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Human immune deficiency virus (HIV) infection of microglial cells in the brain leads to chronic neuroinflammation, which is antecedent to the development of HIV-associated neurocognitive disorders (HAND) in the majority of patients. Productively HIV infected microglia release multiple neurotoxins including proinflammatory cytokines and HIV proteins such as envelope glycoprotein (gp120) and transactivator of transcription (Tat). However, powerful counteracting silencing mechanisms in microglial cells result in the rapid shutdown of HIV expression to limit neuronal damage. Here we investigated whether the Nerve Growth Factor IB-like nuclear receptor Nurr1 (NR4A2), which is a repressor of inflammation in the brain, acts to directly restrict HIV expression. HIV silencing was substantially enhanced by Nurr1 agonists in both immortalized human microglial cells (hµglia) and induced pluripotent stem cells (iPSC)-derived human microglial cells (iMG). Overexpression of Nurr1 led to viral suppression, whereas by contrast, knock down (KD) of endogenous Nurr1 blocked HIV silencing. Chromatin immunoprecipitation (ChIP) assays showed that Nurr1 mediates recruitment of the CoREST/HDAC1/G9a/EZH2 transcription repressor complex to HIV promoter resulting in epigenetic silencing of active HIV. Transcriptomic studies demonstrated that in addition to repressing HIV transcription, Nurr1 also downregulated numerous cellular genes involved in inflammation, cell cycle, and metabolism, thus promoting HIV latency and microglial homoeostasis. Thus, Nurr1 plays a pivotal role in modulating the cycles of proviral reactivation by cytokines and potentiating the proviral transcriptional shutdown. These data highlight the therapeutic potential of Nurr1 agonists for inducing HIV silencing and microglial homeostasis and amelioration of the neuroinflammation associated with HAND.AUTHOR SUMMARYHIV enters the brain almost immediately after infection where it infects perivascular macrophages, microglia and, to a less extent, astrocytes. In previous work using an immortalized human microglial cell model, we observed that integrated HIV constantly underwent cycles of reactivation and subsequent silencing. In the present study, we found that the Nurr1 nuclear receptor is a key mediator of HIV silencing. The functional activation of Nurr1 by specific agonists, or the over expression of Nurr1, resulted in rapid silencing of activated HIV in microglial cells. Global gene expression analysis confirmed that Nurr1 not only repressed HIV expression but also regulated numerous genes involved in microglial homeostasis and inflammation. Thus, Nurr1 is pivotal for HIV silencing and repression of inflammation in the brain and is a promising therapeutic target for treatment of HAND.

Purslane is a widespread wild vegetable with both medicinal and edible properties. It is highly appreciated for its high nutritional value and is also considered as a high-quality feed resource for livestock and poultry. In this study, Sanhuang broilers were used to investigate the effect of feeding purslane diets on the growth performance in broilers and their gut microbiota. A total of 48 birds with good growth and uniform weight were selected and randomly allocated to four treatment groups A (control), B, C and D. Dietary treatments were fed with basal diet without purslane and diets containing 1%, 2% and 3% purslane. The 16S rDNA was amplified by PCR and sequenced using the Illumina HiSeq platform to analyze the composition and diversity of gut microbiota in the four sets of samples. The results showed that dietary inclusion of 2% and 3% purslane could significantly improve the growth performance and reduce the feed conversion ratio. Microbial diversity analysis indicated that the composition of gut microbiota of Sanhuang broilers mainly included Gallibacterium, Bacteroides and Escherichia-Shigella, etc. As the content of purslane was increased, the abundance of Lactobacillus increased significantly, and Escherichia-Shigella decreased. LEfSe analysis revealed that Bacteroides_caecigallinarum, Lachnospiraceae, Lactobacillales and Firmicutes had significant differences compared with the control group. PICRUSt analysis revealed bacteria mainly enriched in carbohydrate metabolism pathway due to the additon of purslane in the diet. These results suggest that the addition of purslane to feed could increase the abundance of Lactobacillus in intestine, modulate the environment of gut microbiota and promote the metabolism of carbohydrates to improve its growth performance. This study indicates that the effect of purslane on the growth-promoting performance of broilers might depend on its modulation on gut microbiota, so as to provide a certain scientific basis for the application of purslane in the feed industry.

HIV-1 infects lymphoid and myeloid cells, which can harbor a latent proviral reservoir responsible for maintaining lifelong infection. Glycolytic metabolism has been identified as a determinant of susceptibility to HIV-1 infection, but its role in the development and maintenance of HIV-1 latency has not been elucidated. By combining transcriptomic, proteomic and metabolomic analysis, we here show that transition to latent HIV-1 infection downregulates glycolysis, while viral reactivation by conventional stimuli reverts this effect. Decreased glycolytic output in latently infected cells is associated with downregulation of NAD+/NADH. Consequently, infected cells rely on the parallel pentose phosphate pathway and its main product, the antioxidant NADPH, fueling antioxidant pathways maintaining HIV-1 latency. Of note, blocking NADPH downstream effectors, thioredoxin and glutathione, favors HIV-1 reactivation from latency in lymphoid and myeloid cellular models. This provides a “shock and kill effect” decreasing proviral DNA in cells from people-living-with-HIV/AIDS. Overall, our data show that downmodulation of glycolysis is a metabolic signature of HIV-1 latency that can be exploited to target latently infected cells with eradication strategies.

Kaposi sarcoma-associated herpesvirus (KSHV) stimulates proliferation, angiogenesis, and inflammation to promote Kaposi sarcoma (KS) tumor growth, which involves various growth factors and cytokines. Previously, we found that KSHV infection of human umbilical vein endothelial cells (HUVECs) induces a transcriptional induction of the proangiogenic and proinflammatory cytokine angiopoietin-2 (Ang-2). Here, we report that KSHV induces rapid release of Ang-2 that is presynthesized and stored in the Weibel-Palade bodies (WPB) of endothelial cells upon binding to its integrin receptors. Blocking viral binding to integrins inhibits Ang-2 release. KSHV binding activates the integrin tyrosine kinase receptor signaling pathways, leading to tyrosine phosphorylation of focal adhesion kinase (FAK), the tyrosine kinase Src, and the Calα2 subunit of the l-type calcium channel to trigger rapid calcium (Ca(2+)) influx. Pretreatment of endothelial cells with specific inhibitors of protein tyrosine kinases inhibits KSHV-induced Ca(2+) influx and Ang-2 release. Inhibition of Ca(2+) mobilization with calcium channel blockers also inhibits Ang-2 release. Thus, the interaction between KSHV and its integrin receptors plays a key role in regulating rapid Ang-2 release from endothelial cells. This finding highlights a novel mechanism of viral induction of angiogenesis and inflammation, which might play important roles in the early event of KS tumor development.

Despite effective antiretroviral therapy (ART), HIV-associated neurocognitive disorders (HAND) are found in nearly one-third of patients. Using a cellular co-culture system including neurons and human microglia infected with HIV (hμglia/HIV), we investigated the hypothesis that HIV-dependent neurological degeneration results from the periodic emergence of HIV from latency within microglial cells in response to neuronal damage or inflammatory signals. When a clonal hμglia/HIV population (HC69) expressing HIV, or HIV infected human primary and iPSC-derived microglial cells, were cultured for a short-term (24 h) with healthy neurons, HIV was silenced. The neuron-dependent induction of latency in HC69 cells was recapitulated using induced pluripotent stem cell (iPSC)-derived GABAergic cortical (iCort) and dopaminergic (iDopaNer), but not motor (iMotorNer), neurons. By contrast, damaged neurons induce HIV expression in latently infected microglial cells. After 48–72 h co-culture, low levels of HIV expression appear to damage neurons, which further enhances HIV expression. There was a marked reduction in intact dendrites staining for microtubule associated protein 2 (MAP2) in the neurons exposed to HIV-expressing microglial cells, indicating extensive dendritic pruning. To model neurotoxicity induced by methamphetamine (METH), we treated cells with nM levels of METH and suboptimal levels of poly (I:C), a TLR3 agonist that mimics the effects of the circulating bacterial rRNA found in HIV infected patients. This combination of agents potently induced HIV expression, with the METH effect mediated by the σ1 receptor (σ1R). In co-cultures of HC69 cells with iCort neurons, the combination of METH and poly(I:C) induced HIV expression and dendritic damage beyond levels seen using either agent alone, Thus, our results demonstrate that the cross-talk between healthy neurons and microglia modulates HIV expression, while HIV expression impairs this intrinsic molecular mechanism resulting in the excessive and uncontrolled stimulation of microglia-mediated neurotoxicity., Author summary Now that HIV patients are living longer due to improved anti-retroviral therapy, the prevalence of HIV-associated neurocognitive disorders (HAND) is rising. HAND is linked to chronic inflammation and microgliosis and is exacerbated by substances of abuse such as methamphetamine (METH). Using co-culture models of neurons and microglia, we demonstrate that healthy neurons can suppress HIV transcription in infected microglia over a short-term (24 h). By contrast, damaged neurons reactivate latent HIV expression. Thus, over a longer term (72 h), neurons lose the ability to suppress HIV expression, resulting in enhanced neural injury. This damaging cycle of latency reversal and neuronal degeneration can be exacerbated by METH and inflammation. Our results support the hypothesis that the dendritic simplification and neuronal injury associated with HAND results from viral reactivation induced by neuronal and microglial cross-talk and exogenous inflammatory stimuli.

