Kathleen Collins - US grants

DESCRIPTION (Verbatim from the applicant's abstract): The telomerase reverse transcriptase adds telomeric DNA simple sequence repeats to chromosome ends by copying a template sequence within its integral RNA component. This de novo addition is required to balance the loss of repeats that occurs with incomplete replication of chromosome ends by conventional DNA-dependent DNA polymerases. Cells that do not produce active telomerase, including most cell types in multicellular organisms, lose telomeric repeats with each round of cell division. When telomeric repeat number reaches a critical minimum, short telomeres signal apoptosis or entry into an irreversible replicative senescence. Cancer cells can escape this limitation of proliferative capacity by activating telomerase. Because telomerase-positive cancer cells appear to require telomerase for continued viability, telomerase inhibitors could prove to be potent, selective and broadly useful anti-cancer therapeutics. Telomerase activators may also be useful in enhancing the proliferative capacity of some human tissues such as blood and skin. The desire to understand telomerase function and regulation is hindered by an incomplete knowledge of proteins associated with the telomerase enzyme and by an even more limited knowledge of what factors govern the telomerase-telomere interaction. By using the ciliate Tetrahymena thermophila as a model system, both of these issues can be addressed. Tetrahymena provides the combination of facile genetic manipulation with a relative abundance of telomeres and telomerase. Affinity purification experiments described in Specific Aim I will identify a complete inventory of telomerase protein components in telomerase RNPs with different biological functions. Specific Aim II describes structural studies of recombinant telomerase that should illuminate the biochemical basis for novel enzyme properties. Specific Aim III examines Tetrahymena telomere structure using microscopy, biochemistry, and molecular genetics, then uses these same techniques to study the molecular regulation and cellular dynamics of telomere-telomerase interaction.

The ability of the human immunodeficiency virus (HIV) to escape immune surveillance in vivo is central to its pathogenicity and potentially limits our ability to develop effective vaccines. In HIV+ individuals, anti-HIV cytolytic T lymphocytes (CTLs) are clearly abundant and play a role in limiting viral loads, but they are unable to eradicate the virus. To better understand this enigma, we studied the efficacy with which anti-HIV CTLs are able to kill infected cells. This required the development of a unique system in which all infected cells could be identified (and quantitated) by the expression of placental alkaline phosphatase (FLAP). We used the PLAP marker to monitor the outcome of infected cells treated with CTLs. We found that HIV encodes at least one factor (nef) that protects infected cells from CTLs. Nef limited the ability of CTLs to kill infected cells by decreasing the density of MHC class I-epitope complexes on the surface of infected cells. In this proposal, we present experiments that will shed more light on the molecular mechanism of nef-mediated MHC class I downmodulation.

The long term objective of this application is to understand how HIV evades the immune response and establishes a chronic infection. Insights into how this occurs may lead to new strategies to augment the ability of the immune response to eradicate the virus. One way that HIV avoids immune recognition is by reducing cell surface levels of major histocompatibility complex class I molecules (MHC-I), thereby protecting HIV-infected cells from recognition by cytotoxic T lymphocytes (CTLs). In addition, we have found that the capacity of cells to process and present antigens varies depending on the MHC-I haplotype and the cell type that is infected. Thus, we will pursue the following aims: (1) We will demonstrate that antigen presenting cells (APCs) and individual MHC-I molecules have unique pathways of antigen processing and presentation that (2) affect their responsiveness to Nef and recognition by CTLs. In addition, we will demonstrate that (3) HIV evades the immune response by remaining latent in long-lived progenitors of these cell types, which can serve as inducible reservoirs of HIV infection in vivo. To accomplish these goals we will use biochemical and cell biological methods to define the trafficking pathways of individual MHC-I molecules in normal and HIV- infected cells. We will also perform studies using patient samples to characterize the capacity of myeloid precursors to support HIV infection in vivo. This proposal is relevant to the mission of NIH in that it seeks to understand basic mechanisms of how HIV establishes a chronic infection. This work has the potential to have a significant impact on public health.

DESCRIPTION (provided by applicant): The disappointing results of the recent Merck vaccine clinical phase IIb trials, which utilized adenovirus-based HIV-1 candidates capable of generating an anti-HIV CTL response (HIV vaccine failure prompts Merck to halt trial. Nature 449, 390 (2007), http://www.hvtn.org/science/1107.html) highlights the importance of understanding the mechanisms through which HIV successfully evades the anti-HIV CTL response. In addition, the inability of current therapies to cure HIV disease underlines the importance of taking new approaches at targeting HIV. Existing drugs effectively prevent new infections, however none of the currently available anti- HIV therapies aid in the eradication of pre-existing infected cells. Thus, it is more clear than ever before, that we need to fully understand the viral mechanisms for immune escape to eradicate infected cells, cure disease and prevent new infection. To this end, the HIV Nef protein is an important and under-developed drug target. HIV-1 Nef has been shown to protect infected primary T cells from CTL recognition and killing in vitro by downmodulating MHC-I protein [7]. There is also substantial in vivo evidence that MHC-I downmodulation is critically important in SIV-infected monkeys for HIV disease pathogenesis [8, 9]. Over the past two funding periods of this grant, we have generated substantial evidence indicating that Nef-mediated MHC-I downmodulation occurs in the following manner: Nef binds to immature forms of MHC-I by targeting hypophosphorylated MHC-I cytoplasmic tail domains in the ER/early Golgi. Binding prevents phosphorylation of the MHC-I cytoplasmic tail and disrupts MHC-I transport from the trans-Golgi network (TGN) to the cell surface. MHC-I is then targeted for degradation in lysosomes. Targeting of MHC-I into the endo-lysosomal pathway from the TGN requires the activity of two cellular co-factors, AP-1 and 2-COP. These cellular trafficking proteins bind the Nef-MHC-I complex at distinct sub-cellular locations and promote targeting into the endosomal and lysosomal pathways respectively. Moreover, we now have a detailed understanding of which amino acids are necessary to form the Nef-MHC-I-AP-1 three-way complex. In the up-coming funding period, we will continue to uncover the mechanism by which Nef functions by determining how Nef is able to bypass the normal cellular regulation of these molecules. In addition, we will begin to apply our assay systems to other Nef targets to compare and contrast the manner in which Nef affects other cellular pathways. Finally, we will demonstrate to what extent these Nef activities are generalized across HIV subtypes.

