K1 protein of human herpesvirus 8 suppresses lymphoma cell Fas-mediated apoptosis.

Expression of the K1 gene of human herpesvirus 8 activates nuclear factor-kappaB and induces lymph node hyperplasia and lymphomas in transgenic mice. To further delineate its role in cell survival, we determined whether K1 altered apoptosis of lymphoma cells. K1 protein is expressed in Kaposi sarcoma and primary effusion lymphoma. We retrovirally transfected BJAB lymphoma, THP-1, U937, and Kaposi sarcoma SLK cells to express K1 and a K1 mutant with the deleted immunoreceptor tyrosine-based activation motif (K1m). We challenged cells with an agonistic anti-Fas antibody, Fas ligand, irradiation, and tumor necrosis factor-related apoptosis-inducing ligand. K1 transfectants but not K1m transfectants exhibited reduced levels of apoptosis induced by the anti-Fas antibody but not apoptosis induced by the tumor necrosis factor-related apoptosis-inducing ligand or irradiation. K1 expression resulted in reduced apoptosis rates as shown in several assays. K1 induced a modest reduction in levels of Fas-associated death domain protein, and procaspase 8 recruited to the death-inducing signaling complex. Finally, K1 transfectants cleaved procaspase 8 at significantly lower rates than did K1m transfectants. K1-transfected mice, compared with vector-transfected mice, showed lower death rates after challenge with anti-Fas antibody. K1 may contribute to lymphoma development by stimulating cell survival by selectively blocking Fas-mediated apoptosis.

Molecular and macromolecular alterations of recombinant adenoviral vectors do not resolve changes in hepatic drug metabolism during infection.

In this report we test the hypothesis that long-term virus-induced alterations in CYP occur from changes initiated by the virus that may not be related to the immune response. Enzyme activity, protein expression and mRNA of CYP3A2, a correlate of human CYP3A4, and CYP2C11, responsive to inflammatory mediators, were assessed 0.25, 1, 4, and 14 days after administration of several different recombinant adenoviruses at a dose of 5.7 x 1012 virus particles (vp)/kg to male Sprague Dawley rats. Wild type adenovirus, containing all viral genes, suppressed CYP3A2 and 2C11 activity by 37% and 39%, respectively within six hours. Levels fell to 67% (CYP3A2) and 79% (CYP2C11) of control by 14 days (p

The recombinant adeno-associated virus vector (rAAV2)-mediated apolipoprotein B mRNA-specific hammerhead ribozyme: a self-complementary AAV2 vector improves the gene expression.

BACKGROUND: In humans, overproduction of apolipoprotein B (apoB) is positively associated with premature coronary artery diseases. To reduce the levels of apoB mRNA, we have designed an apoB mRNA-specific hammerhead ribozyme targeted at nucleotide sequences GUA6679 (RB15) mediated by adenovirus, which efficiently cleaves and decreases apoB mRNA by 80% in mouse liver and attenuates the hyperlipidemic condition. In the current study, we used an adeno-associated virus vector, serotype 2 (AAV2) and a self-complementary AAV2 vector (scAAV2) to demonstrate the effect of long-term tissue-specific gene expression of RB15 on the regulation apoB mRNA in vivo. METHODS: We constructed a hammerhead ribozyme RB15 driven by a liver-specific transthyretin (TTR) promoter using an AAV2 vector (rAAV2-TTR-RB15). HepG2 cells and hyperlipidemic mice deficient in both the low density lipoprotein receptor and the apoB mRNA editing enzyme genes (LDLR-/-Apobec1-/-; LDb) were transduced with rAAV2-TTR-RB15 and a control vector rAAV-TTR-RB15-mutant (inactive ribozyme). The effects of ribozyme RB15 on apoB metabolism and atherosclerosis development were determined in LDb mice at 5-month after transduction. A self-complementary AAV2 vector expressing ribozyme RB15 (scAAV2-TTR-RB15) was also engineered and used to transduce HepG2 cells. Studies were designed to compare the gene expression efficiency between rAAV2-TTR-RB15 and scAAV2-TTR-RB15. RESULTS: The effect of ribozyme RB15 RNA on reducing apoB mRNA levels in HepG2 cells was observed only on day-7 after rAAV2-TTR-RB15 transduction. And, at 5-month after rAAV2-TTR-RB15 treatment, the apoB mRNA levels in LDb mice were significantly decreased by 43%, compared to LDb mice treated with control vector rAAV2-TTR-RB15-mutant. Moreover, both the rAAV2-TTR-RB15 viral DNA and ribozyme RB15 RNA were still detectable in mice livers at 5-month after treatment. However, this rAAV2-TTR-RB15 vector mediated a prolonged but low level of ribozyme RB15 gene expression in the mice livers, which did not produce the therapeutic effects on alteration the lipid levels or the inhibition of atherosclerosis development. In contrast, the ribozyme RB15 RNA mediated by scAAV2-TTR-RB15 vector was expressed immediately at day-1 after transduction in HepG2 cells. The apoB mRNA levels were decreased 47% (p = 0.001), compared to the control vector scAAV2-TTR-RB15-mutant. CONCLUSION: This study provided evidence that the rAAV2 single-strand vector mediated a prolonged but not efficient transduction in mouse liver. However, the scAAV2 double-strand vector mediated a rapid and efficient gene expression in liver cells. This strategy using scAAV2 vectors represents a better approach to express small molecules such as ribozyme.

