasive cellular phenotype by KSHV following de novo infection is an important pathogenic component mediating tumor progression. The metastasis suppressor gene known as Nm23-H1 regulates tumor cell invasiveness, but whether KSHV itself regulates Nm23-H1 expression or subcellular localization, and whether this impacts cell invasiveness, has not been established. We found that KSHV increases expression and nuclear translocation of Nm23-H1 and that nuclear translocation of Nm23-H1 is regulated by the KSHV-encoded latency-associated nuclear antigen (LANA). Moreover, activation of the Ras-BRaf-MAPK (mitogen-activated protein kinase) signal transduction pathway, secretion of promigratory factors associated with this pathway, and cell invasiveness are dependent on KSHV regulation of Nm23-H1. Finally, induction of cytoplasmic overexpression of Nm23-H1 using a pharmacologic inhibitor of DNA methylation reduced KSHV-associated Ras-BRaf-MAPK pathway activation and suppressed KSHV-induced invasiveness. These data provide the first evidence for KSHV regulation of Nm23-H1 as a mechanism for KSHV induction of an invasive cellular phenotype and support the potential utility of targeting Nm23-H1 as a therapeutic approach for the treatment of KS.