Kaposi’s sarcoma-associated herpesvirus (KSHV) is the causal agent for Kaposi’s sarcoma (KS), the most common malignancy in HIV/AIDS patients. Oral transmission through saliva is considered the most common route for spreading the virus among HIV/AIDS patients. However, the role of HIV-specific components in the cotransfection of KSHV is unclear. We demonstrate that exosomes purified from the saliva of HIV-positive patients and secreted by HIV-infected T-cell lines promote KSHV infectivity in immortalized and primary oral epithelial cells. HIV-associated exosomes promote KSHV infection, which depends on HIV trans-activation response element (TAR) RNA and EGFR of oral epithelial cells, which can be targeted for reducing KSHV infection. These results reveal that HIV-associated exosomes are a risk factor for KSHV infection in the HIV-infected population., Kaposi’s sarcoma-associated herpesvirus (KSHV) is the causal agent for Kaposi’s sarcoma (KS), the most common malignancy in people living with human immunodeficiency virus (HIV)/AIDS. The oral cavity is a major route for KSHV infection and transmission. However, how KSHV breaches the oral epithelial barrier for spreading to the body is not clear. Here, we show that exosomes purified from either the saliva of HIV-positive individuals or the culture supernatants of HIV-1-infected T-cell lines promote KSHV infectivity in immortalized and primary human oral epithelial cells. HIV-associated saliva exosomes contain the HIV trans-activation response element (TAR), Tat, and Nef RNAs but do not express Tat and Nef proteins. The TAR RNA in HIV-associated exosomes contributes to enhancing KSHV infectivity through the epidermal growth factor receptor (EGFR). An inhibitory aptamer against TAR RNA reduces KSHV infection facilitated by the synthetic TAR RNA in oral epithelial cells. Cetuximab, a monoclonal neutralizing antibody against EGFR, blocks HIV-associated exosome-enhanced KSHV infection. Our findings reveal that saliva containing HIV-associated exosomes is a risk factor for the enhancement of KSHV infection and that the inhibition of EGFR serves as a novel strategy for preventing KSHV infection and transmission in the oral cavity. IMPORTANCE Kaposi’s sarcoma-associated herpesvirus (KSHV) is the causal agent for Kaposi’s sarcoma (KS), the most common malignancy in HIV/AIDS patients. Oral transmission through saliva is considered the most common route for spreading the virus among HIV/AIDS patients. However, the role of HIV-specific components in the cotransfection of KSHV is unclear. We demonstrate that exosomes purified from the saliva of HIV-positive patients and secreted by HIV-infected T-cell lines promote KSHV infectivity in immortalized and primary oral epithelial cells. HIV-associated exosomes promote KSHV infection, which depends on HIV trans-activation response element (TAR) RNA and EGFR of oral epithelial cells, which can be targeted for reducing KSHV infection. These results reveal that HIV-associated exosomes are a risk factor for KSHV infection in the HIV-infected population.

A high prevalence of Kaposi's sarcoma (KS) is seen in diabetic patients. It is unknown if the physiological conditions of diabetes contribute to KS development. We found elevated levels of viral lytic gene expression when Kaposi's sarcoma-associated herpesvirus (KSHV)-infected cells were cultured in high-glucose medium. To demonstrate the association between high glucose levels and KSHV replication, we xenografted telomerase-immortalized human umbilical vein endothelial cells that are infected with KSHV (TIVE-KSHV cells) into hyperglycemic and normal nude mice. The injected cells expressed significantly higher levels of KSHV lytic genes in hyperglycemic mice than in normal mice. We further demonstrated that high glucose levels induced the production of hydrogen peroxide (H 2 O 2 ), which downregulated silent information regulator 1 (SIRT1), a class III histone deacetylase (HDAC), resulting in the epigenetic transactivation of KSHV lytic genes. These results suggest that high blood glucose levels in diabetic patients contribute to the development of KS by promoting KSHV lytic replication and infection. IMPORTANCE Multiple epidemiological studies have reported a higher prevalence of classic KS in diabetic patients. By using both in vitro and in vivo models, we demonstrated an association between high glucose levels and KSHV lytic replication. High glucose levels induce oxidative stress and the production of H 2 O 2 , which mediates the reactivation of latent KSHV through multiple mechanisms. Our results provide the first experimental evidence and mechanistic support for the association of classic KS with diabetes.

We have developed models of HIV latency using microglia derived from adult human patient brain cortex and transformed with the SV40 T large and hTERT antigens. Latent clones infected by HIV reporter viruses display high levels of spontaneous HIV reactivation in culture. BrainPhys, a medium highly representative of the CNS extracellular environment, containing low glucose and 1% FBS, reduced, but did not prevent, HIV reactivation. We hypothesized that spontaneous HIV reactivation in culture was due to the expression of pro-inflammatory genes, such as TNF-α, taking place in the absence of the natural inhibitory signals from astrocytes and neurons. Indeed, expression and secretion of TNF-α is strongly reduced in HIV-latently infected microglia compared to the subset of cells that have undergone spontaneous HIV reactivation. Whereas inhibitors of NF-κB or of macrophage activation only had a short-term silencing effect, addition of dexamethasone (DEXA), a glucocorticoid receptor (GR) agonist and mediator of anti-inflammation, silenced the HIV provirus in a long-term, and shRNA-mediated knock-down of GR activated HIV. DEXA also decreased secretion of a number of cytokines, including TNF-α. Chromatin immunoprecipitation analysis revealed that DEXA strongly increased GR occupancy at the HIV promoter, and reduced histone 3 acetylated levels. Moreover, TNF-α expression inhibitors in combination with DEXA induced further HIV silencing and increased the histone 3 lysine 27 tri-methylated epigenetic mark of repression at the HIV promoter region. We conclude that GR is a critical repressor of HIV transcription in microglia, and a novel potential pharmacological target to restrict HIV expression in the CNS. Electronic supplementary material The online version of this article (10.1007/s11481-018-9798-1) contains supplementary material, which is available to authorized users.

While T helper (Th) cells play a crucial role in host defense, an imbalance in Th effector subsets due to dysregulation in their differentiation and expansion contribute to inflammatory disorders. Here, we show that Casz1, whose function is previously unknown in CD4+ T cells, coordinates Th differentiation in vitro and in vivo. Casz1 deficiency in CD4+ T cells lowers susceptibility to experimental autoimmune encephalomyelitis, consistent with the reduced frequency of Th17 cells, despite an increase in Th1 cells in mice. Loss of Casz1 in the context of mucosal Candida infection severely impairs Th17 and Treg responses, and lowers the ability of the mice to clear the secondary infection. Importantly, in both the models, absence of Casz1 causes a significant diminution in IFN-γ+IL-17A+ double-positive inflammatory Th17 cells (Th1* cells) in tissues in vivo. Transcriptome analyses of CD4+ T cells lacking Casz1 show a signature consistent with defective Th17 differentiation. With regards to Th17 differentiation, Casz1 limits repressive histone marks and enables acquisition of permissive histone marks at Rorc, Il17a, Ahr, and Runx1 loci. Taken together, these data identify Casz1 as a new Th plasticity regulator having important clinical implications for autoimmune inflammation and mucosal immunity.

