Stephen Goff, Ph.D. - US grants

DESCRIPTION (provided by applicant): This proposal describes the genetic analysis of the Gag and Pol proteins of the Moloney murine leukemia virus and of the cellular host proteins with which they interact. We have used the yeast two-hybrid system to identify novel mammalian proteins that bind to various specific domains of the Gag and Pol gene products. We will now focus our ongoing efforts on several of these new targets: the IQGAP proteins, which interact with the MA portion of Gag; two components of the SUMO transferase system, which interact with the CA portion of Gag; and the ribosomal protein S3a, which binds the RNA pseudoknot at the Gag-Pol border. Biochemical methods will be used to confirm and characterize the interactions between the viral and cellular proteins both in vitro and in vivo. To determine the role of these proteins in virus replication, mutations in the viral genes will be identified that specifically abrogate the interaction with the host protein. These mutations will be introduced into the viral genome and the effects on virus replication in cell culture will be determined. Reversion analysis will be used to further define the requirements for the interaction, and to confirm the importance of the interaction for virus replication. Dominant interfering alleles and RNAi methods will be used to document the requirement for the interaction, and to define the stage of the life cycle at which the proteins act. The generality of the results will be tested by examining the effects of the genes on the replication of a panel of retroviruses of other families. These experiments will significantly extend our understanding of retrovirus replication, and may provide important new targets for antiviral intervention.

This proposal describes experiments using the tools of mouse genetics to determine the in vivo functions of the c-abl proto-oncogene. The c-abl gene product is a cytoplasmic tyrosine kinase important for signal transduction and control of the cell cycle. Knock-out mice deficient in c- abl, generated in the previous funding period, exhibit a number of phenotypes: perinatal lethality, runting, bone abnormalities, and defects in early lymphoid cell lineages, notably a sensitivity to apoptotic stimuli, Several genetic studies of these mice are proposed: First, the osteoporosis causes by the c-abl deficiency will be examined; osteoblasts will be cultured from mutant and control animals, and tested for their ability to respond to a panel of growth factors. Second, new alleles of c- abl will be generated by gene targeting ("knock-in" experiments). One of these alleles will permit the conditional deletions of the gene at selected times in development and in selected tissues; others will express altered forms of c-abl lacking particular domains. Examination of these mice should help determine the functions of each pathway emanating from c- abl. Third, breeding will be used to generate compound mutant mice lacking c-abl and other genes with related functions, including the abi-1 and -2 genes, encoding Abl-interacting proteins, and ret-, a transmembrane receptor kinase under investigation by the Constantini laboratory. Finally, a germ-line mutation of a new member of the Abi family, a mammalian homologue of the yeast Cdc15 gene, will be generate by gene targeting, and the effects of the mutation alone and with other knock-out mutations will be characterize. These studies should help define the many diverse functions of c-abl in mammalian development and physiology.

This study describes experiments to determine the functions of the Abi-l (Abelson interactor-1) protein in oncogenic transformation by the Abelson oncoproteins v-Abl and BCR-ABL. The Abelson gene encodes a cytoplasmic tyrosine kinase; v-Abl is an activated form expressed by the Abelson murine leukemia virus, causing a pre-B lymphoma in mice, which BCR-ABL is a less potent version responsible for chronic myelogenous leukemia (CML) and, sometimes, acute lymphocytic leukemia (ALL) in humans. Abi-l was originally identified using the yeast two-hybrid system as a novel SH3 protein that binds to the proline-rich C terminal tail of v-Abl and suppresses its transforming activity. Genetic and biochemical experiments will be performed to determine which signal transduction pathways are controlled or inhibited by the Abi-1 protein or inhibited by the Abi-1 proteins. Cell lines expressing high levels of either the wild-type or mutant Abi-1 will be examined for activation of downstream target genes (c-myc, DHFR); for activation of downstream target genes (c-myc, DHFR): for activation of known PDGF pathways (Ras, PLCg, PI3K); for stimulation of cell cycle regulatory proteins; and for phosphorylation and activation of Jak/Stat and Crkl proteins. Human tumor samples from CML patients will be tested to determine the level of Abi-1 protein, and the fraction bound to BCR-ABL. Finally, mutants of v- Abl will be generated that transforming activity. The yeast two-hybrid system will be used to isolate mutants that bind to one partner but not to others, and viruses carrying the relevant mutations will then be tested for transforming activity in cell lines and mice. These experiments should help identify which of the various pathways emanating from the Abl kinase are required for oncogenic transformation. These experiments may provide important information about the induction and progression of disease in human CML and ALL.

