Identifying structural risk factors for overdose following incarceration: a concept mapping study.

BACKGROUND: Currently, there are more than two million people in prisons or jails, with nearly two-thirds meeting the criteria for a substance use disorder. Following these patterns, overdose is the leading cause of death following release from prison and the third leading cause of death during periods of incarceration in jails. Traditional quantitative methods analyzing the factors associated with overdose following incarceration may fail to capture structural and environmental factors present in specific communities. People with lived experiences in the criminal legal system and with substance use disorder hold unique perspectives and must be involved in the research process. OBJECTIVE: To identify perceived factors that impact overdose following release from incarceration among people with direct criminal legal involvement and experience with substance use. METHODS: Within a community-engaged approach to research, we used concept mapping to center the perspectives of people with personal experience with the carceral system. The following prompt guided our study: "What do you think are some of the main things that make people who have been in jail or prison more and less likely to overdose?" Individuals participated in three rounds of focus groups, which included brainstorming, sorting and rating, and community interpretation. We used the Concept Systems Inc. platform groupwisdom for our analyses and constructed cluster maps. RESULTS: Eight individuals (ages 33 to 53) from four states participated. The brainstorming process resulted in 83 unique factors that impact overdose. The concept mapping process resulted in five clusters: (1) Community-Based Prevention, (2) Drug Use and Incarceration, (3) Resources for Treatment for Substance Use, (4) Carceral Factors, and (5) Stigma and Structural Barriers. CONCLUSIONS: Our study provides critical insight into community-identified factors associated with overdose following incarceration. These factors should be accounted for during resource planning and decision-making.

Using NMR to Monitor TET-Dependent Methylcytosine Dioxygenase Activity and Regulation.

The active removal of DNA methylation marks is governed by the ten-eleven translocation (TET) family of enzymes (TET1-3), which iteratively oxidize 5-methycytosine (5mC) into 5-hydroxymethycytosine (5hmC), and then 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC). TET proteins are frequently mutated in myeloid malignancies or inactivated in solid tumors. These methylcytosine dioxygenases are α-ketoglutarate (αKG)-dependent and are, therefore, sensitive to metabolic homeostasis. For example, TET2 is activated by vitamin C (VC) and inhibited by specific oncometabolites. However, understanding the regulation of the TET2 enzyme by different metabolites and its activity remains challenging because of limitations in the methods used to simultaneously monitor TET2 substrates, products, and cofactors during catalysis. Here, we measure TET2-dependent activity in real time using NMR. Additionally, we demonstrate that in vitro activity of TET2 is highly dependent on the presence of VC in our system and is potently inhibited by an intermediate metabolite of the TCA cycle, oxaloacetate (OAA). Despite these opposing effects on TET2 activity, the binding sites of VC and OAA on TET2 are shared with αKG. Overall, our work suggests that NMR can be effectively used to monitor TET2 catalysis and illustrates how TET activity is regulated by metabolic and cellular conditions at each oxidation step.

MRE11 liberates cGAS from nucleosome sequestration during tumorigenesis.

Oncogene-induced replication stress generates endogenous DNA damage that activates cGAS-STING-mediated signalling and tumour suppression1-3. However, the precise mechanism of cGAS activation by endogenous DNA damage remains enigmatic, particularly given that high-affinity histone acidic patch (AP) binding constitutively inhibits cGAS by sterically hindering its activation by double-stranded DNA (dsDNA)4-10. Here we report that the DNA double-strand break sensor MRE11 suppresses mammary tumorigenesis through a pivotal role in regulating cGAS activation. We demonstrate that binding of the MRE11-RAD50-NBN complex to nucleosome fragments is necessary to displace cGAS from acidic-patch-mediated sequestration, which enables its mobilization and activation by dsDNA. MRE11 is therefore essential for cGAS activation in response to oncogenic stress, cytosolic dsDNA and ionizing radiation. Furthermore, MRE11-dependent cGAS activation promotes ZBP1-RIPK3-MLKL-mediated necroptosis, which is essential to suppress oncogenic proliferation and breast tumorigenesis. Notably, downregulation of ZBP1 in human triple-negative breast cancer is associated with increased genome instability, immune suppression and poor patient prognosis. These findings establish MRE11 as a crucial mediator that links DNA damage and cGAS activation, resulting in tumour suppression through ZBP1-dependent necroptosis.

Histone H3 proline 16 hydroxylation regulates mammalian gene expression.

