Evaluation and Utilization of Biodiversity in Triticeae for Wheat lmprovement

To adapt new varieties to a wide spectrum of environments breeders and farmers have emphasized the need for broadening the current narrow genetic base of modern varieties of important cereal crops such as wheat and barley. In response to this need, several thousand samples of indigenously cultivated Triticeae species and their wild relatives have been collected from the centers of diversity. However, gene bank collections are of little use if they are not evaluated and the information disseminated widely. Evaluation is essentially the link between conservation and use. Some of the collected material has been evaluated at the International Center for Agricultural Research in the Dry Areas (!CARDA) in Syria. In the past cereal breeders were averse to using germ plasm that after years of work yielded uncertain results. However, in recent years they have begun to successfully utilize non-conventional germplasm (wild/alien and obsolete forms) in their crossing blocks. The substantial progress at !CARDA in the evaluation and utilization of Triticeae germplasm for crop improvement in the low rainfall areas of West Asia and North Africa is described

Human DNA tumor viruses evade uracil-mediated antiviral immunity

It is estimated that approximately 15% of tumors worldwide are caused by viruses [1]. These oncogenic viruses are classified as either RNA (RTVs) or DNA tumor viruses (DTVs) [1]. There are two human RTVs: hepatitis C virus (HCV) and human T-cell lymphotropic virus-1 (HTLV-1), and five human DTVs: human papilloma virus (HPV), hepatitis B virus (HBV), Epstein–Barr virus (EBV), Kaposi sarcoma-associated herpesvirus (KSHV), and Merkel cell polyomavirus (MCPyV) [1]. These tumor viruses (TVs) establish lifelong infection and evade host immunity using several strategies. While not all TV infections cause disease, viral modes of established latency and persistence perturb normal cellular processes, sometimes leading to cancer [1]. Particularly interesting are mechanisms to evade uracil-mediated antiviral immunity, which can be detrimental to the host genome

Transcriptional Regulation of the Kaposi's Sarcoma-Associated Herpesvirus K15 Gene

The K15 gene product of Kaposi's sarcoma-associated herpesvirus (KSHV) is a transmembrane protein that is encoded by the last open reading frame of the KSHV genome. The K15 protein has been implicated in modulation of B-cell signal transduction and activation of the Ras/mitogen-activated protein kinase and NF-κB signal transduction pathways. Here we report the identification of the transcriptional start site of the full-length K15 gene in KSHV-positive BCBL-1 cells. We have mapped the K15 transcriptional start site to a position 152 nucleotides upstream from the translation start site by rapid amplification of cDNA ends and RNase protection assays. We have also characterized the K15 promoter element. To analyze the cis-acting elements necessary to regulate K15 gene expression, a series of 5′ promoter deletion constructs were generated and subcloned upstream of the luciferase reporter gene. Transcriptional assays with these mutant promoters demonstrated that chemical induction in latently infected KSHV-positive BCBL-1 cells activated K15 transcription. In addition, K15 promoter transactivation was also mediated by the viral immediate-early protein Orf50/Rta, suggesting that the K15 gene is actively transcribed during lytic replication

Hsp90 and Hsp40/Erdj3 are required for the expression and anti-apoptotic function of KSHV K1

Kaposi sarcoma-associated herpesvirus (KSHV) is a member of the gammaherpesvirus family. It is the etiological agent of three different human cancers, Kaposi sarcoma (KS), primary effusion lymphoma (PEL), and multicentric Castleman disease (MCD). The far left-end of the KSHV genome encodes a unique transmembrane glycoprotein called K1. K1 possesses the ability to transform rodent fibroblasts and block apoptosis. K1 has also been shown to activate the PI3K/Akt/mTOR pathway in different cellsUsing tandem affinity purification (TAP), we identified heat shock protein 90β (Hsp90β) and endoplasmic reticulum (ER)-associated Hsp40 (Erdj3/DnaJB11), as cellular binding partners of K1. Interactions of K1 with Hsp90β and Hsp40 were confirmed by co-immunoprecipitation in both directions. Furthermore, K1 also interacted with the Hsp90α̣ isoform. We report that siRNAs directed against Hsp90 and Hsp40/Erdj3, as well as pharmacological inhibitors of Hsp90 dramatically reduced K1 expression, suggesting that K1 is a client protein of these chaperones. Additionally, both Hsp90 and Hsp40/Erdj3 were essential for K1’s anti-apoptotic function. Finally, we report that the Hsp90 inhibitors, 17-AAG and 17-DMAG, can suppress the proliferation of KSHV-positive PEL cell lines and exhibited IC50 values of 50nM and below