This core will provide tissue culture-derived reagents for all four projects. The core will make and titer stocks of mutant and wt forms of v-abl and bcr-abl viruses. The core will also use these viruses to transform murine bone marrow cells and fibroblast ell lines in vitro and to infect mice in vivo. The core will also obtain from different projects wt and mutant forms of Jak and Abi-1 proteins and will generate stably transfected lines expressing these proteins in an inducible manner. The core will generate monoclonal antibodies and will provide routine cell culture and freezing for all of the projects.

This proposal describes experiments using the tools of mouse genetics to determine the in vivo functions of the c-abl proto-oncogene. The c-abl gene product is a cytoplasmic tyrosine kinase important for signal transduction and control of the cell cycle. Knock-out mice deficient in c-abl exhibit a number of phenotypes: perinatal lethality, runting, bone abnormalities, and defects in early lymphoid cell lineages. Several genetic studies of these mice are proposed. First, new alleles of c-abl will be generated by gene targeting ("knock-in" experiments). One of these alleles will permit the conditional deletion of the gene at selected times in development and in selected tissues; others will express altered forms of c-abl lacking particular domains responsible for protein localization. Examination of these mice should help determine the functions of each pathway emanating from c-abl; we will be particularly interested to examine the course of B cell development and the repertoire of variable region utilization. Secondly, a germ-line mutation of a new member of the Abi family termed PSTPIP1, a mammalian homologue of the yeast Cdc15 gene, will be generated by gene targeting, and the effects of the mutation alone and with other knock-out mutations will be characterized. Our expectation is that the loss of PSTPIP1 may suppress some of the phenotypes caused by loss of Abl. Thirdly, we will generate and analyze germ line mutations of the UV-DDB proteins, products of the xpe locus in man, and which we have shown are bound and regulated by the Abl kinase. Finally, we will directly catalogue the genes induced in cell lines and in tissues by oxidative stress, comparing the responses in abl mutants with wild-type controls, and thereby identify the Abl-dependent downstream targets. These studies should help define the many diverse functions of c-abl in mammalian development and physiology.

University of Arizona: Richard Jorgensen (PI), Gregory Andrews, Kobus Barnard, Susan Brown, Vicki Chandler, Nirav Merchant, Carolyn Napoli, Sudha Ram, Steven Rounsley
Arizona State University: Daniel Stanzione
Cold Spring Harbor Laboratory: Lincoln Stein, Doreen Ware
Purdue University: Rebecca Doerge,
University of North Carolina at Wilmington: Ann Stapleton

The iPlant Collaborative (iPC) is a new type of organization ? a cyberinfrastructure collaborative for the plant sciences - that enables new conceptual advances through integrative, computational thinking (i.e. thinking at multiple levels of abstraction using a systems-level approach to problem-solving). The iPC is fluid and dynamic, utilizing new computer, computational science and cyberinfrastructure solutions to address an evolving array of grand challenges in the plant sciences. It is community-driven, involving plant biologists, computer and information scientists and engineers, as well as experts from other disciplines, all working in integrated teams. The iPC brings together strengths in plant biology, bioinformatics, statistics, computer science and high throughput computing, as well as innovative approaches to education, outreach, and the study of social networks.

Several key principles guided the development of the iPC. Specifically, the iPC:
? is a cyberinfrastructure collaborative rather than purely a cyberinfrastructure,
? will enable multi-disciplinary teams to address grand challenges in plant science,
? will be an entity that is by, for and of the community,
? will train the next generation in computational thinking, and
? is designed to be able to reinvent itself as needs and technologies change.