Histone post-translational modifications (PTMs) are important for regulating various DNA-templated processes. Here, we report the existence of a histone PTM in mammalian cells, namely histone H3 with hydroxylation of proline at residue 16 (H3P16oh), which is catalyzed by the proline hydroxylase EGLN2. We show that H3P16oh enhances direct binding of KDM5A to its substrate, histone H3 with trimethylation at the fourth lysine residue (H3K4me3), resulting in enhanced chromatin recruitment of KDM5A and a corresponding decrease of H3K4me3 at target genes. Genome- and transcriptome-wide analyses show that the EGLN2-KDM5A axis regulates target gene expression in mammalian cells. Specifically, our data demonstrate repression of the WNT pathway negative regulator DKK1 through the EGLN2-H3P16oh-KDM5A pathway to promote WNT/ß-catenin signaling in triple-negative breast cancer (TNBC). This study characterizes a regulatory mark in the histone code and reveals a role for H3P16oh in regulating mammalian gene expression.

Functional conservation and divergence of the helix-turn-helix motif of E2 ubiquitin-conjugating enzymes.

Polyubiquitination by E2 and E3 enzymes is crucial to cell cycle control, epigenetic regulation, and development. The hallmark of the E2 family is the ubiquitin (Ub)-conjugating (UBC) domain that forms a dynamic thioester conjugate with ubiquitin (E2~Ub). Numerous studies have focused on E2 surfaces, such as the N-terminal and crossover helices, that directly interact with an E3 or the conjugated ubiquitin to stabilize the active, "closed" state of the E2~Ub. However, it remains unclear how other E2 surfaces regulate ubiquitin transfer. Here, we demonstrate the helix-turn-helix (HTH) motif of the UBC tunes the intrinsic polyubiquitination activity through distinct functions in different E2s. Interestingly, the E2HTH motif is repurposed in UBE2S and UBE2R2 to interact with the conjugated or acceptor ubiquitin, respectively, modulating ubiquitin transfer. Furthermore, we propose that Anaphase-Promoting Complex/Cyclosome binding to the UBE2SHTH reduces the conformational space of the flexible E2~Ub, demonstrating an atypical E3-dependent activation mechanism. Altogether, we postulate the E2HTH motif evolved to provide new functionalities that can be harnessed by E3s and permits additional regulation to facilitate specific E2-E3-mediated polyubiquitination.

Visualizing a protonated RNA state that modulates microRNA-21 maturation.

MicroRNAs are evolutionarily conserved small, noncoding RNAs that regulate diverse biological processes. Due to their essential regulatory roles, microRNA biogenesis is tightly regulated, where protein factors are often found to interact with specific primary and precursor microRNAs for regulation. Here, using NMR relaxation dispersion spectroscopy and mutagenesis, we reveal that the precursor of oncogenic microRNA-21 exists as a pH-dependent ensemble that spontaneously reshuffles the secondary structure of the entire apical stem-loop region, including the Dicer cleavage site. We show that the alternative excited conformation transiently sequesters the bulged adenine into a noncanonical protonated A+-G mismatch, conferring a substantial enhancement in Dicer processing over its ground conformational state. These results indicate that microRNA maturation efficiency may be encoded in the intrinsic dynamic ensemble of primary and precursor microRNAs, providing a potential means of regulating microRNA biogenesis in response to environmental and cellular stimuli.

Structural basis of nucleosome-dependent cGAS inhibition.

Cyclic guanosine monophosphate (GMP)-adenosine monophosphate (AMP) synthase (cGAS) recognizes cytosolic foreign or damaged DNA to activate the innate immune response to infection, inflammatory diseases, and cancer. By contrast, cGAS reactivity against self-DNA in the nucleus is suppressed by chromatin tethering. We report a 3.3-angstrom-resolution cryo-electron microscopy structure of cGAS in complex with the nucleosome core particle. The structure reveals that cGAS uses two conserved arginines to anchor to the nucleosome acidic patch. The nucleosome-binding interface exclusively occupies the strong double-stranded DNA (dsDNA)-binding surface on cGAS and sterically prevents cGAS from oligomerizing into the functionally active 2:2 cGAS-dsDNA state. These findings provide a structural basis for how cGAS maintains an inhibited state in the nucleus and further exemplify the role of the nucleosome in regulating diverse nuclear protein functions.