Modulation of Kaposi's Sarcoma-Associated Herpesvirus Interleukin-6 Function by Hypoxia-Upregulated Protein 1

Kaposi's sarcoma-associated herpesvirus (KSHV, also called human herpesvirus 8) is linked to the development of Kaposi's sarcoma (KS), primary effusion lymphoma (PEL), and multicentric Castleman's disease (MCD). KSHV expresses several proteins that modulate host cell signaling pathways. One of these proteins is viral interleukin-6 (vIL-6), which is a homolog of human IL-6 (hIL-6). vIL-6 is able to prevent apoptosis and promote proinflammatory signaling, angiogenesis, and cell proliferation. Although it can be secreted, vIL-6 is mainly an intracellular protein that is retained in the endoplasmic reticulum (ER). We performed affinity purification and mass spectrometry to identify novel vIL-6 binding partners and found that a cellular ER chaperone, hypoxia-upregulated protein 1 (HYOU1), interacts with vIL-6. Immunohistochemical staining reveals that both PEL and KS tumor tissues express significant amounts of HYOU1. We also show that HYOU1 increases endogenous vIL-6 protein levels and that HYOU1 facilitates vIL-6-induced JAK/STAT signaling, migration, and survival in endothelial cells. Furthermore, our data suggest that HYOU1 also modulates vIL-6's ability to induce CCL2, a chemokine involved in cell migration. Finally, we investigated the impact of HYOU1 on cellular hIL-6 signaling. Collectively, our data indicate that HYOU1 is important for vIL-6 function and may play a role in the pathogenesis of KSHV-associated cancers

Disruption of LANA in Rhesus Rhadinovirus Generates a Highly Lytic Recombinant Virus

Rhesus monkey rhadinovirus (RRV) is a gammaherpesvirus that is closely related to human Kaposi's sarcoma-associated herpesvirus (KSHV/HHV-8). RRV is the closest relative to KSHV that has a fully sequenced genome and serves as an in vitro and an in vivo model system for KSHV. The latency-associated nuclear antigen (LANA) protein of both KSHV and RRV plays key roles in the establishment and maintenance of these herpesviruses. We have constructed a RRV recombinant virus (RRVΔLANA/GFP) in which the RRV LANA open reading frame has been disrupted with a green fluorescent protein (GFP) expression cassette generated by homologous recombination. The integrity of the recombinant virus was confirmed by diagnostic PCR, restriction digestion, Southern blot analysis, and whole-genome sequencing. We compared the single-step and multistep replication kinetics of RRVΔLANA/GFP, RRV-GFP, wild-type (WT) RRV H26-95, and a revertant virus using traditional plaque assays, as well as real-time quantitative PCR-based genome quantification assays. The RRVΔLANA/GFP recombinant virus exhibited significantly higher lytic replicative properties compared to RRV-GFP, WT RRV, or the revertant virus. This was observed upon de novo infection and in the absence of chemical inducers such as phorbol esters. In addition, by using a quantitative real-time PCR-based viral array, we are the first to report differences in global viral gene expression between WT and recombinant viruses. The RRVΔLANA/GFP virus displayed increased lytic gene transcription at all time points postinfection compared to RRV-GFP. Moreover, we also examined several cellular genes that are known to be repressed by KSHV LANA and report that these genes are derepressed during de novo lytic infection with the RRVΔLANA/GFP virus compared to RRV-GFP. Finally, we also demonstrate that the RRVΔLANA/GFP virus fails to establish latency in B cells, as measured by the loss of GFP-positive cells and intracellular viral genomes

Signal transducer and activator of transcription 3 (Stat3) regulates host defense and protects mice against herpes simplex virus-1 (HSV-1) infection

Signal transducer and activator of transcription 3 (STAT3) mediates cellular responses to multiple cytokines, governs gene expression, and regulates the development and activation of immune cells. STAT3 also modulates reactivation of latent herpes simplex virus-1 (HSV-1) in ganglia. However, it is unclear how STAT3 regulates the innate immune response during the early phase of HSV-1 lytic infection. Many cell types critical for the innate immunity are derived from the myeloid lineage. Therefore, in this study, we used myeloid-specific Stat3 knockout mice to investigate the role of STAT3 in the innate immune response against HSV-1. Our results demonstrate that Stat3 knockout bone marrow-derived macrophages (BMMs) expressed decreased levels of interferon-α (IFN-α) and interferon-stimulated genes (ISGs) upon HSV-1 infection. In vivo, knockout mice were more susceptible to HSV-1, as marked by higher viral loads and more significant weight loss. Splenic expression of IFN-α and ISGs was reduced in the absence of STAT3, indicating that STAT3 is required for optimal type I interferon response to HSV-1. Expression of TNF-α and IL-12, cytokines that have been shown to limit HSV-1 replication and pathogenesis, was also significantly lower in knockout mice. Interestingly, Stat3 knockout mice failed to expand the CD8+ conventional DC (cDC) population upon HSV-1 infection, and this was accompanied by impaired NK and CD8 T cell activation. Collectively, our data demonstrate that myeloid-specific Stat3 deletion causes defects in multiple aspects of the immune system and that STAT3 has a protective role at the early stage of systemic HSV-1 infection