Covalent addition of a methyl group to the adenosine N6 (m6A) is an evolutionarily conserved and common RNA modification that is thought to modulate several aspects of RNA metabolism. While the presence of multiple m6A editing sites on diverse viral RNAs was reported starting almost 40 years ago, how m6A editing affects virus replication has remained unclear. Here, we used photo-crosslinking-assisted m6A sequencing techniques to precisely map several m6A editing sites on the HIV-1 genome and report that they cluster in the HIV-1 3’ untranslated region (3'UTR). Viral 3'UTR m6A sites or analogous cellular m6A sites strongly enhanced mRNA expression in cis by recruiting the cellular YTHDF m6A “reader” proteins. Reducing YTHDF expression inhibited, while YTHDF overexpression enhanced, HIV-1 protein and RNA expression, and virus replication in CD4+ T cells. These data identify m6A editing, and the resultant recruitment of YTHDF proteins, as major positive regulators of HIV-1 mRNA expression.

N 6 -adenosine methylation (m 6 A) is the most common posttranscriptional RNA modification in mammalian cells. We found that most transcripts encoded by the Kaposi's sarcoma-associated herpesvirus (KSHV) genome undergo m 6 A modification. The levels of m 6 A-modified mRNAs increased substantially upon stimulation for lytic replication. The blockage of m 6 A inhibited splicing of the pre-mRNA encoding the replication transcription activator (RTA), a key KSHV lytic switch protein, and halted viral lytic replication. We identified several m 6 A sites in RTA pre-mRNA crucial for splicing through interactions with YTH domain containing 1 (YTHDC1), an m 6 A nuclear reader protein, in conjunction with serine/arginine-rich splicing factor 3 (SRSF3) and SRSF10. Interestingly, RTA induced m 6 A and enhanced its own pre-mRNA splicing. Our results not only demonstrate an essential role of m 6 A in regulating RTA pre-mRNA splicing but also suggest that KSHV has evolved a mechanism to manipulate the host m 6 A machinery to its advantage in promoting lytic replication. IMPORTANCE KSHV productive lytic replication plays a pivotal role in the initiation and progression of Kaposi's sarcoma tumors. Previous studies suggested that the KSHV switch from latency to lytic replication is primarily controlled at the chromatin level through histone and DNA modifications. The present work reports for the first time that KSHV genome-encoded mRNAs undergo m 6 A modification, which represents a new mechanism at the posttranscriptional level in the control of viral replication.

Tumor associated macrophages (TAMs) promote angiogenesis, tumor invasion and metastasis, and suppression of anti-tumor immunity. These myeloid cells originate from monocytes, which differentiate into TAMs upon exposure to the local tumor microenvironment. We previously reported that Kaposi's sarcoma-associated herpes virus (KSHV) infection of endothelial cells induces the cytokine angiopoietin-2 (Ang-2) to promote migration of monocytes into tumors. Here we report that KSHV infection of endothelial cells induces additional cytokines including interleukin-6 (IL-6), interleukin-10 (IL-10), and interleukin-13 (IL-13) that drive monocytes to differentiate and polarize into TAMs. The KSHV-induced TAMs not only express TAM-specific markers such as CD-163 and legumain (LGMN) but also display a gene expression profile with characteristic features of viral infection. More importantly, KSHV-induced TAMs enhance tumor growth in nude mice. These results are consistent with the strong presence of TAMs in Kaposi's sarcoma (KS) tumors. Therefore, KSHV infection of endothelial cells generates a local microenvironment that not only promotes the recruitment of monocytes but also induces their differentiation and polarization into TAMs. These findings reveal a new mechanism of KSHV contribution to KS tumor development.

Kaposi's sarcoma-associated herpesvirus (KSHV) establishes persistent latent infection in immunocompetent hosts. Disruption of KSHV latency results in viral lytic replication, which promotes the development of KSHV-related malignancies in immunocompromised individuals. While inhibitors of classes I and II histone deacetylases (HDACs) potently reactivate KSHV from latency, the role of class III HDAC sirtuins (SIRTs) in KSHV latency remains unclear. Here, we examined the effects of inhibitors of SIRTs, nicotinamide (NAM) and sirtinol, on KSHV reactivation from latency. Treatment of latently KSHV-infected cells with NAM or sirtinol induced transcripts and proteins of the master lytic transactivator RTA (ORF50), early lytic genes ORF57 and ORF59, and late lytic gene ORF65 and increased the production of infectious virions. NAM increased the acetylation of histones H3 and H4 as well as the level of the active histone H3 trimethyl Lys4 (H3K4me3) mark but decreased the level of the repressive histone H3 trimethyl Lys27 (H3K27me3) mark in the RTA promoter. Consistent with these results, we detected SIRT1 binding to the RTA promoter. Importantly, knockdown of SIRT1 was sufficient to increase the expression of KSHV lytic genes. Accordingly, the level of the H3K4me3 mark in the RTA promoter was increased following SIRT1 knockdown, while that of the H3K27me3 mark was decreased. Furthermore, SIRT1 interacted with RTA and inhibited RTA transactivation of its own promoter and that of its downstream target, the viral interleukin-6 gene. These results indicate that SIRT1 regulates KSHV latency by inhibiting different stages of viral lytic replication and link the cellular metabolic state with the KSHV life cycle. IMPORTANCE Kaposi's sarcoma-associated herpesvirus (KSHV) is the causal agent of several malignancies, including Kaposi's sarcoma, commonly found in immunocompromised patients. While latent infection is required for the development of KSHV-induced malignancies, viral lytic replication also promotes disease progression. However, the mechanism controlling KSHV latent versus lytic replication remains unclear. In this study, we found that class III histone deacetylases (HDACs), also known as SIRTs, whose activities are linked to the cellular metabolic state, mediate KSHV replication. Inhibitors of SIRTs can reactivate KSHV from latency. SIRTs mediate KSHV latency by epigenetically silencing a key KSHV lytic replication activator, RTA. We found that one of the SIRTs, SIRT1, binds to the RTA promoter to mediate KSHV latency. Knockdown of SIRT1 is sufficient to induce epigenetic remodeling and KSHV lytic replication. SIRT1 also interacts with RTA and inhibits RTA's transactivation function, preventing the expression of its downstream genes. Our results indicate that SIRTs regulate KSHV latency by inhibiting different stages of viral lytic replication and link the cellular metabolic state with the KSHV life cycle.

Heparinase I (HepI), which specifically cleaves heparin and heparan sulfate, is one of the most extensively studied glycosaminoglycan lyases. Low productivity of HepI has largely hindered its industrial and pharmaceutical applications. Loss of bacterial HepI enzyme activity through poor thermostability during its expression and purification process in production can be an important issue. In this study, using a thermostabilization strategy combining site-directed mutagenesis and calcium ion addition during its production markedly improved the yield of maltose-binding protein-fused HepI (MBP–HepI) from recombinant Escherichia coli. Substitution of Cys297 to serine in MBP–HepI offered a 30.6 % increase in the recovered total enzyme activity due to a mutation-induced thermostabilizing effect. Furthermore, upon addition of Ca2+ as a stabilizer at optimized concentrations throughout its expression, extraction, and purification process, purified mutant MBP–HepI showed a specific activity of 56.3 IU/mg, 206 % higher than that of the wild type obtained without Ca2+ addition, along with a 177 % increase in the recovered total enzyme activity. The enzyme obtained through this novel approach also exhibited significantly enhanced thermostability, as indicated by both experimental data and the kinetic modeling. High-yield production of thermostable MBP–HepI using the present system will facilitate its applications in laboratory-scale heparin analysis as well as industrial-scale production of low molecular weight heparin as an improved anticoagulant substitute.

Infections by viruses are associated with approximately 12% of human cancer. Kaposi's sarcoma-associated herpesvirus (KSHV) is causally linked to several malignancies commonly found in AIDS patients. The mechanism of KSHV-induced oncogenesis remains elusive, due in part to the lack of an adequate experimental system for cellular transformation of primary cells. Here, we report efficient infection and cellular transformation of primary rat embryonic metanephric mesenchymal precursor cells (MM cells) by KSHV. Cellular transformation occurred at as early as day 4 after infection and in nearly all infected cells. Transformed cells expressed hallmark vascular endothelial, lymphatic endothelial, and mesenchymal markers and efficiently induced tumors in nude mice. KSHV established latent infection in MM cells, and lytic induction resulted in low levels of detectable infectious virions despite robust expression of lytic genes. Most KSHV-induced tumor cells were in a latent state, although a few showed heterogeneous expression of lytic genes. This efficient system for KSHV cellular transformation of primary cells might facilitate the study of growth deregulation mechanisms resulting from KSHV infections.