Streptavidin Promotes DNA Binding and Activation of cGAS to Enhance Innate Immunity.

cGAS/STING signaling plays an essential role in sensing cytosolic DNA. cGAS activity is regulated by posttranslational modifications and binding partners. cGAS interactome largely includes mammalian or viral proteins. Whether and how bacterial proteins bind cGAS to modulate innate immunity remain elusive. Here, we found streptavidin, a secreted bacterial protein, selectively bound cGAS to promote DNA-induced cGAS activation and interferon-ß production. Mechanistically, streptavidin enhanced DNA binding and cGAS phase separation, therefore facilitating cGAS activation. Using an HSV-1-infected mouse model, we found streptavidin nanoparticles facilitated HSV-1 clearance through improving innate immunity. Considering the clinical usage of streptavidin as an immune stimulant and drug delivery vehicle and its biotechnological usage for biotin-labeled protein purification and detection, our studies not only provide an example for a bacterial protein regulating cGAS activity but also suggest caution needs to be taken when using streptavidin in various applications given to its ability to induce innate immunity.

Targeting Cytokine Signaling and Lymphocyte Traffic via Small Molecules in Inflammatory Bowel Disease: JAK Inhibitors and S1PR Agonists.

The inflammatory Bowel diseases (IBDs) are a chronic, relapsing inflammatory diseases of the gastrointestinal tract with heterogeneous behavior and prognosis. The introduction of biological therapies including anti-TNF, anti-IL-12/23, and anti-integrins, has revolutionized the treatment of IBD, but these drugs are not universally effective. Due to the complex molecular structures of biologics, they are uniformly immunogenic. New discoveries concerning the underlying mechanisms involved in the pathogenesis of IBD have allowed for progress in the development of new treatment options. The advantage of small molecules (SMs) over biological therapies includes their lack of immunogenicity, short half-life, oral administration, and low manufacturing cost. Among these, the Janus Kinases (JAKs) inhibition has emerged as a novel strategy to modulate downstream cytokine signaling during immune-mediated diseases. These drugs target various cytokine signaling pathways that participate in the pathogenesis of IBD. Tofacitinib, a JAK inhibitor targeting predominantly JAK1 and JAK3, has been approved for the treatment of ulcerative colitis (UC), and there are other specific JAK inhibitors under development that may be effective in Crohn's. Similarly, the traffic of lymphocytes can now be targeted by another SM. Sphingosine-1-phosphate receptor (S1PR) agonism is a novel strategy that acts, in part, by interfering with lymphocyte recirculation, through blockade of lymphocyte egress from lymph nodes. S1PR agonists are being studied in IBD and other immune-mediated disorders. This review will focus on SM drugs approved and under development, including JAK inhibitors (tofacitinib, filgotinib, upadacitinib, peficitinib) and S1PR agonists (KRP-203, fingolimod, ozanimod, etrasimod, amiselimod), and their mechanism of action.

Cell Trafficking Interference in Inflammatory Bowel Disease: Therapeutic Interventions Based on Basic Pathogenesis Concepts.

After 20 years of successful targeting of pro-inflammatory cytokines for the treatment of IBD, an alternative therapeutic strategy has emerged, based on several decades of advances in understanding the pathogenesis of IBD. The targeting of molecules involved in leukocyte traffic has recently become a safe and effective alternative. With 2 currently approved drugs (ie, natalizumab, vedolizumab) and several others in phase 3 trials (eg, etrolizumab, ozanimod, anti-MAdCAM-1), the blockade of trafficking molecules has firmly emerged as a new therapeutic era for IBD. We discuss the targets that have been explored in clinical trials: chemokines and its receptors (eg, IP10, CCR9), integrins (eg, natalizumab, AJM300, vedolizumab, and etrolizumab), and its endothelial ligands (MAdCAM-1, ICAM-1). We also discuss a distinct strategy that interferes with lymphocyte recirculation by blocking lymphocyte egress from lymph nodes (small molecule sphingosine-phosphate receptor [S1PR] agonists: fingolimod, ozanimod, etrasimod, amiselimod). Strategies on the horizon include additional small molecules, allosteric inhibitors that specifically bind to the active integrin form and nanovectors that allow for the use of RNA interference in the quest to modulate pro-inflammatory leukocyte trafficking in IBD.

Implementation of Mass Cytometry as a Tool for Mechanism of Action Studies in Inflammatory Bowel Disease.