DESCRIPTION (provided by applicant): In dyskeratosis congenita (DC) and many other disease states, patient health and longevity are limited by insufficient hematopoietic renewal. Because bone marrow transplantation has not been an effective DC therapy, new strategies must be devised. This application tests the hypothesis that X-linked DC derives from a specific telomerase RNA deficiency and investigates telomerase function in lymphoid cells, with the long-term goal of understanding disease molecular mechanism(s) and developing effective disease therapies. AIM I: Telomerase function will be restored in X-linked DC patient fibroblasts expressing altered dyskerin, in parallel with telomerase activation in family-matched X-linked DC carrier fibroblasts expressing wild-type dyskerin. DC patient cells rescued for telomerase function will be tested for the acquisition of normal proliferative capacity and telomere length maintenance. These findings will be relevant for understanding disease mechanism and also may provide proof-of-principle for telomerase activation as a clinical therapy. AIM II: Aim I telomerase-activated cell lines expressing DC or normal dyskerin will be used to investigate potential DC dyskerin defects in the biogenesis of RNAs other than telomerase. Unlike currently available DC patient cell cultures, cell lines +TERT have robust, uniform growth and will therefore avoid misleading characterization of RNA biogenesis defects secondary to changes in proliferation rate or the percentage of cells undergoing active proliferation. Dyskerin-associated RNA accumulation and dyskerin enzyme function in ribosomal RNA precursor modification and cleavage will be assayed. AIM III: Mechanisms of telomerase function in normal lymphoid cells will be studied using a cell culture system that resolves telomere length -dependent and -independent roles for telomerase in promoting cell growth and survival. A potential third role for telomerase in protection against cell death will also be evaluated. These studies will reveal new roles for telomerase in the cells that are most significantly affected in DC.

[unreadable] DESCRIPTION (provided by applicant): Support is requested to continue a multidisciplinary program of predoctoral training in cellular, biochemical, and molecular sciences under the auspices of the Department of Molecular and Cell Biology (MCB) at the University of California at Berkeley. MCB is a single Department comprised of 109 faculty in five Divisions: Biochemistry and Molecular Biology; Cell and Developmental Biology; Genetics, Genomics and Development; Immunology; and Neurobiology. Based on this organization, MCB has developed a broad, integrated and interdisciplinary approach to training Ph.D. candidates. The 65 participating faculty listed in this application pursue diverse research problems at the molecular and cellular level, providing research opportunities and appropriate instructional environments for the training of graduate students in: (a) Biochemical sciences (metabolic regulation, enzyme purification, enzymatic catalysis, structure and function of biological macromolecules, especially proteins and nucleic acids); (b) Molecular biology (chromosome structure, regulation of gene expression, DNA replication, recombination, repair, transposition); (c) Cell biology (cellular organization and function, cytoskeletal architecture, cell movement, cell surface receptors, ion channels, signal transduction mechanisms, cell growth control); and (d) Molecular and cellular aspects of development (embryogenesis, pattern formation, cell-cell interactions, neuronal differentiation, apoptosis, and development of the immune system). While enrolled in formal coursework to strengthen and broaden their background, entering students conduct ten-week laboratory research projects in three different faculty laboratories. Based on these experiences, the new trainees select doctoral dissertation mentors. In the second year, students concentrate on research, continue to take lecture and/or seminar classes, acquire teaching experience by serving as Teaching Assistants, and undergo an Oral Qualifying Examination administered by a cross-divisional committee. In years three-to-five, students focus almost exclusively on their thesis research; however, enrollment in at least three advanced seminar courses is required. Weekly divisional colloquia, named lectureships and departmental symposia ensure that eminent scientists are brought to the Berkeley campus to expose trainees to the latest breakthroughs in their fields of interest. Applicants typically have outstanding undergraduate records in the biological, chemical or physical sciences. [unreadable] [unreadable] [unreadable]

