Macrophages infected with a heavy burden of M.tb Erdman undergo a cell death that initially resembles apoptosis but quickly transitions to necrosis. Unlike the previously reported TNF dependent apoptosis induced by avirulent Mycobacterium [1], this form of macrophage cell death is not microbicidal [2]. Microbicidal effects are observed however, when the heavily infected macrophage encounters an uninfected naïve macrophage. My studies describe in part, the crosstalk between the uninfected and infected macrophage that results in the killing of the intracellular M.tb Cell contact between the two cell populations is not necessary for this killing of bacilli to occur and the soluble “signal” of communication between the two cell populations is transferrable, without naïve macrophages present, to newly infected cells also resulting in the reduced viability of the bacilli. We have found that when the IL-1 receptor is absent in the naïve macrophage population that the co-culture antimycobacterial effect is abrogated, suggesting that IL-1 released by the infected dying macrophage is critical for naïve macrophages to respond in a way that results in the decrease in mycobacterial viability. The signaling between the two cell population ultimately converges on activation of iNOS in the infected cell however ROS appears not to be involved.

In the Drosophila brain, memories are processed and stored in two mirrorsymmetrical structures composed of approximately 5,000 neurons called Mushroom Bodies (MB). Depending on their axonal extensions, neurons in the MB can be further classified into three different subgroups: αβ, α’β’ and γ. In addition to the morphological differences between these groups of neurons, there is evidence of functional differences too. For example, it has been previously shown that while neurotransmission from α’β’ neurons is required for consolidation of olfactory memory, output from αβ neurons is required for its later retrieval. To gain insight into the functional properties of these discrete neurons we analyzed whether they were different at the level of gene expression. We generated an intersectional genetic approach to exclusively label each population of neurons and permit their purification. Comparing expression profiles, revealed a large number of potentially interesting molecular differences between the populations. We focused on the finding that the MB αβ neurons, which are the presumed storage site for transcription-dependent long-term memory, express high levels of mRNA for transposable elements and histones suggesting that these neurons likely possess unique genomic characteristics. For decades, transposable elements (TE) were considered to be merely “selfish” DNA elements inserted at random in the genome and that they their sole function was to self-replicate. However, new studies have started to arise that indicate TE contribute more than just “junk” DNA to the genome. Although it is widely believed that mobilization of TE destabilize the genome by insertional mutagenesis, deletions and rearrangements of genes, some rearrangements might be advantageous for the organism. TE mobilization has recently been documented to occur in some somatic cells, including in neuronal precursor cells (NPCs). Moreover, mobilization in NPCs seems to favor insertions within neuronal expressed genes and in one case the insertion elevated the expression. During the last decade, the discovery of the small RNA pathways that suppress the expression and mobilization of TE throughout the animal have helped to uncover new functions that TE play. In this work, we demonstrate that proteins of the PIWI-associated RNA pathway that control TE expression in the germline are also required to suppress TE expression in the adult fly brain. Moreover, we find that they are differentially expressed in subsets of MB neurons, being under represented in the αβ neurons. This finding suggests that the αβ neurons tolerate TE mobilization. Lastly, we demonstrate by sequencing αβ neuron DNA that TE are mobile and we identify >200 de novo insertions into neurally expressed genes. We conclude that this TE generated mosaicism, likely contributes a new level of neuronal diversity making, in theory, each αβ neuron genetically different. In principle the stochastic nature of this process could also render every fly an individual.