Kaposi's sarcoma-associated herpesvirus (KSHV) is causally linked to several acquired immune deficiency syndrome-related malignancies, including Kaposi's sarcoma, primary effusion lymphoma (PEL) and a subset of multicentric Castleman's disease. Control of viral lytic replication is essential for KSHV latency, evasion of the host immune system and induction of tumours. Here, we show that deletion of a 14 microRNA (miRNA) cluster from the KSHV genome significantly enhances viral lytic replication as a result of reduced NF-kappaB activity. The miRNA cluster regulates the NF-kappaB pathway by reducing expression of IkappaBalpha protein, an inhibitor of NF-kappaB complexes. Computational and miRNA seed mutagenesis analyses were used to identify KSHV miR-K1, which directly regulates the IkappaBalpha protein level by targeting the 3'UTR of its transcript. Expression of miR-K1 is sufficient to rescue NF-kappaB activity and inhibit viral lytic replication, whereas inhibition of miR-K1 in KSHV-infected PEL cells has the opposite effect. Thus, KSHV encodes an miRNA to control viral replication by activating the NF-kappaB pathway. These results demonstrate an important role for KSHV miRNAs in regulating viral latency and lytic replication by manipulating the host survival pathway.

Kaposi's sarcoma-associated herpesvirus (KSHV) replication and transcription activator (RTA) encoded by ORF50 is a lytic switch protein for viral reactivation from latency. The expression of RTA activates the expression of downstream viral genes, and is necessary for triggering the full viral lytic program. Using chromatin immunoprecipitation assay coupled with a KSHV whole-genome tiling microarray (ChIP-on-chip) approach, we identified a set of 19 RTA binding sites in the KSHV genome in a KSHV-infected cell line BCBL-1. These binding sites are located in the regions of promoters, introns, or exons of KSHV genes including ORF8, ORFK4.1, ORFK5, PAN, ORF16, ORF29, ORF45, ORF50, ORFK8, ORFK10.1, ORF59, ORFK12, ORF71/72, ORFK14/ORF74, and ORFK15, the two origins of lytic replication OriLyt-L and OriLyt-R, and the microRNA cluster. We confirmed these RTA binding sites by ChIP and quantitative real-time PCR. We further mapped the RTA binding site in the first intron of the ORFK15 gene, and determined that it is RTA-responsive. The ORFK15 RTA binding sequence TTCCAGGAA TTCCTGGAA consists of a palindromic structure of two tandem repeats, of which each itself is also an imperfect inverted repeat. Reporter assay and electrophoretic mobility shift assay confirmed the binding of the RTA protein to this sequence in vitro. Sequence alignment with other RTA binding sites identified the RTA consensus binding motif as TTCCAGGAT(N)0–16TTCCTGGGA. Interestingly, most of the identified RTA binding sites contain only half or part of this RTA binding motif. These results suggest the complexity of RTA binding in vivo, and the involvement of other cellular or viral transcription factors during RTA transactivation of target genes.

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.

Enhanced production of MBP (maltose-binding protein)-heparinase I (HepA) fusion protein in recombinant Escherichia coli was achieved by the optimization of the M9-based culture medium. First, by using the modified expression vector capable of releasing the C-terminus of HepA free to improve the specific activity of MBP–HepA, characteristics of the purified fusion protein were analyzed, which were similar to those of the native HepA except for the decreased affinity towards the substrate. M9-based culture medium was subsequently optimized for the enzyme production by orthogonal experimental design in shake flasks. Three major components were examined, namely glucose, yeast extract and calcium ion. The recombinant E. coli was further cultivated in a fermentor. As a result, total activity of HepA reached 20,650 IU l −1 in the optimized medium by a fed-batch mode in the 5-l fermentor . This study indicated that effective production of MBP–HepA by the present system would facilitate the large scale preparation of low molecular weight heparin (LMWH), which is a useful anticoagulant drug.

Infection by Kaposi's sarcoma-associated herpesvirus (KSHV) is required for the development of Kaposi's sarcoma (KS), a highly inflammatory angiogenic tumor of endothelial cells commonly found in untreated AIDS patients. Angiopoietin 2 (Ang-2) modulates the vasculature during inflammation and angiogenesis, but the mechanism by which KSHV regulates Ang-2 expression has not been investigated. Here, we show that KSHV infection of primary human umbilical vein endothelial cells induced the expression and release of Ang-2, which in turn was required for KSHV-induced paracrine-dependent angiogenesis in vivo. Ang-2 was strongly expressed in small vessels and spindle tumor cells in KS tumors. Mechanistically, KSHV activated the Ang-2 promoter via AP-1 and Ets1 transcriptional factors, which were mediated by ERK, JNK, and p38 mitogen-activated protein kinase (MAPK) pathways. Our findings demonstrate the importance of Ang-2 in KS angiogenesis and define a novel role for AP-1 and MAPK pathways in regulating angiogenesis. This study also illustrates a distinct mechanism by which a tumor virus modulates vasculature to promote tumorigenesis and exemplifies the convergence of oncogenesis and angiogenesis pathways in tumor development.

The genomes of herpesviruses and HIV become silent during latency through multiple chromatin silencing mechanisms including: histone deacetylation, repressive histone methylation, and DNA methylation. Reactivation of the latent virus requires removal of the chromatin silencing marks and their replacement by activating modifications such as histone acetylation and activating histone methylation. In a complementary mechanism, RNA Polymerase II (RNAP II) elongation is regulated by the positive transcription elongation factor b (P-TEFb)-dependent phosphorylation of Ser2 residues on its C-terminal domain. In resting T-cells latently infected by HIV P-TEFb levels are extremely low. We found that a group of short chain fatty acids (SCFAs) produced by oral bacteria not only promote histone acetylation but also changes the histone methylation dynamics by decreasing repressive histone methylation while increasing activating histone methylation. SCFAs also block DNA methylation and activate P-TEFb to enable elongation of stalled RNA polymerase II. Thus these molecules do not simply act as histone deacetylase (HDAC) inhibitors as previously claimed. Instead, they impact multiple complementary epigenetic regulatory mechanisms to promote highly efficient reactivation of latent viruses.

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.

Coordinated expression of viral genes in primary infection is essential for successful infection of host cells. We examined the expression profiles of Kaposi's sarcoma-associated herpesvirus (KSHV) transcripts in productive primary infection of primary human umbilical vein endothelial cells by whole-genome reverse-transcription real-time quantitative PCR. The latent transcripts were expressed early and sustained at high levels throughout the infection while the lytic transcripts were expressed in the order of immediate early, early, and lytic transcripts, all of which culminated before the production of infectious virions. Significantly, transcripts encoding genes with host modulating functions, including mitogenic and cell cycle-regulatory, immune-modulating, and anti-apoptotic genes, were expressed before those encoding viral structure and replication genes, and sustained at high levels throughout the infection, suggesting KSHV manipulation of host environment to facilitate infection. The KSHV transcriptional program in a primary infection defined in this study should provide a basis for further investigation of virus–cell interactions.

HIV patients with severe periodontitis have high levels of residual virus in their saliva and plasma despite effective therapy (HAART). Multiple short chain fatty acids (SCFAs) from periodontal pathogens reactivate HIV-1 in both Jurkat and primary T-cell models of latency. SCFAs not only activate positive transcription elongation factor b (P-TEFb), which is an essential cellular cofactor for Tat, but can also reverse chromatin blocks by inducing histone modifications. SCFAs simultaneously increase histone acetylation by inhibiting class-1/2 histone deacetylases (HDACs) and decrease repressive histone tri-methylation at the proviral LTR by downregulating expression of the class-3 HDAC sirtuin-1 (SIRT1), and the histone methyltransferases enhancer of Zeste homolog 2 (EZH2) and suppressor of variegation 3–9 homolog 1 (SUV39H1). Our findings provide a mechanistic link between periodontal disease and enhanced HIV-1 replication, and suggest that treatment of periodontal disease, or blocking the activities of SCFAs, will have a therapeutic benefit for HIV patients.