Telomerase elongates chromosome ends by addition of tandem telomeric repeats. This new DNA synthesis is required to balance the loss of DNA that is inherent in the incomplete replication of chromosome ends by conventional DNA polymerases. Single-celled eukaryotes constitutively activate telomerase and maintain a homeostasis of telomere length. Surprisingly, human somatic cells do not: they show progressive shortening of the telomeric repeat array with proliferation. Some human cells in the embryo, germline, epithelial tissues, and hematopoietic system have detectable levels of telomerase catalytic activity in cell lysates, but this level of activation is insufficient to prevent an overall loss of telomere length in all human tissues with age. Cumulative loss eventually produces a repeat array that is too short to protect the chromosome end, resulting in a forced exit from the cell cycle. Cancer cells dramatically up-regulate telomerase to permit indefinite growth. For this reason, telomerase inhibitors have great promise as broadly effective anti-cancer therapeutics. Telomerase activators may have equally significant application for expanding the renewal capacity of normal somatic cells with critically short telomeres arising from genetics, disease, age, or environment. The telomerase RNA subunit (TER) is expressed as a precursor that must be processed, folded, and assembled as a stable ribonucleoprotein (RNP) complex in order to accumulate to detectable level in vivo. This RNP then recruits telomerase reverse transcriptase (TERT) to generate the active enzyme. Collins lab efforts in previous funding periods have contributed pioneering insights about the endogenous pathway of human TER precursor processing and RNP assembly and discovered defects in the accumulation of mature telomerase RNP that underlie X-linked and autosomal dominant forms of the bone marrow failure syndrome dyskeratosis congenita. The Specific Aims of the next funding period address remaining gaps in knowledge about human telomerase RNP accumulation and catalytic activation in vivo. Aim 1 exploits methods of transient and stable TER expression in human cells to discover and characterize additional RNA motifs and proteins required for TER maturation and biological stability. Aim 2 applies Collins lab expertise in RNA-protein interaction assays and affinity purification to define the biochemical defects that underlie inherited human diseases of telomerase deficiency. Aim 3 investigates the assembly and activity of telomerase RNP with TERT. In vivo reconstitution methods will be combined with in vitro and in vivo activity assays to define TER motif functions in the catalytic cycle. The physiological specificity of RNA and protein domain interactions within the active RNP will be established. The long-term goal of these studies is to understand telomerase RNP assembly, catalytic activation, and cellular regulation in normal cells and disease and to exploit this understanding for improvement of human health.

DESCRIPTION (provided by applicant): Small molecule chemical inhibitors of biological systems are powerful tools for probing complex biological processes and they also hold the promise of new therapies. Not surprisingly, there has been much effort invested into adapting assays for high throughput screens to identify such molecules that affect important biological processes. In this regard, cell-based phenotypic assays are particularly powerful as they make few assumptions about mechanism and the read out is likely to be biologically relevant. We have developed a high throughput assay system that detects the effect of the HIV protein, Nef, on the cell surface expression of the cellular protein, MHC-I, which is required for immune recognition of infected cells. Nef reduces the expression of this molecule to evade the immune response and establish a chronic infection. Inhibitors of this process would aid efforts to understand the mechanism by which Nef affects MHC-I expression and would have therapeutic potential. PUBLIC HEALTH RELEVANCE: Although currently available anti-HIV drugs effectively prevent the development of disease in many affected individuals, they do not cure disease. It has been proposed that residual cellular reservoirs that are not affected by current therapies are responsible for re-establishing high viral loads. Inhibitors of the Nef protein may improve the treatment of HIV-infected people by allowing the immune system to more efficiently identify and eradicate infected cells.

DESCRIPTION (provided by applicant): HIV causes a chronic infection characterized by depletion of CD4+ T lymphocytes and, at late stages, severe bone marrow abnormalities. Despite the development of drugs that inhibit viral spread, HIV has been difficult to treat because of uncharacterized reservoirs of infected cells that are resistant to highly active antiretroviral therapy and the immune response. We have used CD34+ cells from infected people as well as in vitro studies of wild type HIV to demonstrate infection and killing of CD34+ hematopoietic progenitor cells (HPCs). Importantly, the HPCs that became infected included primitive, multi-potent cells. In some HPCs, we detected latent infection that stably persisted in cell culture until viral gene expression was activated by differentiation factors. This application proposes studies, which will determine whether HIV-1 envelope tropism targets progenitor cells with different developmental capacities and whether HIV-1 infects bona fide stem cells. PUBLIC HEALTH RELEVANCE: The overall goal of this research is to develop improved treatments for HIV-infected people by determining which cell types are responsible for persistent disease.

DESCRIPTION (provided by applicant): There is a fundamental gap in our understanding of which cell types contribute to residual viremia in HAART treated people. Our laboratory's long-range goal is to understand HIV-1 persistence in patients that are optimally treated with highly active anti-retroviral therapy (HAART). We expect that a greater understanding of HIV persistence will inform the design of therapeutic strategies that will result in long-term remissio or cure. As the next step towards this goal, the objective of this application is to identify celluar sources of residual viremia in optimally treated people and to characterize the residual virus. Our central hypothesis is that residual virus is generated in part by latently infected hematopoietic progenitor cells. The rationale of the proposed work is that identifying cellular reservoirs and viral sub-types that contribute to persistence will yield mechanistic insights into the establishment of latency and accelerate the discovery of disease-modifying therapies. We plan to test our central hypothesis and accomplish the objective of this application by pursuing the following four specific aims: (1) Establish the extent to which bone marrow progenitors harbor HIV genomes in optimally treated people on HAART. We hypothesize, based on preliminary studies, that HSPCs are an inducible reservoir of HIV that can spread infection to T cells. We will test this by measuring the amount of reactivatable HIV proviral DNA in HSPCs from a cohort of optimally treated donors on HAART, including donors who have been on HAART since primary HIV infection. (2) Identify which chemokine receptor is primarily utilized by viruses to enter HSPCs. Our working hypothesis, based on preliminary data, is that HSPCs from HAART-treated donors will harbor dual or CXCR4-tropic HIVs. To test this, we will characterize full-length viral envelopes from these cells and use functional assays to assign tropism. (3) Characterize the contribution of infected HSPCs to residual viremia in optimally treated people on HAART. Our preliminary data led us to hypothesize that reactivated virus from infected HSPCs will seed the periphery. To test this, we will isolate and sequence residual virus and determine based on sequence comparisons to what extent HSPCs versus other possible reservoirs can be identified as the cellular source of this virus. (4) We will determine the mechanisms by which quiescent viral DNA in HSPCs can be reactivated to eliminate latently infected cells. Viral latency in HSPCs has not been well characterized. We will examine the cellular and viral factors that contribute to latency in HSPCs to aid in the selection or development of drugs that can purge latently infected HSPCs. This contribution will be significant because identifying the relevant cellular reservoirs and defining the mechanisms of latency is an important initial step toward the development of better treatment strategies for HIV infected people. If our hypothesis is proven correct, this work will refocus efforts to identify therapeutic targets for eradication of cellular reservoirs to long-lived hematopoietic progenitor cells. PUBLIC HEALTH RELEVANCE: The proposed research is relevant to public health because HIV is an incurable, pandemic virus that has infected millions of people globally and that continues to infect nearly 40,000 people each year in the United States. The outcomes of the proposed research are expected to have an important positive impact by the identification of cellular reservoirs from which latent HIV-1 can cause resurgent viremia and by the identification of potential therapeutic targets for new drug development.