Activation of Src kinase Lyn by the Kaposi sarcoma-associated herpesvirus K1 protein: implications for lymphomagenesis.

The K1 gene of Kaposi sarcoma-associated herpesvirus (KSHV) encodes a transmembrane glycoprotein bearing a functional immunoreceptor tyrosine-based activation motif (ITAM). Previously, we reported that the K1 protein induced plasmablastic lymphomas in K1 transgenic mice, and that these lymphomas showed enhanced Lyn kinase activity. Here, we report that systemic administration of the nuclear factor kappa B (NF-kappaB) inhibitor Bay 11-7085 or an anti-vascular endothelial growth factor (VEGF) antibody significantly reduced K1 lymphoma growth in nude mice. Furthermore, in KVL-1 cells, a cell line derived from a K1 lymphoma, inhibition of Lyn kinase activity by the Src kinase inhibitor PP2 decreased VEGF induction, NF-kappaB activity, and the cell proliferation index by 50% to 75%. In contrast, human B-cell lymphoma BJAB cells expressing K1, but not the ITAM sequence-deleted mutant K1, showed a marked increase in Lyn kinase activity with concomitant VEGF induction and NF-kappaB activation, indicating that ITAM sequences were required for the Lyn kinase-mediated activation of these factors. Our results suggested that K1-mediated constitutive Lyn kinase activation in K1 lymphoma cells is crucial for the production of VEGF and NF-kappaB activation, both strongly implicated in the development of KSHV-induced lymphoproliferative disorders.

Cryo-electron tomography of Kaposi's sarcoma-associated herpesvirus capsids reveals dynamic scaffolding structures essential to capsid assembly and maturation.

Kaposi's sarcoma-associated herpesvirus (KSHV) is a recently discovered DNA tumor virus that belongs to the gamma-herpesvirus subfamily. Though numerous studies on KSHV and other herpesviruses, in general, have revealed much about their multilayered organization and capsid structure, the herpesvirus capsid assembly and maturation pathway remains poorly understood. Structural variability or irregularity of the capsid internal scaffolding core and the lack of adequate tools to study such structures have presented major hurdles to earlier investigations employing more traditional cryo-electron microscopy (cryoEM) single particle reconstruction. In this study, we used cryo-electron tomography (cryoET) to obtain 3D reconstructions of individual KSHV capsids, allowing direct visualization of the capsid internal structures and systematic comparison of the scaffolding cores for the first time. We show that B-capsids are not a structurally homogenous group; rather, they represent an ensemble of "B-capsid-like" particles whose inner scaffolding is highly variable, possibly representing different intermediates existing during the KSHV capsid assembly and maturation. This information, taken together with previous observations, has allowed us to propose a detailed pathway of herpesvirus capsid assembly and maturation.

Unique structures in a tumor herpesvirus revealed by cryo-electron tomography and microscopy.

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.

Direct visualization of the putative portal in the Kaposi's sarcoma-associated herpesvirus capsid by cryoelectron tomography.

Genetic and biochemical studies have suggested the existence of a bacteriophage-like, DNA-packaging/ejecting portal complex in herpesviruses capsids, but its arrangement remained unknown. Here, we report the first visualization of a unique vertex in the Kaposi's sarcoma-associated herpesvirus (KSHV) capsid by cryoelectron tomography, thus providing direct structural evidence for the existence of a portal complex in a gammaherpesvirus. This putative KSHV portal is an internally localized, umbilicated structure and lacks all of the external machineries characteristic of portals in DNA bacteriophages.