Periodontal pathogens such as Porphyromonas gingivalis and Fusobacterium nucleatum produce five different short-chain fatty acids (SCFAs) as metabolic by-products. We detect significantly higher levels of SCFAs in the saliva of patients with severe periodontal disease. The different SCFAs stimulate lytic gene expression of Kaposi's sarcoma-associated herpesvirus (KSHV) dose dependently and synergistically. SCFAs inhibit class-1/2 histone deacetylases (HDACs) and downregulate expression of silent information regulator-1 (SIRT1). SCFAs also downregulate expression of enhancer of zeste homolog2 (EZH2) and suppressor of variegation 3-9 homolog1 (SUV39H1), which are two histone N-lysine methyltransferases (HLMTs). By suppressing the different components of host epigenetic regulatory machinery, SCFAs increase histone acetylation and decrease repressive histone trimethylations to transactivate the viral chromatin. These new findings provide mechanistic support that SCFAs from periodontal pathogens stimulate KSHV replication and infection in the oral cavity and are potential risk factors for development of oral Kaposi's sarcoma (KS). IMPORTANCE About 20% of KS patients develop KS lesions first in the oral cavity, while other patients never develop oral KS. It is not known if the oral microenvironment plays a role in oral KS tumor development. In this work, we demonstrate that a group of metabolic by-products, namely, short-chain fatty acids, from bacteria that cause periodontal disease promote lytic replication of KSHV, the etiological agent associated with KS. These new findings provide mechanistic support that periodontal pathogens create a unique microenvironment in the oral cavity that contributes to KSHV replication and development of oral KS.

In this study, we assessed the effects of in vivo hypoxia on the expression of Tie-2 receptors and angiopoietins in various organs of conscious rats and correlated these effects with the expression of hypoxia-inducible factor-1 (HIF-1). RT-PCR and Southern blotting were used to amplify mRNA expression of angiopoietin-1, -2, and -3, Tie-2, and HIF-1α in tissues of normoxic and hypoxic (fraction of inspired oxygen of 9–10% for either 12 or 48 h) rats. Hypoxia provoked a decline in angiopoietin-1 mRNA and Tie-2 mRNA, protein, and phosphorylation levels in the lung, liver, cerebellum, and heart but not in the kidney and diaphragm. In comparison, hypoxia raised the levels of angiopoietin-2 mRNA in the cerebellum and angiopoietin-3 mRNA in the lung, kidney, and diaphragm. HIF-1α mRNA was abundant in most organs of normoxic rats but was significantly induced in the kidney and diaphragm of hypoxic rats. We conclude that in vivo hypoxia exerts inhibitory effects on the activity of the angiopoietin-1/Tie-2 receptor pathway through reduction of angiopoietin-1 and upregulation of angiopoietin-2 and -3. Induction of angiopoietin-3 in the kidney and diaphragm of hypoxic rats could be mediated through the HIF-1 transcription factor.

Genomes of Spiroplasma citri strains have rearranged frequently during their evolution, partly due to multiple integrated sequences of spiroplasma viruses. To understand better the role of viral se...

Kaposi sarcoma-associated herpesvirus (KSHV) is a tumor virus encoding several proto-oncogenes. However, the roles of these viral genes in KSHV-induced tumorigenesis have not been defined. In this study, we used a recently developed model of KSHV-induced cellular transformation and tumorigenesis combining with a reverse genetic system to examine the role of a KSHV latent gene vCyclin (ORF72), a cellular Cyclin D2 homolog, in KSHV-induced oncogenesis. Deletion of vCyclin did not affect cell proliferation and cell cycle progression at a low-density condition, when cells were at an active proliferation state. However, vCyclin mutant cells were contact-inhibited and arrested at G 1 phase at a high-density condition. As a result, vCyclin mutant cells formed less and smaller colonies in soft agar assay. Nude mice inoculated with vCyclin mutant cells had reduced tumor incidence and extended tumor latency and survival compared with mice inoculated with wild-type (WT) virus-infected cells. WT but not mutant virus effectively induced Cyclin-dependent kinase inhibitor p27/Kip1 Ser10 phosphorylation and cytoplasmic relocalization. shRNA knockdown of p27 released the blockage of the mutant cells from cell cycle arrest at G 1 phase at a high-density condition. Together, these results indicate that vCyclin primarily functions to enhance cellular transformation and tumorigenesis by promoting cell cycle progression and cell proliferation at a contact-inhibited condition.

Genetic variations in the plant pathogen, Spiroplasma citri strain BR3, were characterized through physical genome mapping of the original isolate, BR3-3X, and two derivatives, BR3-T and BR3-G, obtained after several years of different maintenance conditions. BR3-T was transmitted from plant to plant via its natural insect vector, the leafhopper Circulifer tenellus, while BR3-G was maintained only in plants by periodic grafting and has lost its ability to be insect transmitted. By pulsed field gel electrophoresis (PFGE) analysis and DNA hybridization, extensive changes in chromosomal DNA restriction patterns relative to the parent, BR3-3X, were observed in both BR3-T and BR3-G, each of which also had a larger genome size than the parent line. Genetic organization was relatively conserved between BR3-T and BR3-3X. In contrast, a large chromosomal inversion and deletions of approximately 10 kb near each of the inversion borders were observed in BR3-G. One of the deletions, which included several possibly functional genes, was closely linked to a SpV1-related transposase gene. The locations of the deletion borders were also determined. The results of this study demonstrated remarkable genome instability of spiroplasmas.

Four lines of Spiroplasma citri strain BR3, derived from different maintenance conditions over several years and differing in their ability to be transmitted by the beet leafhopper Circulifer tenellus were characterized. The lines included BR3-T (transmissible), maintained in turnip by leafhopper transmission; BR3-G (now nontransmissible), maintained in plants by periodic graft transmission; BR3-P (now rarely transmissible), subcultured in artificial medium over 130 times; and BR3-M (transmissible), subcultured 43 times. Although all four lines had similar overall protein profiles, the two transmissible lines each contained two proteins missing in the non- or rarely transmitted lines. In addition, one protein was unique to BR3-M and another was unique to BR3-P. Spiralin, a major S. citri membrane protein, had dual mobility in line BR3-G only. Patterns of extrachromosomal DNA and restricted total DNA also were similar, although differences occurred among the four lines. The genome of line BR3-G was larger than those of the other lines and unique restriction bands occurred in this line. Protein and DNA profiles of six to eight individual clones of each line also were compared. Protein patterns within each clone were indistinguishable except for a difference in the migration rate of spiralin in clones of BR3-G. Restricted total DNA showed differential patterns among clones of each line, possibly reflecting differences in extrachromosomal DNA. Molecular differences among the spiroplasma lines may reflect the selection pressures of the different environments in which they were maintained and suggest genes and proteins that may be involved in the biological phenotypes of these lines.Key words: spiroplasma, Mollicutes, insect transmission.

Background Biologically active inclusion bodies (IBs) have gained much attention in recent years. Fusion with IB-inducing partner has been shown to be an efficient strategy for generating active IBs. To make full use of the advantages of active IBs, one of the key issues will be to improve the activity yield of IBs when expressed in cells, which would need more choices on IB-inducing fusion partners and approaches for engineering IBs. Green fluorescent protein (GFP) has been reported to aggregate when overexpressed, but GFP fusion has not been considered as an IB-inducing approach for these fusion proteins so far. In addition, the role of linker in fusion proteins has been shown to be important for protein characteristics, yet impact of linker on active IBs has never been reported. Results Here we report that by fusing GFP and acid phosphatase PhoC via a linker region, the resultant PhoC-GFPs were expressed largely as IBs. These IBs show high levels of specific fluorescence and specific PhoC activities (phosphatase and phosphotransferase), and can account for up to over 80% of the total PhoC activities in the cells. We further demonstrated that the aggregation of GFP moiety in the fusion protein plays an essential role in the formation of PhoC-GFP IBs. In addition, PhoC-GFP IBs with linkers of different flexibility were found to exhibit different levels of activities and ratios in the cells, suggesting that the linker region can be utilized to manipulate the characteristics of active IBs. Conclusions Our results show that active IBs of PhoC can be generated by GFP fusion, demonstrating for the first time the potential of GFP fusion to induce active IB formation of another soluble protein. We also show that the linker sequence in PhoC-GFP fusion proteins plays an important role on the regulation of IB characteristics, providing an alternative and important approach for engineering of active IBs with the goal of obtaining high activity yield of IBs.