? DESCRIPTION: HIV-1 causes a persistent infection due to long-lived reservoirs of latently infected cells that present a barrier to achieving a cure. There are active efforts ongoing to eliminate the latently infected cells by using drugs that antagonize latency and activate viral gene expression. To date, the antagonists that have been identified tend to reactivate viral gene expression without efficiently killing the infected cells and clearing the reservoirs. Therefore, i may be necessary to develop immunotherapeutic approaches to ultimately clear the virus and cure disease. Cytotoxic T lymphocytes, which can recognize actively infected cells may aid in the clearance of reservoirs treated with latency antagonists. However, CTLs are limited in the extent to which they can effectively clear infection. Our long-term goal is to understand how to enable the immune response to more effectively recognize and eradicate latently infected cells that have been treated with latency antagonists. The overall objective of this application, which is the next step toward attainment of our long-term goal, is to identify compounds that maximize viral antigen presentation in HIV-infected cells. We hypothesize that while CTLs have activity against latently infected cells that have been induced to express HIV-1 genes, HIV-1 Nef, which down modulates MHC-I, limits the extent to which induced cells can be killed. Preliminary studies indicate that Nef inhibitors found in natural product extracts can reverse the effects of Nef and sensitize infected cells to CTL killing. These studies have led to the hypothesis that combination therapy with latency antagonists plus Nef inhibitors could act synergistically to clear reservoirs. The R21 phase of this proposal aims to complete the identification of inhibitory factors in high priority natural product extracts that contain anti-Nef activity. Upon successful completion of the R21 phase, The R33 phase will involve (1) determining the mechanism by which each factor inhibits Nef-mediated MHC-1 down modulation and (2) conducting lead compound optimization to improve the pharmaceutical properties and identify a leading drug candidate for development.

? DESCRIPTION (provided by applicant): Our laboratory's long-range goal is to develop approaches to eradicate HIV. Unlike most viral infections, HIV is able to overcome the host innate and adaptive immune response to establish a life long infection in nearly every infected person. Moreover, standard approaches to develop vaccines and antiviral drugs have not succeeded in providing protection from initial infection or cure. Thus, novel approaches are needed that will more effectively counteract the unique ability of HIV to establish a persistent infection. To achieve this goal, we must first acquire a better understanding of how HIV evades the host innate and adaptive immune responses. We expect that a greater understanding of these viral strategies will inform the design of therapeutic approaches that will result in prevention of infection, long-term remission or cure. As the next step towards this goal, the objective of this application is to understand how the HIV accessory protein, Vpr, allows HIV to evade innate immune responses. Based on strong preliminary data, our central hypothesis is that the innate immune system fails to contain and eradicate HIV because HIV Vpr counteracts host innate immune defense strategies. This hypothesis springs from data demonstrating that Vpr limits the ability of the target macrophage to sense infection and respond to counteract viral spread. In particular, Vpr counteracts a macrophage-specific innate immune response mechanism that recognizes HIV and activates a type I interferon response against virally infected cells. Thus, the rationale of the proposed work is that a greater understanding of how HIV evades the innate immune response will inform approaches that aim to activate the innate immune response to clear infection. By identifying the key viral mechanisms that are needed for persistence, we will better understand which approaches are likely to be effective. Thus, we plan to use the genetic, biochemical and primary cell culture systems we have established to test our central hypothesis and accomplish the objective of this application, which are summarized in following specific aims: 1) Define the restriction factor that limits viral spread in the absence of Vpr; 2) Determine how IFN activates the restriction that is counteracted by Vpr; and 3) Determine how HIV is sensed in the absence of Vpr, which will inform approaches to determine how Vpr limits sensing. At the completion of these studies we expect to establish that Vpr functions to counteract host cell recognition of viral infection by hijacking host DNA repair mechanisms that limit the accumulation of HIV replication intermediates in primary cell culture. Thus, we expect to demonstrate that Vpr prevents the activation of downstream pathways that would otherwise potently inhibit infection. These outcomes are expected to have an important positive impact by identifying the pathways that are most important to target for anti-HIV drug development. Targeting the mechanisms by which the virus disrupts immune pathways will allow the development of safer, more specific treatment approaches that will improve the care of HIV-infected people.