Long-term antiviral antibody responses provide protection from re-infection and recurrence of persistent viruses. Using a polyomavirus (PyV) mouse model, our lab has shown that MyD88-deficient mice generate low levels of virus-specific IgG after the acute phase of infection and that these IgG responses have a skewed isotype distribution with low levels of IgG2a/c. Moreover MyD88-deficient mice have reduced numbers of long-lived plasma cells in the bone marrow. These studies suggest an important role of MyD88-mediated signaling in long-term antiviral responses. Our lab has shown that T cell-deficient mice can also maintain long-term virus-specific IgG responses following PyV infection. The goal of this thesis is to evaluate the role of innate signaling pathways in maintaining serological memory to persistent virus infection and to elaborate on how long-term antiviral responses can be maintained in an immunocompetent or partially immune compromised, T cell-deficient host. Regarding T cell-dependent B cell responses, I set out to investigate the upstream and downstream components of the MyD88-mediated pathways required for normal antibody isotype and long-term humoral responses. IgG2a is a predominant immunoglobulin isotype in most virus infections. Wild type mice, in response to PyV infection, primarily induce antiviral IgG2a with some IgG1. MyD88-deficient mice in response to PyV infection display attenuated levels of virus-specific IgG2a, but normal levels of IgG1. Using Unc93B1 mutant mice (3d mice), which are defective in TLRs 3, 7 and 9 signaling, I show that 3d mice also generated low levels of virus-specific IgG2a following PyV infection. Studies in individual TLR3-/-, TLR7-/- or TLR9-/- mice displayed PyV-specific IgG2a responses similar to wild type responses. TLR7 and TLR9 double deficient mice generated similar skewed antibody isotype responses, where virus-specific IgG2a was reduced compared to wild type mice. This shows that TLR7 and TLR9-MyD88 mediated pathways are important in regulating IgG2a responses during a PyV infection. To investigate what components downstream of MyD88 are involved in mediating IgG2a responses, I worked with IRF5-deficient mice. IRF5 is a transcription factor that is activated upon stimulation of TLR7 or TLR9-MyD88-mediated pathways. Moreover, IRF5-deficient mice cannot generate autoantibodies specifically of the IgG2a isotype in a mouse lupus model, suggesting that IRF5 plays an important function in mediating class switching to IgG2a. In vitro studies where IRF5-/- B cells were stimulated with TLR7 or TLR9 ligands also generated low levels of γ2a germ-line transcripts, suggesting a B cell-intrinsic role for IRF5 in regulating γ2a germ-line transcription. PyV infection of IRF5-deficient mice resulted in similar skewed isotypes as observed in MyD88-deficient and 3d mice. To investigate a B cell-intrinsic role for IRF5 in regulating IgG2a responses in vivo upon PyV infection, I transferred IRF5-/- B cells and WT T cells into RAG KO mice prior to infection and compared the responses of these mice with mice reconstituted with wild type B6 B and T cells. Diminished numbers of IgG2a+ B cells and reduced levels of virus-specific IgG in mice reconstituted with IRF5-/- B cells were seen compared to mice reconstituted with wild type B cells. Regarding the defect in long-term IgG production in MyD88-/- mice upon PyV infection, I conducted studies in IRF5-/-, 3d, single TLR3-/-, TLR7-/-, TLR9-/- and TLR7/9 double deficient mice. These studies reveal an important and redundant role for TLR7- and TLR9-MyD88 signaling in maintaining long-term anti-PyV IgG responses. To determine how MyD88 signaling affects the generation of long-lived plasma cells and memory B cells, I investigated germinal center (GC) responses in MyD88-deficient mice. A defect in GC B cell numbers is observed in MyD88-deficient mice after the acute phase of infection. The GC reaction is essential for the generation and maintenance of long-lived plasma cells and memory B cells. T follicular helper (TFH) cells are absolutely required to generate normal GC. l found reduced numbers of TFH cells in MyD88-deficient mice. Lower numbers of T FH cells suggests that poor T cell help may contribute to the diminished number of GC B cells. However, interaction with B cells is required for the formation of fully differentiated TFH cells. Along with B cell function, MyD88 signaling can affect T cell and dendritic cell function as