Enzymatic degradation of heparin has great potential as an ecological and specific way to produce low molecular weight heparin. However, the commercial use of heparinase I (HepA), one of the most important heparin lyases, has been hampered by low productivity and poor thermostability. Fusion with green fluorescent protein (GFP) or maltose-binding protein (MBP) has shown potential in facilitating the industrial use of HepA. Thus, tripartite fusion of GFP, MBP and HepA would be a promising approach. Therefore, in the present study, the tripartite fusion strategy was systematically studied, mainly focusing on the fusion order and the linker sequence, to obtain a fusion protein offering one-step purification and real-time detection of HepA activity by fluorescence as well as high HepA activity and thermostability. Our results show that fusion order is important for MBP binding affinity and HepA activity, while the linker sequences at domain junctions have significant effects on protein expression level, HepA activity and thermostability as well as GFP fluorescence. The best tripartite fusion was identified as MBP-(EAAAK)(3)-GFP-(GGGGS)(3)-HepA, which shows potential to facilitate the production of HepA and its application in industrial preparation of low molecular weight heparin.

Kaposi sarcoma (KS) tumors often contain a wild-type p53. However, the function of this tumor suppressor in KS tumor cells is inhibited by both MDM2 and latent nuclear antigen (LANA) of Kaposi sarcoma-associated herpes virus (KSHV). Here, we report that MDM2 antagonist Nutlin-3 efficiently reactivates p53 in telomerase-immortalized human umbilical vein endothelial cells (TIVE) that had been malignantly transformed by KSHV as well as in KS tumor cells. Reactivation of p53 results in a G 1 cell cycle arrest, leading to inhibition of proliferation and apoptosis. Nutlin-3 inhibits the growth of "KS-like" tumors resulting from xenografted TIVE-KSHV cells in nude mice. In addition, Nutlin-3 strongly inhibits expression of the pro-angiogenic and pro-inflammatory cytokine angiopoietin-2 (Ang-2). It also disrupts viral latency by inducing expression of KSHV lytic genes. These results suggest that Nutlin-3 might serve as a novel therapy for KS.

Ubiquitination, a post-translational modification, mediates diverse cellular functions including endocytic transport of molecules. Kaposi's sarcoma-associated herpesvirus (KSHV), an enveloped herpesvirus, enters endothelial cells primarily through clathrin-mediated endocytosis. Whether ubiquitination and proteasome activity regulates KSHV entry and endocytosis remains unknown. We showed that inhibition of proteasome activity reduced KSHV entry into endothelial cells and intracellular trafficking to nuclei, thus preventing KSHV infection of the cells. Three-dimensional (3-D) analyses revealed accumulation of KSHV particles in a cytoplasmic compartment identified as EEA1+ endosomal vesicles upon proteasome inhibition. KSHV particles are colocalized with ubiquitin-binding proteins epsin and eps15. Furthermore, ubiquitination mediates internalization of both KSHV and one of its receptors integrin β1. KSHV particles are colocalized with activated forms of the E3 ligase c-Cbl. Knock-down of c-Cbl or inhibition of its phosphorylation reduced viral entry and intracellular trafficking, resulting in decreased KSHV infectivity. These results demonstrate that ubiquitination mediates internalization of both KSHV and one of its cognate receptors integrin β1, and identify c-Cbl as a potential E3 ligase that facilitates this process., Author Summary Ubiquitination, a post-translational modification, mediates important cellular functions including endocytic transport of molecules. Kaposi's sarcoma-associated herpesvirus (KSHV) is a gammaherpesvirus linked to the development of Kaposi's sarcoma, an endothelial malignancy commonly found in AIDS patients, and several other malignancies. KSHV enters endothelial cells primarily through clathrin-mediated endocytosis. In this study, we show that the proteasome activity is required for KSHV entry into endothelial cells and intracellular trafficking to nuclei. Inhibition of proteasome activity reduced KSHV infectivity and led to the accumulation of KSHV particles in EEA1+ early endosomal vesicles. Furthermore, we show that ubiquitination mediates the internalization of both KSHV and one of its receptors integrin β1. KSHV particles are colocalized with ubiquitin-binding proteins epsin and eps15, as well as activated forms of the E3 ligase c-Cbl. Knock-down of c-Cbl or inhibition of its phosphorylation blocked KSHV entry and trafficking, thus preventing KSHV infection of endothelial cells. Together, these results illustrate the essential role of ubiquitination during the internalization of KSHV and its cognate receptor integrin β1. The identification of an E3 ligase that mediates the ubiquitination of KSHV and its cognate receptor integrin β1 leading to viral entry provide a potential therapeutic target for this oncogenic virus.

Kaposi's sarcoma-associated herpesvirus (KSHV) establishes a latent infection in the host following an acute infection. Reactivation from latency contributes to the development of KSHV-induced malignancies, which include Kaposi's sarcoma (KS), the most common cancer in untreated AIDS patients, primary effusion lymphoma and multicentric Castleman's disease. However, the physiological cues that trigger KSHV reactivation remain unclear. Here, we show that the reactive oxygen species (ROS) hydrogen peroxide (H2O2) induces KSHV reactivation from latency through both autocrine and paracrine signaling. Furthermore, KSHV spontaneous lytic replication, and KSHV reactivation from latency induced by oxidative stress, hypoxia, and proinflammatory and proangiogenic cytokines are mediated by H2O2. Mechanistically, H2O2 induction of KSHV reactivation depends on the activation of mitogen-activated protein kinase ERK1/2, JNK, and p38 pathways. Significantly, H2O2 scavengers N-acetyl-L-cysteine (NAC), catalase and glutathione inhibit KSHV lytic replication in culture. In a mouse model of KSHV-induced lymphoma, NAC effectively inhibits KSHV lytic replication and significantly prolongs the lifespan of the mice. These results directly relate KSHV reactivation to oxidative stress and inflammation, which are physiological hallmarks of KS patients. The discovery of this novel mechanism of KSHV reactivation indicates that antioxidants and anti-inflammation drugs could be promising preventive and therapeutic agents for effectively targeting KSHV replication and KSHV-related malignancies., Author Summary Kaposi's sarcoma-associated herpesvirus (KSHV) is the etiologic agent of all clinical forms of Kaposi's sarcoma (KS) and several other malignancies. The life cycle of KSHV consists of latent and lytic phases. While establishment of viral latency is essential for KSHV to evade host immune surveillances, viral lytic replication promotes KSHV-induced malignancies. In this study, we show that the reactive oxygen species (ROS) hydrogen peroxide (H2O2) induces KSHV reactivation from latency. Furthermore, induction of KSHV reactivation by oxidative stress, hypoxia, and proinflammatory and proangiogenic cytokines, which are physiological hallmarks in all clinical forms of KS patients, is mediated by H2O2. Significantly, antioxidants inhibit H2O2-induced KSHV lytic replication in culture and in a mouse model of KSHV-induced lymphoma. These results show that ROS is likely an important physiological cue that triggers KSHV replication. The discovery of this novel mechanism of KSHV reactivation indicates that antioxidants and anti-inflammation drugs might be promising preventive and therapeutic agents for effectively targeting KSHV replication and KSHV-related malignancies.

The life cycle of Kaposi's sarcoma-associated herpesvirus (KSHV) consists of latent and lytic replication phases. During latent infection, only a limited number of KSHV genes are expressed. However, this phase of replication is essential for persistent infection, evasion of host immune response, and induction of KSHV-related malignancies. KSHV reactivation from latency produces a wide range of viral products and infectious virions. The resultingde novoinfection and viral lytic products modulate diverse cellular pathways and stromal microenvironment, which promote the development of Kaposi's sarcoma (KS). The mechanisms controlling KSHV latency and reactivation are complex, involving both viral and host factors, and are modulated by diverse environmental factors. Here, we review the cellular and molecular basis of KSHV latency and reactivation with a focus on the most recent advancements in the field.

