RNA interference in marine and freshwater sponges

Background: The marine sponge Tethya wilhelma and the freshwater sponge Ephydatia muelleri are emerging model organisms to study evolution, gene regulation, development, and physiology in non-bilaterian animal systems. Thus far, functional methods (i.e., loss or gain of function) for these organisms have not been available. Results: We show that soaking developing freshwater sponges in double-stranded RNA and/or feeding marine and freshwater sponges bacteria expressing double-stranded RNA can lead to RNA interference and reduction of targeted transcript levels. These methods, first utilized in C. elegans, have been adapted for the development and feeding style of easily cultured marine and freshwater poriferans. We demonstrate phenotypic changes result from ‘knocking down’ expression of the actin gene. Conclusion: This technique provides an easy, efficient loss-of-function manipulation for developmental and gene regulatory studies in these important non-bilaterian animals

The interaction of murine gammaherpesvirus 68 with the innate and adaptive immune systems of the host

Herpesviruses establish life-long infections that represent a détente with the immune system of the host. These large enveloped DNA viruses have two distinct phases of their life cycle. During lytic infection, most viral genes are expressed and infectious virions are produced for dissemination. In contrast, viral gene expression is tightly controlled during the latent stage of infection, and the virus is not easily targeted by the immune system. Gammaherpesviruses evade clearance by the immune response to gain access to B lymphocytes, the primary reservoir of latent infection. In the context of immune suppression, control of the infection is lost, increasing the risk of virus-driven malignancies. Our laboratory seeks to understand the mechanisms by which gammaherpesviruses overcome innate and adaptive immune clearance and hijack immune cells for latency. We use a natural pathogen of murid rodents, murine gammaherpesvirus 68 (MHV68), to probe the interactions between the virus and the host. T cells of the adaptive immune system that recognize both lytic and latent viral antigens control long-term infection. I determined that T cells lacking the negative regulators Suppressor of TCR signaling-1 and -2 (Sts-1 and Sts-2) better respond to gammaherpesvirus-infected cells in vitro. However, hyper-responsiveness in the T cell compartment did not impact pathogenesis in vivo. With regard to innate immunity, the inflammasome is an intracellular surveillance system that can detect pathogen-associated molecules. I found that Caspase1-mediated inflammasome signaling did not contribute to the control of MHV68 pathogenesis; but in vitro investigations uncovered a reduction in the pro-inflammatory molecule IL-1beta upon infection of primary macrophages. Lastly, I sought to examine the role of IKKalpha-dependent non-canonical NF-kappaB signaling pathway during gammaherpesvirus pathogenesis. Although IKKalpha-mediated signaling was dispensable for virus production, IKKalpha signaling was dampened upon extrinsic signaling during lytic infection. In summary, my studies determined that enhancement of T cell responses did not lead to improved clearance of the virus and that MHV68 infection evades activation of pro-inflammatory responses and alternative NF-kappaB signaling. These studies emphasize the complexity of gammaherpesvirus interactions with the innate and adaptive immune arms of the host and uncover novel mechanisms by which a gammaherpesvirus subverts these responses. | Herpesviruses establish life-long infections that represent a détente with the immune system of the host. These large enveloped DNA viruses have two distinct phases of their life cycle. During lytic infection, most viral genes are expressed and infectious virions are produced for dissemination. In contrast, viral gene expression is tightly controlled during the latent stage of infection, and the virus is not easily targeted by the immune system. Gammaherpesviruses evade clearance by the immune response to gain access to B lymphocytes, the primary reservoir of latent infection. In the context of immune suppression, control of the infection is lost, increasing the risk of virus-driven malignancies. Our laboratory seeks to understand the mechanisms by which gammaherpesviruses overcome innate and adaptive immune clearance and hijack immune cells for latency. We use a natural pathogen of murid rodents, murine gammaherpesvirus 68 (MHV68), to probe the interactions between the virus and the host. T cells of the adaptive immune system that recognize both lytic and latent viral antigens control long-term infection. I determined that T cells lacking the negative regulators Suppressor of TCR signaling-1 and -2 (Sts-1 and Sts-2) better respond to gammaherpesvirus-infected cells in vitro. However, hyper-responsiveness in the T cell compartment did not impact pathogenesis in vivo. With regard to innate immunity, the inflammasome is an intracellular surveillance system that can detect pathogen-associated molecules. I found that Caspase1-mediated inflammasome signaling did not contribute to the control of MHV68 pathogenesis; but in vitro investigations uncovered a reduction in the pro-inflammatory molecule IL-1beta upon infection of primary macrophages. Lastly, I sought to examine the role of IKKalpha-dependent non-canonical NF-kappaB signaling pathway during gammaherpesvirus pathogenesis. Although IKKalpha-mediated signaling was dispensable for virus production, IKKalpha signaling was dampened upon extrinsic signaling during lytic infection. In summary, my studies determined that enhancement of T cell responses did not lead to improved clearance of the virus and that MHV68 infection evades activation of pro-inflammatory responses and alternative NF-kappaB signaling. These studies emphasize the complexity of gammaherpesvirus interactions with the innate and adaptive immune arms of the host and uncover novel mechanisms by which a gammaherpesvirus subverts these responses. | 227 page