well. Thus, it is not clear at this point whether the requirement for intact MyD88 signaling for the formation and maintenance of long-term B cell populations is completely B cell-intrinsic. Some viruses can induce T cell-independent B cell responses, perhaps due to their complex arrays of repetitive antigenic epitopes on virions, coupled with the induction of innate cytokines. Nevertheless, T cell help is usually necessary for generating long-term antibody responses in the form of long-lived plasma cells and memory B cells. In contrast, our lab has found that T cell-deficient mice infected with PyV develop long-lasting, protective antiviral IgG responses. I questioned whether these mice could generate TI B cell memory cells or long-lived plasma cells. I show that long-lasting anti-PyV antibody in T cell-deficient mice was not due to the presence of long-lived plasma cells or memory B cell responses. TCRβδ deficient mice, which lack both CD4 and CD8 T cells, had ~10 a times higher virus load persisting in various organs. Therefore, I hypothesized that the high level of persistent PyV antigen, in completely T cell-deficient mice, may activate naïve B cell populations continuously, thereby maintaining the long-lasting IgG responses. Prior to PyV infection, T cell-deficient mice received wild type CD8 T cells, which reduced PyV loads, and this was associated with decreased levels of antiviral serum IgG over time. As in TCRβδ deficient mice, high PyV loads were detected in the bone marrow, which is the site for B cell lymphopoiesis, I questioned how B cells develop in the presence of PyV antigen and still stay responsive to PyV, generating long-term antiviral IgG responses in the periphery. Studies have shown that self-antigens that trigger both B cell receptor signaling and TLR-MyD88 signaling pathways in the bone marrow lead to the breaking of B cell tolerance and production of autoantibody in the periphery. Thus, we hypothesized that high PyV levels in the bone marrow signal through both B cell-receptors and TLRs, allowing continuous antiviral antibody production by B cells. Using mice that are deficient in T cells and MyD88 signaling, I found that PyV-specific TI IgG levels gradually decreased, supporting this hypothesis. Thus, high PyV loads and innate signaling together can break B cell tolerance. During a persistent virus infection this can result in sustaining long-term protective T cell-independent IgG responses.

Small RNA silencing pathways regulate development, viral defense, and genomic integrity in all kingdoms of life. An Argonaute (Ago) protein, guided by a tightly bound, small RNA or DNA, lies at the core of these pathways. Argonaute uses its small RNA or DNA to find its target sequences, which it either cleaves or stably binds, acting as a binding scaffold for other proteins. We used Co-localization Single-Molecule Spectroscopy (CoSMoS) to analyze target binding and cleavage by Ago and its guide. We find that both eukaryotic and prokaryotic Argonaute proteins re-shape the fundamental properties of RNA:RNA, RNA:DNA, and DNA:DNA hybridization: a small RNA or DNA bound to Argonaute as a guide no longer follows the well-established rules by which oligonucleotides find, bind, and dissociate from complementary nucleic acid sequences. Counter to the rules of nucleic acid hybridization alone, we find that mouse AGO2 and its guide bind to microRNA targets 17,000 times tighter than the guide without Argonaute. Moreover, AGO2 can distinguish between microRNA-like targets that make seven base pairs with the guide and the products of cleavage, which bind via nine base pairs: AGO2 leaves the cleavage products faster, even though they pair more extensively. This thesis presents a detailed kinetic interrogation of microRNA and RNA interference pathways. We discovered sub-domains within the previously defined functional domains created by Argonaute and its bound DNA or RNA guide. These sub-domains have features that no longer conform to the well-established properties of unbound oligonucleotides. It is by re-writing the rules for nucleic acid hybridization that Argonautes allow oligonucleotides to serve as specificity determinants with thermodynamic and kinetic properties more typical of RNA-binding proteins than that of RNA or DNA. Taken altogether, these studies further our understanding about the biology of small RNA silencing pathways and may serve to guide future work related to all RNA-guided endonucleases.