Background: Novel therapeutics for inflammatory bowel disease (IBD) are under development, yet mechanistic readouts at the tissue level are lacking. Techniques to assess intestinal immune composition could represent a valuable tool for mechanism of action (MOA) studies of novel drugs. Mass cytometry enables analysis of intestinal inflammatory cell infiltrate and corresponding molecular fingerprints with unprecedented resolution. Here, we aimed to optimize the methodology for isolation and cryopreservation of cells from intestinal tissue to allow for the potential implementation of mass cytometry in MOA studies. Methods: We investigated key technical issues, including minimal tissue requirements, cell isolation protocols, and cell storage, using intestinal biopsies and peripheral blood from healthy individuals. High-dimensional mass cytometry was employed for the analyses of biopsy-derived intestinal cellular subsets. Results: Dithiothreitol and mechanical dissociation decreased epithelial cell contamination and allowed for isolation of adequate cell numbers from 2 to 4 colonic or ileal biopsies (6 × 104±2 × 104) after a 20-minute collagenase digestion, allowing for reliable detection of most major immune cell subsets. Biopsies and antibody-labeled mononuclear cells could be cryopreserved for later processing and acquisition (viability > 70%; P < 0.05). Conclusions: Mass cytometry represents a unique tool for deep immunophenotyping intestinal cell composition. This technique has the potential to facilitate analysis of drug actions at the target tissue by identifying specific cellular subsets and their molecular signatures. Its widespread implementation may impact not only IBD research but also other gastrointestinal conditions where inflammatory cells play a role in pathogenesis.

CD301b/MGL2+ Mononuclear Phagocytes Orchestrate Autoimmune Cardiac Valve Inflammation and Fibrosis.

BACKGROUND: Valvular heart disease is common and affects the mitral valve (MV) most frequently. Despite the prevalence of MV disease (MVD), the cellular and molecular pathways that initiate and perpetuate it are not well understood. METHODS: K/B.g7 T-cell receptor transgenic mice spontaneously develop systemic autoantibody-associated autoimmunity, leading to fully penetrant fibroinflammatory MVD and arthritis. We used multiparameter flow cytometry, intracellular cytokine staining, and immunofluorescent staining to characterize the cells in inflamed K/B.g7 MVs. We used genetic approaches to study the contribution of mononuclear phagocytes (MNPs) to MVD in this model. Specifically, we generated K/B.g7 mice in which either CX3CR1 or CD301b/macrophage galactose N-acetylgalactosamine-specific lectin 2 (MGL2)-expressing MNPs were ablated. Using K/B.g7 mice expressing Cx3Cr1-Cre, we conditionally deleted critical inflammatory molecules from MNPs, including the Fc-receptor signal-transducing tyrosine kinase Syk and the cell adhesion molecule very late antigen-4. We performed complementary studies using monoclonal antibodies to block key inflammatory molecules. We generated bone marrow chimeric mice to define the origin of the inflammatory cells present in the MV and to determine which valve cells respond to the proinflammatory cytokine tumor necrosis factor (TNF). Finally, we examined specimens from patients with rheumatic heart disease to correlate our findings to human pathology. RESULTS: MNPs comprised the vast majority of MV-infiltrating cells; these MNPs expressed CX3CR1 and CD301b/MGL2. Analogous cells were present in human rheumatic heart disease valves. K/B.g7 mice lacking CX3CR1 or in which CD301b/MGL2-expressing MNPs were ablated were protected from MVD. The valve-infiltrating CD301b/MGL2+ MNPs expressed tissue-reparative molecules including arginase-1 and resistin-like molecule α. These MNPs also expressed the proinflammatory cytokines TNF and interleukin-6, and antibody blockade of these cytokines prevented MVD. Deleting Syk from CX3CR1-expressing MNPs reduced their TNF and interleukin-6 production and also prevented MVD. TNF acted through TNF receptor-1 expressed on valve-resident cells to increase the expression of vascular cell adhesion molecule-1. Conditionally deleting the vascular cell adhesion molecule-1 ligand very late antigen-4 from CX3CR1-expressing MNPs prevented MVD. CONCLUSIONS: CD301b/MGL2+ MNPs are key drivers of autoimmune MVD in K/B.g7 mice and are also present in human rheumatic heart disease. We define key inflammatory molecules that drive MVD in this model, including Syk, TNF, interleukin-6, very late antigen-4, and vascular cell adhesion molecule-1.

Engineering Cellular Resistance to HIV-1 Infection In Vivo Using a Dual Therapeutic Lentiviral Vector.

We described earlier a dual-combination anti-HIV type 1 (HIV-1) lentiviral vector (LVsh5/C46) that downregulates CCR5 expression of transduced cells via RNAi and inhibits HIV-1 fusion via cell surface expression of cell membrane-anchored C46 antiviral peptide. This combinatorial approach has two points of inhibition for R5-tropic HIV-1 and is also active against X4-tropic HIV-1. Here, we utilize the humanized bone marrow, liver, thymus (BLT) mouse model to characterize the in vivo efficacy of LVsh5/C46 (Cal-1) vector to engineer cellular resistance to HIV-1 pathogenesis. Human CD34+ hematopoietic stem/progenitor cells (HSPC) either nonmodified or transduced with LVsh5/C46 vector were transplanted to generate control and treatment groups, respectively. Control and experimental groups displayed similar engraftment and multilineage hematopoietic differentiation that included robust CD4+ T-cell development. Splenocytes isolated from the treatment group were resistant to both R5- and X4-tropic HIV-1 during ex vivo challenge experiments. Treatment group animals challenged with R5-tropic HIV-1 displayed significant protection of CD4+ T-cells and reduced viral load within peripheral blood and lymphoid tissues up to 14 weeks postinfection. Gene-marking and transgene expression were confirmed stable at 26 weeks post-transplantation. These data strongly support the use of LVsh5/C46 lentiviral vector in gene and cell therapeutic applications for inhibition of HIV-1 infection.