The driving force behind the iPC is the nature of the grand challenge questions in plant sciences, and all facets of the collaborative are organized around those selected questions. The act of selecting these questions will be community-driven, and to facilitate that, the Collaborative will host a series of workshops, each focused on a specific area of plant biology, but with participants cutting across the spectrum of the computational and biological sciences. The goal of each workshop will be to identify the grand challenge questions in that field, as well as the necessary strategies and approaches that will be needed to solve the question(s). Self-forming Grand Challenge Teams from the community will then work with iPC personnel to develop a ?Discovery Environment? (DE), which will be a cyberinfrastructure for open-access research and education focused on a grand challenge question. Over time, the DEs designed for different grand challenges will overlap and coalesce into a comprehensive cyberinfrastructure for discovery and learning.

The cyberinfrastructure created by the iPC will provide the community with two main capabilities: it will provide access to world-class physical infrastructure ? for example persistent storage, and compute power via local and national resources, and it will provide services that promote interactions, communications and collaborations and that advance the understanding and use of computational thinking in plant biology. Through these capabilities, the iPC will catalyze progress in targeted areas of plant biology, and more broadly advance the whole of plant science through new, creative, synthesis activities, and training the next generation of scientists in computational (and collaborative) thinking.

The broader impacts of the iPC project will not be limited merely to creating the tools for solution of currently intractable grand challenge questions, because at its core the iPC is actually a community building and educational enterprise designed to facilitate education and outreach. Grand Challenge teams and iPC staff will work together to educate students (K-12, undergraduate, and graduate, including members of underrepresented groups) through the use and development of Discovery Environments. Thus, education and outreach efforts will permeate the iPlant Collaborative.

The molecular mechanisms responsible for aging remain unclear. Proposed theories ascribe aging to a buildup of deleterious mutations or damaged macromolecules, to the differential advantage of some genes early in life that are later detrimental, or to an age-related trade-off between metabolic energy used early in life for reproduction versus energy used for repair and maintenance. These models are not mutually exclusive and each may play a role in the aging process. From studies of model organisms it is known that aging is associated with a decrease in energy metabolism, decreased rates of protein translation, lower protein turnover, and a buildup of damaged proteins. This protein damage is proposed to be a natural by-product of metabolism and lifespan may be inversely correlated with metabolic rate. The Principal Investigator (PI) proposes a novel idea about the molecular basis of aging. The PI hypothesizes that damaged proteins that accumulate with aging are increasingly difficult for cells to degrade and eliminate. Cells respond to the accumulation of damaged proteins by enhancing the expression of protein metabolism genes encoding chaperonins involved in protein folding and enzymes involved in protein degradation. The enhanced protein folding capability is proposed to decrease a cellular protein quality control mechanism that minimizes expression of unstable proteins, thus creating a positive feedback loop that causes even more damaged or unfolded proteins to be created. Regulation of the system could be at either the breakdown of the protein and its mRNA, or at the level of gene expression assuming epigenetic mechanisms have evolved to down-regulate mRNAs encoding unstable proteins. The proposed working model is that controlled expression of unstable or weakly folding proteins (i.e., allele-specific gene expression) is a critical quality-control component of the youthful state that is lost in aging individuals, making the aged individual less vigorous and more susceptible to disease. This aging model predicts that allele-specific gene expression and protein quality control will be maintained during lifespan extension by caloric restriction as well as in worm and fruit fly mutants with extended lifespan. The model also predicts that identification of allele-specific gene expression experimentally coupled with computational analysis of relative protein stability will serve as an efficient assay to monitor and assess the progression of aging. This molecular model can be readily tested by combined computational and wet-lab approaches focused on mice, worms, and flies. Proof-of-concept of this novel theory has important broader impacts, leading to the development of new computational approaches for addressing the aging process which may provide diagnostic and computational approaches to mitigate the impact of inherited deleterious genes in humans.