DNA damage induces mitotic exit delays through a process that requires the spindle assembly checkpoint (SAC), which blocks the metaphase to anaphase transition in the presence of unaligned chromosomes. Using time-lapse confocal microscopy in syncytial Drosophila embryos, we show that DNA damage leads to arrest during prometaphase and anaphase. In addition, functional GFP fusions to the SAC components MAD2 and Mps1, and the SAC target Cdc20 relocalize to kinetochore through anaphase arrest, and a null mad2mutation blocks damage induced prometaphase and anaphase arrest. We also show that the DNA damage signaling kinase Chk2 is required for damage induced metaphase and anaphase arrest, and that a functional GFP-Chk2 fusion localizes to kinetochores and centrosomes through mitosis. In addition, in the absence of Chk2, we find that DNA damage sufficient to fragment centromere DNA does not delay mitotic exit. We conclude that DNA damage signaling through Chk2 triggers Mad2-dependent delays in mitotic progression, both before or after the metaphase-anaphase transition.

Newcastle disease virus (NDV), an avian paramyxovirus, enters the host cell by fusion of viral and host cell membranes. The fusion of two membranes is mediated by the viral fusion (F) protein. The F protein, like other class I fusion proteins, is thought to undergo major conformational changes during the fusion process. The exact mechanism that leads to major refolding of F protein is not clear. Recently, it has been proposed that disulfide bond reduction in the fusion protein of some viruses may be involved in the conformational changes in fusion proteins. In some viruses, the reduction of disulfide bonds in the fusion protein is mediated by host cell disulfide isomerases belonging to the protein disulfide isomerase (PDI) family. In this study, the role of disulfide bond isomerization in the entry of NDV was analyzed. Using inhibitors of thiol-disulfide isomerases, we found that blocking the reduction of disulfide bonds in the fusion protein inhibited cell-cell fusion as well as virus entry into the host cell. Also, over-expression of isomerases belonging to the PDI family significantly enhanced cell-cell fusion. Taken together, these results suggest that free thiols play an important role in fusion mediated by NDV glycoproteins. Using a thiol specific, membrane impermeable biotin, MPB, we found that free thiols are produced in cell surface-expressed NDV F protein. The production of free thiols was inhibited by inhibitors of thiol-disulfide isomerases. Over-expression of isomerases belonging to the PDI family enhanced detection of free thiols in F protein. In F protein, present in virions or in virus-like particles, free thiols were detected only after the particles were attached to target cells. Taken together, these results suggest that free thiols are produced in F protein and the production of free thiols is mediated by host cell thiol-disulfide isomerases. Using conformation sensitive antibodies, we also studied the conformation of cell surface-expressed F protein in the presence ofthiol-disulfide isomerase inhibitors or in cells over-expressing thiol-disulfide isomerases. In the presence of thiol-disulfide isomerase inhibitors, the cell surface-expressed F protein was in a prefusion conformation while in cells over-expressing thiol-disulfide isomerases the F protein was in a post-fusion conformation. We also correlated the production of free thiols to the conformational changes in F protein. Using temperature-arrested intermediates or F protein with mutations in heptad repeat domains, which are defective in attaining intermediate conformations, we found that free thiols are produced before any of the proposed conformational changes in F protein. Also, the production of free thiols in F protein was found to be independent of its activation by hemagglutinin-neuraminidase (HN) protein. These results suggest that free thiols are probably required for the activation of F protein during membrane fusion.