Our laboratory?s long-range goal is to understand how HIV-1 persists despite optimal antiretroviral therapy and to develop new approaches to clear the infection. Virus isolated from patient blood encodes vpr, which is packaged into virions and expressed at late stages of viral infection. Vpr is needed for optimal infection of macrophages, tonsillar tissue and SIV model systems, however its actions in T cells are largely detrimental to infection and a requirement for Vpr for HIV infection is not observed in pure T cell systems. There is currently a critical gap in our understanding of why Vpr is conserved in the virus despite its unique role in macrophages, which are not known to be crucial for HIV transmission or persistence. Our preliminary data strongly indicate that Vpr is responsible for downmodulation of a macrophage-specific protein that interferes with Env expression. Thus, the objectives of this application are (1) to determine the extent to which Vpr-mediated downmodulation of the macrophage protein alleviates restriction of Env and spread of HIV in macrophages, (2) to determine the domain of Env required for restriction in macrophages and (3) to identify viral populations from in vivo samples that manifest these phenotypes. Our central hypothesis is that a macrophage protein binds residues on Env and targets it to degradative and/or antigen presentation compartments to restrict spread and enhance antigen presentation in infected and bystander myeloid cells; Vpr counteracts this pathway by reducing levels of the restricting protein through direct infection and fusion of Vpr-containing virions with bystander myeloid cells. The rationale for the proposed work is that a greater understanding of how HIV overcomes cellular antiviral mechanisms to establish a persistent infection in macrophages will facilitate efforts to clear infection in the CNS and other reservoirs. Thus, we plan to test our central hypothesis and accomplish the objective of this application by pursuing the following specific aims: 1. Determine the extent to which the macrophage protein we have identified accounts for restriction of Env in HIV infected macrophages lacking Vpr. 2. Define the domain of Env proteins needed for susceptibility to restriction by macrophages. 3. Identify viral populations from in vivo samples that target the restriction factor. At the completion of these studies we expect to determine the extent to which; (1) macrophages restrict viral infection by targeting a specific domain on Env, (2) sensitivity to this restriction is an important characteristic of viruses that form reservoirs in people, (3) Vpr functions to counteract this restriction by downmodulating the macrophage protein. An understanding of the macrophage restriction factor and identification of Env domains responsible for sensitivity to the restriction may provide new approaches to limit spread of HIV and enhance the immune response by promoting antigen presentation. Targeting the mechanisms by which the virus disrupts the immune system may allow the development of safer, more specific treatment approaches that will promote the clearance of HIV-1 infected cells.

Our laboratory's long-range goal is to develop new approaches to eradicate HIV-1. Unlike most viral infections, HIV-1 is able to overcome the host innate and adaptive immune response to establish a life long infection in nearly every infected person. Moreover, standard approaches to develop vaccines have not succeeded in providing protection from initial infection and antiviral drugs have not yet provided a cure. Thus, novel approaches are needed that will more effectively counteract the unique ability of HIV-1 to establish a persistent infection in nearly every host. We expect that a greater understanding of viral strategies will inform the design of therapeutic approaches that will result in prevention of infection, long-term remission or cure. As the next step towards this goal, the objective of this application is to understand how the HIV-1 accessory protein, Vpr, allows HIV-1 to evade innate immune responses. We have shown that Vpr counteracts a macrophage-specific Env degradation pathway in primary monocyte derived macrophages (MDM) and MDM-T cell co-cultures and that Vpr limits induction of interferon (IFN) gene expression following infection. Determining how Vpr evades these responses will provide crucial knowledge that may reveal approaches that will help clear infection. To accomplish this goal, we will test our central hypothesis and accomplish the objective of this application by pursuing the following specific aims: (1) Identify the IFN-induced macrophage-specific restriction factor that promotes Envelope degradation and (2) Determine how Vpr allows HIV to evade the innate immune response in macrophages. At the completion of these studies we expect to have identified the IFN-induced restriction factor and we expect to establish that Vpr interacts with host DNA repair proteins to limit sensing of DNA intermediates and innate immune responses in MDM. These outcomes are expected to have an important contribution to the field by identifying the pathways that are most important to target for anti-HIV drug development.

The reverse transcriptase telomerase elongates chromosome 3' ends by additional telomeric repeats, compensating for repeat loss during conventional DNA replication. Restrictions of telomerase function set an upper limit on human somatic tissue renewal, with consequences including immune deficiencies from chronic infection and diseases of bone marrow failure, pulmonary fibrosis, and other disorders from inherited telomerase subunit mutations. Inversely, constitutive over-activation of telomerase is almost universally required for human cancer progression and metastasis. Telomerase is unique among polymerases in its reiterative copying of a hard-wired internal template in the enzyme's integral RNA subunit. Also, unlike all other templated DNA polymerases, telomerase must release product that is single-stranded rather than duplex in order to regenerate the template and allow complementary-strand telomere synthesis. The elaborate catalytic cycle of repeat synthesis required to support telomerase specialization is accomplished by intimate co-folding and functional collaboration of telomerase reverse transcriptase (TERT) and telomerase RNA (TER). In addition, telomerase activity at telomeres and telomerase cellular regulation require numerous other subunits of cellular telomerase holoenzymes that are in general poorly characterized due to scarcity. The long-term objective of Collins lab NIGMS research funding is to determine the molecular and biochemical principles that underlie telomerase enzyme mechanism and cellular action. These goals inform fundamental principles of protein-nucleic acid interaction, ribonucleoprotein biogenesis and function, nucleic acid synthesis, cellular proliferation control, genome stability, and tumorigenesis. The strong Collins lab track record of insights supported by NIGMS funding emerges from parallel studies of telomerase in two enabling model systems: the ciliate Tetrahymena and cultured human cells. Our recent efforts have accomplished innovative telomerase reconstitutions that enable dissection of enzyme mechanism; field-shifting discoveries of cellular telomerase holoenzyme subunits and their functions; and truly landmark determinations of Tetrahymena and human telomerase holoenzyme structures by cryo-EM, made possible by a wide range of accumulated expertise. Future studies will build from this foundation towards the ultimate goal of enabling telomerase manipulation for clinical therapeutics. In the near term, expanded cryo-EM studies of human telomerase throughout its catalytic cycle, combined with other structural and biochemical assays, will identify the determinants of dynamic nucleic acid handling necessary for telomeric repeat synthesis. We will also investigate the mechanism of telomerase activation at telomeres and how telomerase synthesis of single-stranded DNA is coupled to the complementary strand synthesis necessary for telomere stability.