CHARACTERIZATION OF GCN5 HISTONE ACETYLTRANSFERASE ACTIVITY AND BROMODOMAIN ACETYL-LYSINE BINDING FUNCTION

In eukaryotes, DNA is packaged into chromatin, which consists of the DNA wrapped around an octameric core of histone proteins (H2A, H2B, H3, and H4) which collectively comprise the nucleosome. Gene activation and silencing determined by the access of transcriptional machinery to DNA can be dictated in part by dynamic post-translational modifications (PTMs) on histone proteins. These PTMs can be added, removed, and interpreted by chromatin effector complexes that interact with chromatin. In yeast, the highly conserved histone acetyltransferase (HAT) Gcn5 associates with Ada2 and Ada3 to form the catalytic module of the ADA and SAGA transcriptional coactivator complexes. Gcn5 also contains an acetyl-lysine binding bromodomain that has been implicated in regulating nucleosomal acetylation in vitro, as well as at gene promoters in cells. However, the contribution of the Gcn5 bromodomain in regulating site specificity of HAT activity remains unclear. Here, we used a combined acid-urea gel and quantitative mass spectrometry approach to compare the HAT activity of wild-type and Gcn5 bromodomain-mutant ADA subcomplexes (Gcn5-Ada2-Ada3). Wild-type ADA subcomplex acetylated H3 lysines with the following specificity; H3K14 > H3K23 > H3K9 ≈ H3K18 > H3K27 > H3K36. However, when the Gcn5 bromodomain was defective in acetyl-lysine binding, the ADA subcomplex demonstrated altered site-specific acetylation on free and nucleosomal H3, with H3K18ac being the most severely diminished. H3K18ac was also severely diminished on H3K14R, but not H3K23R, substrates in wild-type HAT reactions, further suggesting that Gcn5-catalyzed acetylation of H3K14 and bromodomain binding to H3K14ac are important steps preceding H3K18ac. Consistent with our in vitro results, when the Gcn5 bromodomain was impaired in vivo we observed a global decrease in H3K18 acetylation. In sum, this work details a previously uncharacterized cross-talk between the Gcn5 bromodomain "reader" function and enzymatic HAT activity that might ultimately affect gene expression. Through our development of in vitro assays that contain selectively modified histones and intact epigenetic complexes and modules, we can gain biologically relevant insight into the activity and chromatin interactions of effector protein complexes. Future studies of how mutations in bromodomains or other histone post-translational modification readers can affect chromatin-templated enzymatic activities will yield unprecedented insight into a potential "histone/epigenetic code.

RNA interference in marine and freshwater sponges: actin knockdown in Tethya wilhelma and Ephydatia muelleri by ingested dsRNA expressing bacteria

Background: The marine sponge Tethya wilhelma and the freshwater sponge Ephydatia muelleri are emerging model organisms to study evolution, gene regulation, development, and physiology in non-bilaterian animal systems. Thus far, functional methods (i.e., loss or gain of function) for these organisms have not been available. Results: We show that soaking developing freshwater sponges in double-stranded RNA and/or feeding marine and freshwater sponges bacteria expressing double-stranded RNA can lead to RNA interference and reduction of targeted transcript levels. These methods, first utilized in C. elegans, have been adapted for the development and feeding style of easily cultured marine and freshwater poriferans. We demonstrate phenotypic changes result from `knocking down' expression of the actin gene. Conclusion: This technique provides an easy, efficient loss-of-function manipulation for developmental and gene regulatory studies in these important non-bilaterian animals

Identification of preexisting adaptive immunity to Cas9 proteins in humans

The CRISPR-Cas9 system is a powerful tool for genome editing, which allows the precise modification of specific DNA sequences. Many efforts are underway to use the CRISPR-Cas9 system to therapeutically correct human genetic diseases1-6. The most widely used orthologs of Cas9 are derived from Staphylococcus aureus and Streptococcus pyogenes5,7. Given that these two bacterial species infect the human population at high frequencies8,9, we hypothesized that humans may harbor preexisting adaptive immune responses to the Cas9 orthologs derived from these bacterial species, SaCas9 (S. aureus) and SpCas9 (S. pyogenes). By probing human serum for the presence of anti-Cas9 antibodies using an enzyme-linked immunosorbent assay, we detected antibodies against both SaCas9 and SpCas9 in 78% and 58% of donors, respectively. We also found anti-SaCas9 T cells in 78% and anti-SpCas9 T cells in 67% of donors, which demonstrates a high prevalence of antigen-specific T cells against both orthologs. We confirmed that these T cells were Cas9-specific by demonstrating a Cas9-specific cytokine response following isolation, expansion, and antigen restimulation. Together, these data demonstrate that there are preexisting humoral and cell-mediated adaptive immune responses to Cas9 in humans, a finding that should be taken into account as the CRISPR-Cas9 system moves toward clinical trials