Primate lentiviruses encode four “accessory proteins” including Vif, Vpu, Nef, and Vpr/ Vpx. Vif and Vpu counteract the antiviral effects of cellular restrictions to early and late steps in the viral replication cycle. The functions of Vpx/ Vpr are not well understood. This study presents evidence that the Vpx proteins of HIV-2/ SIVSMpromote HIV-1 infection by antagonizing an antiviral restriction in myeloid cells. Fusion of macrophages in which Vpx was essential for virus infection, with COS cells in which Vpx was dispensable for virus infection, generated heterokaryons that supported infection by wild-type SIV but not Vpx-deleted SIV. The restriction potently antagonized infection of macrophages by HIV-1, and expression of Vpx in macrophages in transovercame the restriction to HIV-1 and SIV infection. Similarly, the cellular restriction is the obstacle to transduction of macrophages by MLV. Neutralization of the restriction by Vpx rendered macrophages permissive to MLV infection. Vpx was ubiquitylated and both ubiquitylation and the proteasome regulated the activity of Vpx. The ability of Vpx to counteract the restriction to HIV-1 and SIV infection was dependent upon the HIV-1 Vpr interacting protein, damaged DNA binding protein 1 (DDB1), and DDB1 partially substituted for Vpx when fused to Vpr. This study further demonstrates that this restriction prevents transduction of quiescent monocytes by HIV-1. Although terminally differentiated macrophages are partially permissive to HIV-1, quiescent monocytes, which are macrophage precursors, are highly refractory to lentiviral infection. Monocyte-HeLa heterokaryons were resistant to HIV-1 infection, while heterokaryons formed between monocytes and HeLa cells expressing Vpx were permissive to HIV-1 infection, suggesting the resistance of quiescent monocytes to HIV-1 transduction is governed by a restriction factor. Encapsidation of Vpx within HIV-1 virions conferred the ability to infect quiescent monocytes. Introduction of Vpx into monocytes by pre-infection also rendered quiescent monocytes permissive to HIV-1 infection. Infection of monocytes by HIV-1 either with or without Vpx did not have an effect on temporal expression of CD71. In addition, Vpx increased permissivity of CD71– and CD71+cells to HIV-1 infection with no apparent bias. These results confirm that Vpx directly renders undifferentiated monocytes permissive to HIV-1 transduction without inducing their differentiation. The introduction of Vpx did not significantly alter APOBEC3G complex distribution, suggesting a restriction other than APOBEC3G was responsible for the resistance of monocytes to HIV-1. Collectively our results indicate that macrophages and monocytes harbor a potent antiviral restriction that is counteracted by the Vpx protein. The relative ability of primate lentiviruses and gammaretroviruses to transduce non-dividing myeloid-cells is dependent upon their ability to neutralize this restriction.

The central dogma of biology asserts that DNA is transcribed into RNA and RNA is translated into protein. However, this overtly simplistic assertion fails to portray the highly orchestrated and regulated mechanisms of transcription and translation. During the process of transcription, RNA provides the template for translation and protein synthesis as well as the structural and sequence specificity of many RNA and protein-based machines. While only 1-5% of the genome will escape the nucleus to be translated as mRNAs, complex, parallel, highly-conserved mechanisms have evolved to regulate specific mRNAs. Trans-acting factors bind cis-elements in both the 5" and 3" untranslated regions of mRNA to regulate their stability, localization, and translation. While a few salient examples have been elucidated over the last few decades, mRNA translation can be reversibly regulated by the shortening and lengthening of the 3" polyadenylate tail of mRNA. CPEB, an important factor that nucleates a complex of proteins to regulate the polyadenylate tail of mRNA, exemplifies a major paradigm of translational control during oocyte maturation and early development. CPEB function is also conserved in neurons and somatic foreskin fibroblasts where it plays an important role in protein synthesis dependent synaptic plasticity and senescence respectively. Focusing on the function of CPEB and its role in mRNA polyadenylation during human cellular senescence, the following dissertation documents the important finding that CPEB is required for the normal polyadenylation of p53 mRNA necessary for its normal translation and onset of senescence. Cells that lack CPEB have abnormal levels of mitochondria and ROS production, which are demonstrated to arise from the direct result of hypomorphic p53 levels. Finally, in an attempt to recapitulate the model of CPEB complex polyadenylation in human somatic cells, I unexpectedly find that Gld-2, a poly(A) polymerase required for CPEB-mediated polyadenylation in Xenopus laevis oocytes, is not required for p53 polyadenylation, but instead regulates the stability of a microRNA that in turn regulates CPEB mRNA translation. Furthermore, I demonstrate that CPEB requires Gld-4 for the normal polyadenylation and translation of p53 mRNA.