DESCRIPTION (provided by applicant): With Highly Active AntiRetroviral Therapy (HAART), HIV-1 replication can be controlled to maintain a plasma level of viral RNA below detection by conventional means. However, the continued presence of silent HIV-1 proviral DNA in memory CD4+ T cells is a major barrier to the eradication of the virus in AIDS patients. Multiple mechanisms have been reported by which HIV-1 proviruses are maintained in a silent state in these cells. The major block to active viral replication is believed to be at the transcriptional level. However, viral RNAs can be detected in long-term HAART-treated patients by sensitive detection methods, suggesting that transcriptional control may not be the only mechanism underlying the maintenance of viral latency. This project now aims to understand the post-transcriptional control of HIV-1 gene expression mediated by three regulatory systems: the Nonsense Mediated Decay (NMD) system; a repressor of HIV translation (RVB2) which we have recently identified; and the Zinc- finger Antiviral Protein (ZAP) which silences and triggers degradation of many viral RNAs. We will specifically explore the possible role of each of these systems in the maintenance of HIV-1 latency. We will examine the levels of HIV-1 RNA and protein expression in CD4-positive memory T cells from HIV-1 infected patients after manipulating the functions of each of these regulatory systems using siRNA knockdown technology. Discovery of the major mechanisms controlling post-transcriptional regulation of HIV-1 gene expression is an essential first step toward the release of repression and the activation of viral replication. This activation will permit the detection of virus-infected cells y the immune system and facilitate the clearance of infected cells by therapies that target viral proteins expressed in and on the surface of memory T cells.

iPlant is a new kind of virtual organization, a cyberinfrastructure (CI) collaborative created to catalyze progress in computationally-based discovery in plant biology. iPlant has created a comprehensive and widely used CI, driven by community needs, and adopted by a number of large-scale informatics projects and thousands of individual users. The project has laid a strong foundation to build an increasingly more capable CI and is poised to have an even greater impact on the plant sciences and a number of related fields, with a new focus on addressing computational bottlenecks for a broad number of life science researchers.

In the next five years, iPlant will continue to enhance the capabilities of a comprehensive CI and will also expand the scope to cover a number of new fields of inquiry. In iPlant's initial phase, Grand Challenge projects were defined to shape community requirements for the design of the CI. The two projects, Genotype-to-Phenotype and the Tree of Life, led to new analytical tools and competences for genomics and evolutionary biology. Future work will advance these capabilities and expand into capture and modeling of phenotypic, environmental, and ecological data. As before, this growth will be motivated by community needs and accomplished by community collaboration.

iPlant will continue to actively partner with other large CI development efforts and will coordinate CI development where feasible, appropriate, and mutually beneficial. iPlant will continue to be the underlying infrastructure provider for a number of projects that provide a variety of bioinformatics services. While continuing to support plant biology discovery research, iPlant will expand scope beyond the plant sciences, in coordination with nascent animal-centered efforts. The project will continue to adapt to the rapidly changing needs of the life sciences community and the rapidly changing technological landscape faced by researchers.

The intellectual merit of the project is in advancing the state of modern biology. Without question, research progress in the plant sciences, and in life sciences more generally, is increasingly limited by data and computational challenges. As knowledge of plant biology increases, the field will progress from informatics-based discovery to predictive modeling and eventually to synthetic biology. A comprehensive CI that eliminates the bottlenecks of data management, data standards, file formats, analysis, efficient collaboration, and knowledge dissemination will be a necessary underlying enabler to achieve this vision, and iPlant is positioned to be this enabling infrastructure.

The broader impacts of the project are numerous. The CI currently supports thousands of end users through its data storage, cloud, and online analytical capabilities. As a service provider, iPlant underlies a number of other online biological information resources. The project will continue its wide-ranging and successful education and outreach efforts, and will teach computational skills to learners at all levels, with particular focus on faculty to enable a sustained culture change that incorporates these advanced skills into the teaching of biology.