This award will support attendance by graduate students and postdoctoral researchers at the 2019 Gordon Research Seminar (GRS) on Nucleic Acids to be held June 1-2, 2019 at the Grand Summit Hotel at Sunday River in Newry, ME just prior to the associated Gordon Research Conference. The topical theme of the GRS meeting is "From organization and regulation to structure and mechanisms". The meeting is organized and co-chaired by two senior graduate students, thus providing them with valuable professional development experience. The format of the meeting will be a combination of talks and posters, aimed at encouraging scientific exchange in both formal and informal settings. Participants will benefit from a dedicated education, career and mentorship session featuring a keynote lecture from an expert in science education and a panel discussion with program directors in Federal funding agencies.

The scientific focus of this meeting and the accompanying Gordon Research Conference will be on multiple aspects of nucleic acids biology including research on both RNA and DNA. Specific topics will include telomeres, mRNA splicing, CRISPR technologies, 3D genome organization/dynamics/repair, translation regulation, RNA and DNA modifications, RNA quality control, and therapeutics. Emphasis on cutting-edge, unpublished research across many biological systems offers participants the opportunity to explore how new findings can be leveraged for their own work. The meeting will provide an excellent forum for junior investigators, including those from underrepresented groups, to network with each other and to have the opportunity to present and receive feedback on their own research.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

The goal of the University of Michigan Medical Scientist Training Program (MSTP) is to train physician scientists primarily for careers in academic medicine with a focus on translational and basic biomedical research directly related to clinical medicine. Such individuals are uniquely positioned to bridge the gap between basic and clinical medicine, and hence to connect basic discoveries to improvements in human health. We request 32 predoctoral positions. The total training is typically 8 years. The University of Michigan Medical Center is one of the world's largest one-site complexes devoted to health education, research and patient care, with over 4 million sq ft of space dedicated to education and laboratory research. The MSTP provides an integrated curriculum of MD/PhD training. Matriculants have a strong history of academic success and research experience, and are graduates of outstanding colleges from all parts of the US. There are currently 96 students and 238 graduates. The MSTP strives to include a diverse body of students. During the past 5 years, 24% of matriculants have been Underrepresented Minorities (URMs), and the entire student body currently is 17% URM. The curriculum typically begins with the first two years of medical school, which include a ?basic science trunk? year, and a ?clinical trunk? year during which students undertake the core clinical clerkships. A graduate level biochemistry course is taken as part of the 1st year of medical school. Trainees undertake a research rotation after the 1st year of medical school and one or two rotations after the 2nd year. The MSTP provides guidance to all trainees in the selection of research mentors, and carefully monitors progress and provides guidance through all stages of training. Trainees typically select a PhD field during the 2nd year of medical school; core participating departments include Bioinformatics, Biological Chemistry, Cancer Biology, Cell & Developmental Biology, Cellular & Molecular Biology, Human Genetics, Immunology, Microbiology & Immunology, Molecular & Cellular Pathology, Molecular & Integrative Physiology, Neuroscience, and Pharmacology. Other fields are possible; for example, currently there are trainees in our Schools of Engineering and Public Health, as well as in our Departments of Anthropology, Economics, and History. In the typical progression, students complete the M1 and M2 years and take USMLE Step 1 prior to continuing graduate studies full time. During these research years, trainees participate in clinical preceptorships to maintain and enhance their clinical skills and knowledge. Upon successful completion of the thesis defense, trainees complete their clinical training. All trainees take a clinical refresher tutorial shortly before the return to the clinical rotations. There is flexibility in the timing of return to medical school, and the requirements in the final year of medical school are decreased. The academic and clinical training are complemented by monthly program activities including academic seminars, career development activities, social events, and a 3-day annual off-site scientific retreat.