Cytochrome P450 2D18 in the mammalian brain: Recombinant expression, purification, and characterization of xenobiotic and endobiotic metabolism

The cytochromes P450 (P450) comprise a superfamily of hemoproteins that function in concert with NADPH-cytochrome P450 reductase (P450-reductase) to metabolize both endogenous and exogenous compounds. Many pharmacological agents undergo phase I metabolism by this P450 and P450-reductase monooxygenase system. Phase I metabolism ensures that these highly hydrophobic xenobiotics are made more hydrophilic, and hence easier to extrude from the body. While the majority of phase I metabolism occurs in the liver, metabolism in extrahepatic organ-systems like the intestine, kidney, and brain can have important roles in drug metabolism and/or efficacy. ^ While P450-mediated phase I metabolism has been well studied, investigators have only recently begun to elucidate what physiological roles P450 may have. One way to approach this question is to study P450s that are highly or specifically expressed in extrahepatic tissues. In this project I have studied the role of a recently cloned P450 family member, P450 2D18, that was previously shown to be expressed in the rat brain and kidney, but not in the liver. To this end, I have used the baculovirus expression system to over-express recombinant P450 2D18 and purified the functional enzyme using nickel and hydroxylapatite chromatography. SDS-PAGE analysis indicated that the enzyme was purified to electrophoretic homogeneity and Western analysis showed cross-reactivity with rabbit anti-human P450 2D6. Carbon monoxide difference spectra indicated that the purified protein contained no denatured P450 enzyme; this allowed for further characterization of the substrates and metabolites formed by P450 2D18-mediated metabolism. ^ Because P450 2D18 is expressed in brain, we characterized the activity toward several psychoactive drugs including the antidepressants imipramine and desipramine, and the anti-psychotic drugs chlorpromazine and haloperidol. P450 2D18 preferentially catalyzed the N-demethylation of imipramine, desipramine, and chlorpromazine. This is interesting given the fact that other P450 isoforms form multiple metabolites from such compounds. This limited metabolic profile might suggest that P450 2D18 has some unique function, or perhaps a role in endobiotic metabolism. ^ Further analysis of possible endogenous substrates for P450 2D18 led to the identification of dopamine and arachidonic acid as substrates. It was shown that P450 2D18 catalyzes the oxidation of dopamine to aminochrome, and that the enzyme binds dopamine with an apparent KS value of 678 μM, a value well within reported dopamine concentration in brain dopaminergic systems. Further, it was shown that P450 2D18 binds arachidonic acid with an apparent KS value of 148 μM, and catalyzes both the ω-hydroxylation and epoxygenation of arachidonic acid to metabolites that have been shown to have vasoactive properties in brain, kidney, and heart tissues. These data provide clues for endogenous roles of P450 within the brain, and possible involvement in the pathogenesis of Parkinson's disease. ^

Characterization of recombinant class I MHC alloantigen action upon the survival of disparate heart allografts

Previous studies have led to the development of allochimeric class I MHC proteins as agents that effectively induce donor-specific transplantation tolerance in a rat system with or without additional immunosuppression. Within the α1-helical region of RT1.Au, an epitope that conferred immunologic tolerance was discovered. Studies presented herein were designed to test our central hypothesis that allochimeric proteins onfer tolerance in a mouse model. To test this hypothesis, portal vein (PV) injection of wild-type H2Kd and H2Dd proteins were produced in a bacterial expression system and found to specifically prolong the survival of BALB/c (H2d) heart allografts in C57BL/10 (H2b) recipients. Although a single PV injection of 50 μg α1–α 3 H2Kd alone was ineffective, 50 μg α1 –α3 alone slightly prolonged BALB/c heart allograft survivals. In contrast, the combination of 25 μg α1–α 3 H2Kd and 25 μg α1–α 3 H2Dd proteins prolonged BALB/c graft survivals to 20.2 ± 6.4 days (p < 0.004). The effect was donor-specific, since a combination of 25 μg α1–α3 H2Kd and 25 μg α1–α3 H2Dd proteins failed to affect survivals of third-party C3H (H2k k) heart allografts, namely 9.0 ± 0.0 days in treated versus 7.8 ± 0.5 days in untreated hosts. Thus, the combination of two H2K d and H2Dd proteins is more effective in prolonging allograft survival than a single protein produced in a bacterial expression system. A single PV injection (day 0) of 25 μg α1–α 2 H2Kd and 25 μg α1–α 2 H2Dd proteins to C57BL/10 mice prolonged the survival of BALB/c heart allografts to 22.4 ± 4.5 days. Within a WF to ACI rat heart allograft system, a single PV injection of 20 μg 70–77 u-RT1.Aa induced specific tolerance of allografts. This therapy could be combined with CsA to induce transplantation tolerance. However, combination of 70–77u-RT1.Aa with CTLA4Ig, rapamycin, or AG-490 effectively blocked the induction of transplantation tolerance. Tolerance generated by allochimeric protein could be adoptively transferred to naive recipients. Intragraft cytokine mRNA levels showed a bias towards a Th2-type phenotype. Additionally, studies of cytokine signaling and activation of transcription factors revealed a requirement that these pathways remain available for signaling in order for transplantation tolerance to occur. These studies suggest that the generation of regulatory cells are required for the induction of transplantation tolerance through the use of allochimeric proteins. ^