RNA polymerase II-mediated transcription of HIV-1 genes depends on positive transcription elongation factor b (P-TEFb), the complex of cyclin T1 and CDK9. Recent evidence suggests that regulation of transcription by P-TEFb involves chromatin binding and modifying factors. To determine how P-TEFb may connect chromatin remodeling to transcription, we investigated the relationship between P-TEFb and histone H1. We show that P-TEFb interacts with H1 and that H1 phosphorylation in cell culture correlates with P-TEFb activity. Importantly, P-TEFb also directs H1 phosphorylation during Tat transactivation and wild type HIV-1 infection. Our results also show that P-TEFb phosphorylates histone H1.1 at a specific C-terminal site. Expression of a mutant H1.1 that cannot be phosphorylated by P-TEFb disrupts Tat transactivation as well as transcription of the c-fos and hsp70 genes in HeLa cells. P-TEFb phosphorylation of H1 also plays a role in the expression of muscle differentiation marker genes in the skeletal myoblast cell line C2C12. Additionally, ChIP experiments demonstrate that H1 dissociates from the HIV-1 LTR in MAGI cells, stress-activated genes in HeLa cells, and muscle differentiation marker genes in C2C12 cells under active P-TEFb conditions. Our results overall suggest a new role for P-TEFb in both cellular and HIV-1 transcription through chromatin.

Erythropoietin (Epo) modulates red blood cell production (erythropoiesis) by binding to its receptor and activating STAT5, a Signal Transducer and Activator of Transcription (STAT) protein implicated in both basal and stress erythropoiesis. Epo concentration in serum changes over three orders of magnitude, as it regulates basal erythropoiesis and its acceleration during hypoxic stress. However, it is not known how STAT5 translates the changes in Epo concentration into the required erythropoietic rates. We addressed this question by studying STAT5 phosphorylation, at the single cell level, in developing erythroblasts. We divided erythroid progenitors in tissue into several flow-cytometric subsets and found that each of them exhibited distinct modes of Stat5 activation, based on their developmental stage. STAT5 activation is bistable in mature erythroblasts, resulting in a binary (or digital), low-intensity STAT5 phosphorylation signal (p-Stat5). In early erythroblasts, and in response to stress levels of Epo, the low intensity bistable p-Stat5 signal is superseded by a high-intensity graded, or analog, signal. The gradual shift from high-intensity graded signaling in early erythroblasts to low intensity binary signaling in mature erythroblasts is due to a decline in STAT5 expression with maturation. We were able to convert mature, digital transducing erythroblasts into analog transducers simply by expressing high levels of exogenous STAT5. We found that EpoR-HM mice, expressing a mutant EpoR that lacks STAT5 docking sites, generate the binary, but not the analog, STAT5 signal. Unlike Stat5-null mice, which die perinatally, the EpoR-HM mice are viable but deficient in their response to stress, demonstrating that while binary STAT5 signaling is sufficient to support basal erythropoiesis, analog signaling is required for the stress response. Bistable systems contain a positive loop, which is important for flipping the switch between the two stable ‘on’ or ‘off’ states. We show that bistable activation is the result of an autocatalytic loop in which active STAT5 promotes further STAT5 activation. The isolated STAT5 N-terminal domain, which is not required for STAT5 phosphorylation, enhanced autocatalysis, converting a high intensity graded signal into a high intensity binary response. The N-terminal domain is known to participate in a radical conformational reorientation of STAT5 dimers inherent in STAT5 activation. We propose that the N-terminal domains of active STAT5 dimers facilitate the conformational reorientation of inactive dimers, in a prion-like autocatalytic interaction that underlies bistability and binary signaling. Together, bistable STAT5 activation, combined with a graded response allow erythropoietic rate to faithfully reflect a wide Epo concentration range, while preventing aberrant signaling.