Abstract With the development of triple combination antiretroviral therapy, routine HIV treatment eliminates nearly all actively infected cells. Nevertheless, the small reservoir of latently infected cells, which can remain dormant for long periods of time before becoming active and producing new virus particles, represents a crucial barrier to completely curing the disease. Identifying markers that identify latently infected cells or the biochemical factors that control latency activation could enable the effective use of a ?shock and kill? strategy, where specific targeting or activation of latently infected cells eliminates the viral reservoir. Our recent work suggests that the global transcriptomic and epigenomic changes during hematopoietic differentiation affect viral latency and activation. Additionally, we recently found that global inhibition of histone deacetylase activity increases viral activation in these cells, further implicating epigenomic changes in activation. These results raise fundamental questions: What are the markers of latently infected cells? How do the transcriptomic and epigenomic state of a cell affect latency and activation? How does differentiation state relate to viral latency? Here, we leverage our experimental platform for identifying latently and actively infected cells, single cell transcriptome and epigenome sequencing, and our recently developed computational integration methods to investigate these questions. Our interdisciplinary team combines expertise in HIV basic science, HIV clinical treatment, and bioinformatics to develop an experimental and computational framework for integrated gene expression, chromatin accessibility, and lineage into a single picture of viral latency and activation. Specifically, this project will (1) use single-cell RNA-seq and single-cell ATAC-seq to map diversity of infected cells, (2) investigate the relationship between hematopoietic differentiation state and viral activation, (3) determine viral integration sites through single-cell RNA-seq, (4) computationally integrate single cell transcriptome and epigenome profiles, and (5) computationally infer cell lineage relationships among viral genomes and infected cells. To accomplish these goals, we will carry out the following aims: (1) Characterize lineage, transcriptomic and epigenomic diversity of single latently and actively infected primary cells. (2) Investigate latency and activation during in vitro differentiation. (3) Survey single cell diversity of re-activated and in vitro infected cells from cART-suppressed patients. Together, these aims will produce a comprehensive, integrated transcriptomic and epigenomic atlas of the HIV reservoir, identify DNA and RNA biomarkers of latency, and characterize clonal expansion patterns. Our work also develops a broadly applicable experimental and computational framework, laying a foundation for the discovery of novel insights into HIV latency and activation.

Current combined antiretroviral therapies (cART) suppress viral levels in the blood but do not eradicate reservoirs of cells harboring integrated copies of HIV proviral genomes. These cells persist in part because the provirus maintains a latent state that evades the immune response and viral cytopathic effect. Approaches to clear reservoirs by reactivating latent cells have provided evidence that latency can be reversed in vivo, however reversal of latency alone has not been sufficient to reduce latent reservoirs. Efforts are now in place to couple latency reactivation with strategies to eradicate the infected cells ? such as by design and activation of more efficacious anti-HIV cytotoxic T lymphocytes (CTLs). Another key player is Nef, an accessory protein encoded by HIV, which is a primary focus of our proposed research. Because Nef inhibits the activity of anti- HIV CTLs, a potent inhibitor of this protein would help achieve HIV eradication. One of the main functions of Nef is the down-modulation of major histocompatibility complex class I encoded proteins (MHC-I), masking infection from the host immune system and allowing HIV infected cells to persist. Combination therapy with latency antagonists plus Nef inhibitors could act synergistically to clear HIV reservoirs. To date, no Nef inhibitor has achieved potent restoration of MHC-I in the presence of Nef. We developed a high-throughput assay to identify inhibitors of Nef-mediated MHC-I downregulation, and a screen of natural product extracts (NPEs) yielded 10 hits with Nef inhibitory activity. We identified a number of related compounds, as the active component in several of these extracts. The pure natural products potently restore surface expression of MHC- I in the presence of Nef without inhibiting its other activities. We tested a number of structurally related compounds within this natural product family and identified two that possess pM to nM potencies in human primary cells. Based on this strong preliminary data, we believe that further enhancing the Nef inhibitory activity of these molecules through analog development will yield a safe anti-Nef drug. Therefore, we plan to (A) optimize these inhibitors by further separating and characterizing the anti-Nef effect from off-target activities to identify a lead drug candidate for development and (B) determine the mechanism by which the inhibitor disrupts Nef-mediated MHC-I downmodulation so that optimization can be conducted more intelligently. These goals will be achieved through the following specific aims: (1) Conduct lead compound structural optimization to improve pharmaceutical properties. (2) Perform a detailed functional analysis of all promising analogs to identify ideal lead compounds and (3) Determine the mechanism by which the natural product-derived inhibitor disrupts Nef-mediated MHC-I downmodulation including target identification and biochemical studies. From this work, we expect to generate a new class of compounds that are potent Nef inhibitors with high pharmaceutical potential. The addition of Nef inhibitory compounds to current cART cocktails is expected to enhance immune clearance of viral reservoirs, leading to the long-elusive HIV cure.

Antiretroviral medications suppress viral replication but do not eradicate cellular sources of integrated proviral genomes that are a major barrier to a cure. CD4+ hematopoietic stem and progenitor cells (HSPCs) have the capacity for life-long survival, self-renewal and the generation of daughter cells. They are also susceptible to HIV infection in vitro and in vivo. Combination antiretroviral therapy effectively suppresses viremia in HIV- infected people. However, residual plasma virus (RPV) can be detected with very sensitive assays. Recently published studies demonstrate that clusters of identical proviruses from HSPCs and their likely progeny often match RPV and are sometimes infectious. A higher proportion of these sequences matched RPV than proviral genomes from peripheral blood mononuclear cells that lacked evidence of clonal expansion. Furthermore, we provide examples of proviral genomes from progenitors that were latent in peripheral blood T cells while simultaneously contributing to RPV. The cellular source of RPV in these cases is not known but is unlikely to be peripheral blood T cells, which required latency reactivation for gene expression. We have developed a model based on these data. In this model, we propose that heterogeneous differentiated progeny of infected progenitors can support either active or latent infection depending on progeny epigenetic and transcriptional programs. The overall objective of this application is to test this hypothesis by comprehensively characterizing intact HIV in peripheral blood and tissue reservoirs. A secondary objective is to determine whether infected HSPCs are required for clonal provirus and RPV and to identify any alternative proliferative sources of non- HSPC generated clonal genomes. To accomplish this, we will: 1. Analyze intact near full length viral genomes to identify sources of clonally amplified proviral genomes in peripheral blood and to determine their relationship to proliferative sources such as HSPCs; 2. use viral outgrowth assays to confirm relationships amongst sources of infectious virus; and 3. determine the active and latently infected tissue sources of infectious virus and their relationships to proliferative sources such as HSPCs. Results from these aims will comprehensively identify sources of functional virus and RPV across multiple disparate tissue sites. They will determine the extent to which multipotent and/or restricted progenitors or other proliferative sources serve as the source for clonally expanded HIV proviral genomes present in the peripheral blood and tissue sites. These studies will provide important new information that has the potential to change the way we think about the source of functionally relevant HIV reservoirs.