Kaposi's sarcoma-associated herpesvirus expresses an array of viral microRNAs in latently infected cells

MicroRNAs (miRNAs) are an endogenously encoded class of small RNAs that have been proposed to function as key posttranscriptional regulators of gene expression in a range of eukaryotic species, including humans. The small size of miRNA precursors makes them potentially ideal for use by viruses as inhibitors of host cell defense pathways. Here, we demonstrate that the pathogenic human herpesvirus Kaposi's sarcoma-associated herpesvirus (KSHV) encodes an array of 11 distinct miRNAs, all of which are expressed at readily detectable levels in latently KSHV infected cells. Individual KSHV miRNAs were expressed at up to 2,200 copies per cell. The KSHV miRNAs are expressed from what appears to be a single genetic locus that largely coincides with an ≈4-kb noncoding sequence located between the KSHV v-cyclin and K12/Kaposin genes, both of which are also expressed in latently infected cells. Computer analysis of potential mRNA targets for these viral miRNAs identified a number of interesting candidate genes, including several mRNAs previously shown to be down-regulated in KSHV-infected cells. We hypothesize that these viral miRNAs play a critical role in the establishment and/or maintenance of KSHV latent infection in vivo and, hence, in KSHV-induced oncogenesis

Latent Kaposi's Sarcoma-Associated Herpesvirus Infection of Monocytes Downregulates Expression of Adaptive Immune Response Costimulatory Receptors and Proinflammatory Cytokines

Kaposi's sarcoma-associated herpesvirus (KSHV) infection is associated with the development of Kaposi's sarcoma, primary effusion lymphoma, and multicentric Castleman's disease. We report the establishment of a monocytic cell line latently infected with KSHV (KSHV-THP-1). We profiled viral and cytokine gene expression in the KSHV-THP-1 cells compared to that in uninfected THP-1 cells and found that several genes involved in the host immune response were downregulated during latent infection, including genes for CD80, CD86, and the cytokines tumor necrosis factor alpha (TNF-α) and interleukin-1β (IL-1β). Thus, KSHV minimizes its immunological signature by suppressing key immune response factors, enabling persistent infection and evasion from host detection

Identification and Characterization of the Orf49 Protein of Kaposi's Sarcoma-Associated Herpesvirus

Kaposi's sarcoma-associated herpesvirus (KSHV) is the etiological agent of Kaposi's sarcoma, primary effusion lymphoma, and multicentric Castleman's disease. Kaposi's sarcoma is the most common neoplasm among human immunodeficiency virus-positive individuals. Like other herpesviruses, KSHV is able to establish a predominantly latent, life-long infection in its host. The KSHV lytic cycle can be triggered by a number of stimuli that induce the expression of the key lytic switch protein, the replication and transcription activator (RTA) encoded by Orf50. The expression of Rta is necessary and sufficient to trigger the full lytic program resulting in the ordered expression of viral proteins, release of viral progeny, and host cell death. We have characterized an unknown open reading frame, Orf49, which lies adjacent and in the opposite orientation to Orf50. Orf49 is expressed during the KSHV lytic cycle and shows early transcription kinetics. We have mapped the 5′ and 3′ ends of the unspliced Orf49 transcript, which encodes a 30-kDa protein that is localized to both the nucleus and the cytoplasm. Interestingly, we found that Orf49 was able to cooperate with Rta to activate several KSHV lytic promoters containing AP-1 sites. The Orf49-encoded protein was also able to induce transcriptional activation through c-Jun but not the ATF1, ATF2, or CREB transcription factor. We found that Orf49 could induce phosphorylation and activation of the transcription factor c-Jun, the Jun N-terminal kinase (JNK), and p38. Our data suggest that Orf49 functions to activate the JNK and p38 pathways during the KSHV lytic cycle