Kaposi's sarcoma (KS) is a vascular tumor of proliferative endothelial cells caused by infection of Kaposi's sarcoma-associated herpesvirus (KSHV). Aberrant vascular permeability is a hallmark of KS manifested as multifocal edematous skin and visceral lesions with dysregulated angiogenesis and vast inflammatory infiltrations. In this study, we showed that KSHV infection increased the permeability of confluent endothelial monolayers to serum albumin, blood-derived cells, KSHV-infected cells and KSHV virions. KSHV-induced permeability was associated with the disruption of adherens junctions and degradation of vascular endothelial (VE)-cadherin protein. Ultraviolet irradiation that inactivated KSHV virions and cycloheximide that blocked de novo protein synthesis failed to reverse KSHV-induced disruption of adherens junctions. However, soluble heparin that blocked KSHV entry into cells completely inhibited KSHV-induced permeability. Furthermore, KSHV-induced degradation of VE-cadherin was dose-dependent on the internalized virus particles. Together, these results indicate that KSHV infection induces vascular permeability by inducing VE-cadherin degradation during virus entry into cells. KSHV-induced aberrant vascular permeability could facilitate virus spread, promote inflammation and angiogenesis, and contribute to the pathogenesis of KSHV-induced malignancies.

Spiroplasma citri, transmitted by phloem-feeding leafhoppers, moves from the gut lumen through the gut wall, hemolymph, and salivary glands and multiplies in insect tissues. Nontransmissible lines were deficient in their ability to cross these barriers. Molecular analysis revealed extensive chromosomal rearrangements between the transmissible and nontransmissible spiroplasma lines including a large chromosomal inversion and deletions of about 10 kb at each inversion border. One open reading frame of the deleted region, cloned from the transmissible strain BR3-3X, encodes an integral membrane protein of 58 kDa that shares limited sequence similarity with major adhesin proteins of two zoopathogenic mycoplasmas. Adhesion of spiroplasmas to cultured leafhopper cells was inhibited by proteases, suggesting that adherence to host cells is mediated by spiroplasma membrane protein(s). A hypothetical model for insect transmission of phytopathogenic mollicutes is presented.

Kaposi's sarcoma (KS) is a vascular tumor of proliferative endothelial cells caused by KS-associated herpesvirus (KSHV) infection. Aberrant vascular permeability is a hallmark of KS manifested as multifocal edematous skin and visceral lesions with dysregulated angiogenesis and vast inflammatory infiltrations. In this study, we showed that KSHV infection increased the permeability of confluent endothelial monolayers to serum albumin, blood-derived cells, KSHV-infected cells, and KSHV virions. KSHV-induced permeability was associated with the disruption of adherens junctions and the degradation of vascular endothelial cadherin (VE-cadherin) protein. Both the inactivation of KSHV virions by UV irradiation and the blockage of de novo protein synthesis with cycloheximide failed to reverse the KSHV-induced disruption of adherens junctions. However, soluble heparin that blocked KSHV entry into cells completely inhibited KSHV-induced permeability. Furthermore, the KSHV-induced degradation of VE-cadherin was dose dependent on the internalized virus particles. Together, these results indicate that KSHV infection induces vascular permeability by inducing VE-cadherin degradation during virus entry into cells. KSHV-induced aberrant vascular permeability could facilitate virus spread, promote inflammation and angiogenesis, and contribute to the pathogenesis of KSHV-induced malignancies.

Following primary infection, KSHV establishes a lifelong persistent latent infection in the host. The mechanism of KSHV latency is not fully understood. The latent nuclear antigen (LANA or LNA) encoded by ORF73 is one of a few viral genes expressed during KSHV latency, and is consistently detected in all KSHV-related malignancies. LANA is essential for KSHV episome persistence, and regulates the expression of viral lytic genes through epigenetic silencing, and inhibition of the expression and transactivation function of the key KSHV lytic replication initiator RTA (ORF50). In this study, we used a genetic approach to examine the role of LANA in regulating KSHV lytic replication program. Deletion of LANA did not affect the expression of its adjacent genes vCyclin (ORF72) and vFLIP (ORF71). In contrast, the expression levels of viral lytic genes including immediate-early gene RTA, early genes MTA (ORF57), vIL-6 (ORF-K2) and ORF59, and late gene ORF-K8.1 were increased before and after viral lytic induction with 12-O-tetradecanoyl-phorbol-13-acetate and sodium butyrate. This enhanced expression of viral lytic genes was also observed following overexpression of RTA with or without simultaneous chemical induction. Consistent with these results, the LANA mutant cells produced more infectious virions than the wild-type virus cells did. Furthermore, genetic repair of the mutant virus reverted the phenotypes to those of wild-type virus. Together, these results have demonstrated that, in the context of viral genome, LANA contributes to KSHV latency by regulating the expression of RTA and its downstream genes.

Lytic replication of Kaposi's sarcoma-associated herpesvirus (KSHV) promotes the progression of Kaposi's sarcoma (KS), a dominant malignancy in patients with AIDS. While 12-O-tetradecanoyl-phorbol-13-acetate (TPA)-induced KSHV reactivation from latency is mediated by the protein kinase C δ and MEK/ERK mitogen-activated protein kinase (MAPK) pathways, we have recently shown that the MEK/ERK, JNK and p38 MAPK pathways modulate KSHV lytic replication during productive primary infection of human umbilical vein endothelial cells [Pan, H., Xie, J., Ye, F., Gao, S.J., 2006. 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. J. Virol. 80 (11), 5371–5382]. Here, we report that, besides the MEK/ERK pathway, the JNK and p38 MAPK pathways also mediate TPA-induced KSHV reactivation from latency. The MEK/ERK, JNK and p38 MAPK pathways were constitutively activated in latent KSHV-infected BCBL-1 cells. TPA treatment enhanced the levels of activated ERK and p38 but not those of activated JNK. Inhibitors of all three MAPK pathways reduced TPA-induced production of KSHV infectious virions in BCBL-1 cells in a dose-dependent fashion. The inhibitors blocked KSHV lytic replication at the early stage(s) of reactivation, and reduced the expression of viral lytic genes including RTA, a key immediate-early transactivator of viral lytic replication. Activation of MAPK pathways was necessary and sufficient for activating the promoter of RTA. Furthermore, we showed that the activation of RTA promoter by MAPK pathways was mediated by their downstream target AP-1. Together, these findings suggest that MAPK pathways might have general roles in regulating the life cycle of KSHV by mediating both viral infection and switch from viral latency to lytic replication.

KSHV has been established as the causative agent of KS, PEL, and MCD, malignancies occurring more frequently in AIDS patients. The aggressive nature of KSHV in the context of HIV infection suggests that interactions between the two viruses enhance pathogenesis. KSHV latent infection and lytic reactivation are characterized by distinct gene expression profiles, and both latency and lytic reactivation seem to be required for malignant progression. As a sophisticated oncogenic virus, KSHV has evolved to possess a formidable repertoire of potent mechanisms that enable it to target and manipulate host cell pathways, leading to increased cell proliferation, increased cell survival, dysregulated angiogenesis, evasion of immunity, and malignant progression in the immunocompromised host. Worldwide, approximately 40.3 million people are currently living with HIV infection. Of these, a significant number are coinfected with KSHV. The complex interplay between the two viruses dramatically elevates the risk for development of KSHV-induced malignancies, KS, PEL, and MCD. Although HAART significantly reduces HIV viral load, the entire T-cell repertoire and immune function may not be completely restored. In fact, clinically significant immune deficiency is not necessary for the induction of KSHV-related malignancy. Because of variables such as lack of access to therapy noncompliance with prescribed treatment, failure to respond to treatment and the development of drug-resistant strains of HIV, KSHV-induced malignancies will continue to present as major health concerns.