Engineered type 1 regulatory T cells designed for clinical use kill primary pediatric acute myeloid leukemia cells

Type 1 regulatory (Tr1) T cells induced by enforced expression of IL-10 (LV-10) are being developed as a novel treatment for chemotherapy-resistant myeloid leukemias. In vivo, LV-10 cells do not cause graft vs host disease while mediating graft vs leukemia (GvL) effect against adult acute myeloid leukemia (AML). Since pediatric AML (pAML) and adult AML are different on a genetic and epigenetic level, we investigate herein whether LV-10 cells also efficiently kill pAML cells. We show that the majority of primary pAML are killed by LV-10 cells, with different levels of sensitivity to killing. Transcriptionally, pAML sensitive to LV-10 killing expressed a myeloid maturation signature. Overlaying the signatures of sensitive and resistant pAML onto the public NCI TARGET pAML dataset revealed that sensitive pAML clustered with M5 monocytic pAML and pAML with MLL rearrangement. Resistant pAML clustered with myelomonocytic leukemias and those bearing the core binding factor translocations inv(16) or t(8;21)(RUNX1-RUNX1T1). Furthermore, resistant pAML upregulated the membrane glycoprotein CD200, which binds to the inhibitory receptor CD200R1 on LV-10 cells. To examine if CD200 expression on target cells can impair LV-10 cell function, we overexpressed CD200 in myeloid leukemia cell lines ordinarily sensitive to LV-10 killing. Indeed, LV-10 cells degranulated less and killed fewer CD200-overexpressing cells compared to controls, indicating that pAML can utilize CD200 expression for immune evasion. Altogether, the majority of pAML are killed by LV-10 cells in vitro, supporting further LV-10 cell development as an innovative cell therapy for pAML

Apple stem pitting virus

NYS IPM Type: Fruits IPM Fact SheetApple stem pitting virus (ASPV) is a latent virus of pome fruits with worldwide distribution. In addition to apple, ASPV can affect pear and quince. Similarly to other latent viruses of pome fruits, infections with ASPV are often symptomless and therefore go mostly undetected

Grape Leafroll Disease

NYS IPM Type: Fruits IPM Fact SheetLeafroll is one of the most important virus diseases of grapevine. It occurs in every major grape-growing region of the world. Grapevine leafroll disease can affect all native and Vitis vinifera cultivars, hybrids, and rootstocks, although symptoms are not always expressed on infected vines. The disease was described as early as the 19th century in Europe, but its graft-transmissibility was not demonstrated until 1937. In 1979, a specific type of flexuous and filamentous virus was reported in a leafroll-affected vine. Shortly thereafter, in 1983, the capacity of mealybugs to transmit one of the viruses associated with this disease was shown

Grapevine Red Blotch Disease

NYS IPM Type: Fruits IPM Fact SheetGrapevine red blotch disease (GRBD) is a recently recognized viral disease. It was first described on Vitis vinifera cv. Cabernet Sauvignon at the University of California Oakville Research Field Station in 2008, but the causative agent was not characterized until 2012. The disease, GRBD, is caused by a DNA virus named Grapevine red blotch-associated virus (GRBaV). Infected red wine grapevines exhibit symptoms similar to those associated with leafroll disease, with red blotches forming on leaves during the later summer months. Fruit ripening issues have been reported in grapevines diagnosed with GRBD. The symptom similarity to leafroll disease and abiotic factors such as nutrient deficiencies, insect damage, or physical injuries makes visual diagnosis difficult and explains the delay in recognition and characterization of the disease

Apple chlorotic leaf spot virus

NYS IPM Type: Fruits IPM Fact SheetApple chlorotic leaf spot virus (ACLSV) infects pome and stone fruits. It can elicit diverse symptoms although, in most cultivars the virus is latent, which means that infected trees do not manifest observable symptoms. Apple chlorotic leaf spot virus is often detected in co-infection with other latent viruses such as Apple stem pitting virus (ASPV) and Apple stem grooving virus. Apple chlorotic leaf spot virus can have a devastating effect on apple growth and productivity