DESCRIPTION (provided by applicant): Upon entry into cells, many diverse viruses exploit their hosts' cytoskeletal transport networks to reach their sub-cellular site of replication, and nw viral progeny use these same networks to return to the cell surface and spread. Viruses frequently move along actin at the cell periphery before transitioning onto host microtubule (MT) networks that mediate long-range intracellular transport. MTs are highly dynamic heteropolymers of ¿/¿ tubulin (half-lives <5 min), which radiate from the perinuclear MT Organization Center (MTOC) towards the cell surface. Subsets of MTs become stabilized (half-life >1h) in response to various environmental and developmental signals, and are thought to act as specialized networks for vesicle transport during events such as cell polarization and motility. MT dynamics and stabilization are controlled by a number of highly specialized regulators, including actin-MT crosslinking factors and MT plus-end binding proteins (+TIPs), whose accumulation at MT ends is facilitated by the MT plus-end tracking protein, EB1. Movement of cargos on these MT networks involves motor proteins; generally, dynein directs minus-end and kinesins direct plus-end transport. However, our understanding of the role of MTs, their regulators and motors in the movement of viral particles during infection is severely limited. This Program Project Grant (PPG) nucleates expertise in cytoskeletal regulation, motors and MT-based motility, cell signaling and infection by diverse viruses to address these fundamental questions in mechanistic detail in a variety of systems. As a group, our interactions to date have established that both RNA and DNA viruses cause distinct MT modifications and require a range of specialized MT regulatory factors for efficient infection, including actin-MT crosslinkers, +TIPs and EB1, as well as identifying specific host motors used for virion traffickin to the nucleus. In this PPG we aim to determine the mechanistic details underlying these highly dynamic interactions between MT subsets, motors and invading virions, including the use of state-of-the-art dual-color imaging to analyze these events in real time. This integrated and interactive approach not only greatly enhances each individual project's potential by leveraging the strengths of other members, but also serves to focus our cumulative expertise on addressing key aspects of the Overall Aims of this PPG, Microtubule Networks and Virus Trafficking. This efficient group approach has the potential to uncover fundamental new insights in MT function and regulation during viral infection that will likely be important in broaer biological contexts and may lead to the development of novel therapeutic approaches.

Moloney murine leukemia virus (MLV) is a prototypical gammaretrovirus that replicates to high titer in nearly all mitotic rodent cells, causes a persistent viremia in infected mice, and induces a T-cell leukemia at a high incidence through insertional activation of host protooncogenes. Much of what we know about retrovirus replication was first learned through the study of the simple viruses such as the MLVs. In the early phases of infection, these viruses enter the cell through specific receptors, synthesize a DNA copy of the viral RNA genome by reverse transcription in the cytoplasm, direct the movement of the resulting preintegration complex (PIC) into the nucleus, and integrate the viral DNA into the host genome to form the provirus. In the late phases this DNA is expressed to form viral RNAs and proteins, and progeny virions are assembled at the plasma membrane and released to begin a new infectious cycle. In this project we propose to examine the early post-entry events of infection, the most poorly characterized portion of the viral life cycle, focusing on a key host protein, IQGAP, microtubules, and dynein motors. We have previously identified IQGAPI as a major host protein interacting with the Moloney MuLV Gag matrix protein (MA), and have documented the critical importance of that interaction for virus replication. The IQGAPs are large cytoskeletal scaffolding proteins involved in the regulation of cell motility and morphology, and are noteworthy in binding and regulating both actin and microtubule networks. They integrate multiple inputs (especially from small GTPases) and produce multiple outputs, including stabilization of microtubules and capture of microtubule ends. We will determine the role ofthe IQGAPs in MLV infection, and the precise time and step in the life cycle at which they act. Using live-cell imaging of fluorescence-tagged virions, we will examine the trafficking of MLV mutants that do not bind IQGAP, and of wild-type virus blocked by dominant-negative fragments of IQGAP, to determine whether virions fail to be properly delivered to microtubules. We will test for the importance of phosphorylation of IQGAP, thought to be mediated by PKCe, in normal virus trafficking. Finally, we will test the key subunits of the dynein motor and dynactin for their roles in movement of the MLV PICs along both stable and dynamic microtubules. With the help of our cell biologist colleagues in this program, we hope to fill in a major gap in our current understanding of the MLV life cycle. RELEVANCE (See instructions): Retroviruses are agents of serious human diseases, including leukemias and AIDS, and conversely hold out great promise as tools for gene therapy. Recent work has shown that these viruses are critically dependent on the cytoskeleton and microtubules (MTs) for their intracellular trafficking. In this proposal we aim to define the role of particular MT regulators and motors in early steps of MLV infection. Deeper understanding of these processes will provide new insights into retrovirus replication, potentially define new antiviral targets and increase our knowledge of trafficking of cargos on MTs.