Latent nuclear antigen (LNA) is implicated in Kaposi's sarcoma-associated herpesvirus (KSHV) episome persistence. LNA colocalizes with KSHV episomes on chromosomes in metaphase, and it maintains the stability and replication of KSHV terminal repeat-containing plasmids. In this study, we examined the function of LNA in episome persistence in the context of full-length KSHV genome by mutagenesis analysis. We generated a KSHV mutant, BAC36-ΔLNA, with LNA disrupted by transposon-based mutagenesis with a KSHV BAC clone, BAC36, as a template. Immunofluorescence antibody staining revealed that the insertion of a transposon cassette into LNA disrupted its expression but had no effect on the expression of two adjacent genes, the vCyclin and vFLIP genes. Using a green fluorescent protein (GFP) cassette as a tracking marker for the KSHV episome, we found 8.7-fold-fewer GFP-positive cells in BAC36-ΔLNA cultures than in wild-type BAC36 cultures at the early stage following episome delivery into 293 cells by transfection, which could be partially rescued by cotransfection with a LNA expression plasmid but not a control plasmid. Cells harboring BAC36-ΔLNA with or without transient complementation rapidly lost episomes and became virus-free after 2 weeks of culture based on GFP expression and Gardella gel analysis and quantitative PCR assays for detecting KSHV genomes. In contrast, BAC36 episomes were stably maintained during the same period. Stable cultures with close to 100% of cells harboring KSHV episomes were readily established by hygromycin selection for BAC36 but not for BAC36-ΔLNA. These results conclusively indicate that LNA is essential for the establishment and persistence of KSHV episomes in mammalian cells.

Reactivation of Kaposi sarcoma-associated herpesvirus (KSHV) from latency for lytic replication plays a pivotal role in the development of KS tumors. However, the physiological factors of KSHV reactivation in KS patients remain undefined. Two recent studies independently discovered that the reactive oxygen species (ROS) H(2)O(2) induces KSHV reactivation in latently infected cells, which can be inhibited by H(2)O(2)-specific antioxidants. H(2)O(2) not only directly induces KSHV reactivation but also is involved in spontaneous lytic replication as well as reactivation stimulated by TPA, hypoxia, and cytokines. Furthermore, in a xenograft-based primary effusion lymphoma (PEL) mouse model, in vivo KSHV reactivation is also H(2)O(2)-dependent and can be suppressed by antioxidants. Mechanistically, H(2)O(2) primarily activates the MAPK pathways to induce viral lytic gene expression and replication. This new finding defines a novel role of H(2)O(2) in KS tumorigenesis and highlights great potentials of using antioxidants and anti-inflammatory drugs for the prevention and treatment of KS tumors.

Dynein and kinesin are the main microtubule-dependent motors that mediate intracellular movement in eukaryotic organisms. We have cloned a full-length cDNA encoding rat dynein light chain protein, robl/LC7-like (class 1), from visual cortex. We found that rat robl/LC7-like gene is highly expressed in neocortex and displays the unusual feature of being rapidly down-regulated by sensory stimulation. This effect was seen at both mRNA and protein levels in visual cortex, being detectable in as little as 45 min after the onset of visual stimulation. Down-regulation by sensory stimulation was also found within ocular dominance columns of area V1 in monocularly deprived monkeys. Our results suggest a high turnover rate of the robl/LC7-like protein and the presence of a repressor mechanism in neurons that is tightly coupled to synaptic stimulation.

Publisher Summary This chapter overviews the techniques pertinent to the production and the characterization of zebrafish/mouse cell hybrids. Transfer of zebrafish chromosomes to cells of a foreign species has been accomplished successfully with three different fish cell lines-ZF4, LFF, and AB9—as donor cells, and with the mouse B78 cells or hamster Wg3H cells as recipients. The mouse B78 melanoma cell line was chosen as recipient in whole-cell fusion experiments because it was previously shown to fuse very easily with other cell lines. Furthermore, the cells have a fast growth rate and are easy to maintain, characteristics that were often retained after fusion. Zebrafish chromosomes are tagged with the neo resistance gene. Mouse B78 cells are not deficient in any enzyme—therefore, the aminoglycoside phosphotransferase (neo) gene, conferring resistance to the aminoglycoside drug G418, is introduced into zebrafish chromosomes to select against unfused recipient mouse cells and to ensure retention of zebrafish chromosomes in hybrids.

The genomes of various Spiroplasma citri strains were digested with several restriction enzymes, and the fragments were analyzed by pulsed-field gel electrophoresis. Polymorphism of the restriction patterns was observed. The genome sizes of the strains obtained when we added the restriction fragment sizes ranged from 1,650 to 1,910 kbp. Physical and genetic maps of 12 strains were constructed by using several DNA probes as genetic markers. The relative positions of mapped loci were conserved in most of the strains; the main differences were differences in the order and number of restriction sites and differences in the sizes of certain fragments. The distribution of viral sequences, which occur at multiple sites in the 5. citri genome and are homologous to the sequences of S. citri virus SpV1 strains R8A2B and S102, was also studied.

The development of Kaposi’s sarcoma (KS), the most common malignancy in AIDS patients, results from Kaposi’s sarcoma-associated herpesvirus (KSHV) infection of endothelial cells and subsequent induction of proliferation, angiogenesis, and inflammation. Previously, we demonstrated that KSHV infection of human umbilical vein endothelial cells (HUVEC) induced a transcriptional induction of Angiopoietin-2 (Ang-2), a pro-angiogenic and pro-inflammatory cytokine that is highly present in KS tumors. This transcriptional up-regulation of Ang-2 started at 12 h and peaked at 54 h post-infection. In difference to our previous data, here we demonstrate that KSHV infection of HUVEC induces rapid release of Ang-2 within minutes of viral contact. Pre-made Ang-2 is stored in the Weibel-Palade body in endothelial cells and is released through regulated exocytosis. KSHV binding to HUVEC is responsible for rapid Ang-2 release because blockade of viral binding inhibits this cytokine exocytosis. We find that KSHV binding to its integrin receptors on endothelial cells activates the integrin tyrosine kinase receptors signaling pathways, including tyrosine phosphorylation of the kinases FAK and Src, and triggers rapid calcium mobilization. This mobilization likely plays a key role in mediating Ang-2 release, as its inhibition by various calcium chelators and calcium channel blockers substantially reduces Ang-2 release. We also demonstrate a direct interaction and association of the kinase Src with the alpha1C subunit of L-type calcium channel. Indeed, specific inhibitors of protein tyrosine phosphorylation not only disrupt this interaction but also abolish Ang-2 release. Finally, preliminary data from in vitro cell adhesion assays suggest that this rapidly released Ang-2 enhances migration and adhesion of monocytes to the infected endothelial cells. To our knowledge, this is the first demonstration of interaction between KSHV and its integrins receptors in regulating rapid cytokine release. This study also uncovers a novel mechanism of KSHV induction of angiogenesis and inflammation, which is much faster, and could likely play important roles in the early event of KS tumor development.

A 5.6-kbp fragment of Spiroplasma citri DNA containing the dnaA gene has been cloned and sequenced. Nucleotide sequence analysis shows that this fragment harbors the genes for the replication initiator protein (dnaA), the beta subunit of DNA polymerase III (dnaN), and the DNA gyrase subunits A and B (gyrA and gyrB). The arrangement of these genes, dnaA-dnaN-gyrB-gyrA, is similar to that found in all Gram-positive bacterial genomes studied so far, except that no recF gene was found between dnaN and gyrB. Several DnaA-box consensus sequences were found upstream of dnaA and in the dnaA-dnaN intergenic region. The dnaA region with the flanking DnaA-boxes and the tetracycline resistance determinant, tetM, were linked into a circular recombinant DNA. This DNA was able to replicate autonomously when introduced by electroporation into S. citri cells. These experiments show that the dnaA region with the DnaA-boxes is the origin of replication of S. citri and can be used to construct gene vectors.

Kaposi's sarcoma-associated herpesvirus (KSHV) latency is central to the evasion of host immune surveillances and induction of KSHV-related malignancies. The mechanism of KSHV latency remains unclear. Here, we show that the KSHV latent gene vFLIP promotes viral latency by inhibiting viral lytic replication. vFLIP suppresses the AP-1 pathway, which is essential for KSHV lytic replication, by activating the NF-κB pathway. Thus, by manipulating two convergent cellular pathways, vFLIP regulates both cell survival and KSHV lytic replication to promote viral latency. These results also indicate that the effect of the NF-κB pathway on KSHV replication is determined by the status of the AP-1 pathway and hence provide a mechanistic explanation for the contradictory role of the NF-κB pathway in KSHV replication. Since the NF-κB pathway is commonly activated during infection of gammaherpesviruses, these findings might have general implications for the control of gammaherpesviral latency.