ABSTRACT Human genome engineering has widely anticipated promise as a healthcare strategy, but current technologies are unlikely to provide the safe, efficient, and broadly useful implementation of transgene introduction essential to complete the next big leap forward for gene therapy. CRISPR-based approaches for transgene integration have major impediments, including the need for donor DNA delivery, the propensity of that DNA to undergo non-specific integration, and the low efficiency of repair by homologous recombination relative to sloppy rejoining of the broken DNA ends. Also severely limiting is the fact that slowly proliferating cells are rarely in a cell cycle phase favorable for homologous recombination, and just the presence of a DNA break can be toxic. The alternative approach of adeno-associated virus introduction of a transgene also has limitations, among others including the small transgene size permitted by the virus capsid and the challenges of engineering virus uptake into different cell types. It remains an unmet need to have a non-mutagenic, non-toxic approach for gene introduction to the human genome. Therapy for many loss-of-function pathologies hinges on this missing technology. Also, only transgene introduction offers the opportunity for non-native control of protein expression, isoform selectivity, and myriad other clinically useful outcomes. Starkly missing from current efforts to develop transgene introduction techniques is an approach exploiting the gene insertion strategy widespread endogenously across eukaryotes: cDNA synthesis. The ancestral, evolutionarily persistent type of eukaryotic LINE/non-LTR retroelement integrates by nick-primed reverse transcription that is rigorous both it its sequence specificity of target site selection and in its specificity for use of an RNA transcript with the retroelement 3? UTR as template. The biochemical activities required for target site selection, introduction of precisely positioned nick, and cDNA synthesis are carried out by a single protein. Any RNA sequence flanked by 5? and 3? regions of the retroelement genome should assemble with a favorably modified retroelement protein, and this RNP would then seek its native insertion site. Because several LINE/non-LTR retroelement families target highly conserved, repetitive sequences invariant across multicellular eukaryotes, there is no need to re-engineer DNA site-specificity of these retroelement proteins, although that may become of interest to undertake. The simple architecture of the non-LTR retroelements begs to be exploited for developing an approach to human genome supplementation with genes of therapeutic impact. The novelty of this approach demands continuous innovation and obliges high risk of failure to reach the goal of delivering an engineered RNP capable of transgene introduction into human cells. Success of this strategy would usher in a new modality of therapeutic treatment for loss-of-function diseases.

Abstract The goal of proposed experiments in this application is to determine how HIV infection disrupts intestinal barrier function. It is now appreciated that microbial translocation across an impaired epithelial barrier leads to circulating LPS, persistent immune activation and chronic inflammation in people living with HIV (PLWH). These HIV associated effects are important contributors to premature development of neurocognitive disorders, cardiovascular disease, metabolic syndrome and bone abnormalities even in PLWH on optimal combination antiretroviral therapy (cART). Untreated infection is characterized by the production of proinflammatory cytokines such as interleukin (IL)-1?, IL-6 and tumor necrosis factor (TNF?). Following therapy, cytokine levels decline but chronic inflammation continues. Prevention of inflammation-induced comorbidities requires the development of more specific therapeutics targeting the underlying cause. However, a gap exists in our understanding of the underlying molecular pathways involved. This proposal will capitalize on an established collaboration between investigators with expertise in HIV biology and intestinal barrier function/pathobiology. We have generated strong preliminary data that provides a framework for understanding the underlying link between disrupted intestinal epithelial barrier function and HIV infection. While the overall chronic inflammatory manifestations of HIV infection are likely to be multi-factorial, our exciting results support an overarching hypothesis that lamina propria HIV-1 infected primary human CD4+T lymphocytes that closely interact with intestinal epithelial initiate a process leading to enhanced production of pro-inflammatory cytokines that negatively impact epithelial homeostasis resulting in a leaky intestinal barrier. Given these important new insights, funding is requested to support a major collaborative effort between established investigators in the areas of HIV biology and intestinal inflammation/barrier disruption to determine the mechanism(s) through which primary human intestinal epithelial cells (IECs) and HIV-infected primary T cells synergize to cause intestinal pathobiology. Specifically, we will determine the HIV-dependent mechanisms that alter T-cell function and disrupt the intestinal barrier. In addition, we will identify the pathways altered in IECs exposed to HIV infected T-cells that lead to barrier dysfunction. Findings generated from these studies will allow a better understanding of the mechanisms underlying HIV related enteropathy that is known to be a major source of morbidity and mortality in HIV-infected individuals and will lead to development of new strategies to improve the health of HIV infected people. 1