This proposal describes genetic and biochemical analyses of host factors regulating the expression of the Moloney murine leukemia virus, the prototype of the simple mammalian retroviruses. We are especially focused on characterizing the mechanism of action of specific factors that we have identified that limit or restrict virus expression in embryonic cell types. We will characterize the mechanisms by which murine embryonic stem (ES) cells transcriptionally silence proviral DNAs and maintain the integrity of the germ line. We will study three parallel pathways ? a rapid and highly efficient mechanism targeting a specific DNA element of the Moloney provirus, the tRNApro Primer Binding Site (PBS)? a less potent one acting at a conserved site, the negative control region (NCR) present on the proviral DNA of many retroviruses;? and a newly-­identified one also acting broadly on many retroviruses. We will characterize the DNA-­binding host proteins that mediate the silencing (ZFP809, YY1, and NP220) and determine how these silencing mechanisms are regulated so as to be specifically active in ES cells. The study will involve examination of ubiquitin ligases, SUMO transferases, and protein-­protein interactions needed to form the large complex that binds to the viral DNA and induces silencing by making repressive histone modifications. We will also examine the mechanism by which one of these factors uniquely activates, rather than silences, one member of the retrovirus family (HTLV-1). Because the expression of retroviral DNAs is so closely correlated with expression of host genes during embryonic development, these experiments will provide important new information about the properties that define ?stemness? ? the pluripotent state of ES cells. These aspects of control of retroviruses by host factors will provide new targets for antiviral therapy. Most importantly, these experiments will significantly extend our understanding of fundamental aspects of retrovirus replication, and of new cell biological processes that impact on these important viruses.

We will explore the role of a novel host factor in retrovirus replication, heme oxygenase 2 (HO-2), which we discovered binds N-terminally myristoyled proteins and regulates their trafficking to the plasma membrane. We have generated cell lines deficient in HO-2 by shRNA methods, or entirely lacking HO-2 by CRISPR-directed knockout (KO), and documented dramatic increases in retrovirus virion production. We will now characterize the intracellular transport of the myristoylated murine leukemia virus (MLV) Gag protein with and without HO-2 by examination of fixed sections by immunofluorescence, and by live cell imaging. We will study the size of intracellular complexes containing Gag by sedimentation analysis of extracts, followed by Western blots probed with anti-Gag antibodies. We will follow the course of Gag multimerization by co-immunoprecipitation experiments. We will look for alterations in Gag association with its known trafficking partners (including ABCE1, DDX6, and MOV10), and with proteins known to be important in the process of virion assembly and release (notably the ESCRT and CHMP proteins). We will assess the course of Gag interaction with viral RNA by RNA-immunoprecipitation (RIP) assays. These experiments should give us a comprehensive view of the role of HO-2 in controlling Gag localization and function in virion assembly. The work will define a new potential target for antiviral therapies against retroviruses, and other viruses encoding myristoylated proteins. In addition, we will explore the role of HO-2 in trafficking of selected cellular myristoylated proteins. We have developed an extensive hit list of host proteins bound by HO-2 and will focus on two of these proteins: Toll-like receptor adaptor molecule 2 (TRAM), involved in innate immune signaling;? and reticulon 2, a key protein in intracellular trafficking and in promoting membrane curvature. We will characterize the role of HO-2 in controlling the localization of TRAM, the association of TRAM with the TLR4 receptor, and the signaling through TRAM in response to lipopolysaccharide (LPS). We will similarly test for the role of HO-2 in the localization of reticulon 2 to the endoplasmic reticulum. Because of its potential role in membrane bending, we will test for the effect of reticulon 2 KO on ER structure and virion budding. These experiments will reveal new functions of HO-2 in regulating those specific host functions that mediate virus restriction and impact virion assembly. The findings have great potential to reveal entirely new approaches to regulate or inhibit virus replication.