Molecular crowders and cosolutes promote folding cooperativity of RNA under physiological ionic conditions.

Folding mechanisms of functional RNAs under idealized in vitro conditions of dilute solution and high ionic strength have been well studied. Comparatively little is known, however, about mechanisms for folding of RNA in vivo where Mg(2+) ion concentrations are low, K(+) concentrations are modest, and concentrations of macromolecular crowders and low-molecular-weight cosolutes are high. Herein, we apply a combination of biophysical and structure mapping techniques to tRNA to elucidate thermodynamic and functional principles that govern RNA folding under in vivo-like conditions. We show by thermal denaturation and SHAPE studies that tRNA folding cooperativity increases in physiologically low concentrations of Mg(2+) (0.5-2 mM) and K(+) (140 mM) if the solution is supplemented with physiological amounts (∼ 20%) of a water-soluble neutral macromolecular crowding agent such as PEG or dextran. Low-molecular-weight cosolutes show varying effects on tRNA folding cooperativity, increasing or decreasing it based on the identity of the cosolute. For those additives that increase folding cooperativity, the gain is manifested in sharpened two-state-like folding transitions for full-length tRNA over its secondary structural elements. Temperature-dependent SHAPE experiments in the absence and presence of crowders and cosolutes reveal extent of cooperative folding of tRNA on a nucleotide basis and are consistent with the melting studies. Mechanistically, crowding agents appear to promote cooperativity by stabilizing tertiary structure, while those low molecular cosolutes that promote cooperativity stabilize tertiary structure and/or destabilize secondary structure. Cooperative folding of functional RNA under physiological-like conditions parallels the behavior of many proteins and has implications for cellular RNA folding kinetics and evolution.

Preclinical safety and efficacy of an anti-HIV-1 lentiviral vector containing a short hairpin RNA to CCR5 and the C46 fusion inhibitor.

Gene transfer has therapeutic potential for treating HIV-1 infection by generating cells that are resistant to the virus. We have engineered a novel self-inactivating lentiviral vector, LVsh5/C46, using two viral-entry inhibitors to block early steps of HIV-1 cycle. The LVsh5/C46 vector encodes a short hairpin RNA (shRNA) for downregulation of CCR5, in combination with the HIV-1 fusion inhibitor, C46. We demonstrate here the effective delivery of LVsh5/C46 to human T cell lines, peripheral blood mononuclear cells, primary CD4(+) T lymphocytes, and CD34(+) hematopoietic stem/progenitor cells (HSPC). CCR5-targeted shRNA (sh5) and C46 peptide were stably expressed in the target cells and were able to effectively protect gene-modified cells against infection with CCR5- and CXCR4-tropic strains of HIV-1. LVsh5/C46 treatment was nontoxic as assessed by cell growth and viability, was noninflammatory, and had no adverse effect on HSPC differentiation. LVsh5/C46 could be produced at a scale sufficient for clinical development and resulted in active viral particles with very low mutagenic potential and the absence of replication-competent lentivirus. Based on these in vitro results, plus additional in vivo safety and efficacy data, LVsh5/C46 is now being tested in a phase 1/2 clinical trial for the treatment of HIV-1 disease.

Colocalization of fast and slow timescale dynamics in the allosteric signaling protein CheY.

It is now widely recognized that dynamics are important to consider for understanding allosteric protein function. However, dynamics occur over a wide range of timescales, and how these different motions relate to one another is not well understood. Here, we report an NMR relaxation study of dynamics over multiple timescales at both backbone and side-chain sites upon an allosteric response to phosphorylation. The response regulator, Escherichia coli CheY, allosterically responds to phosphorylation with a change in dynamics on both the microsecond-to-millisecond (µs-ms) timescale and the picosecond-to-nanosecond (ps-ns) timescale. We observe an apparent decrease and redistribution of µs-ms dynamics upon phosphorylation (and accompanying Mg(2+) saturation) of CheY. Additionally, methyl groups with the largest changes in ps-ns dynamics localize to the regions of conformational change measured by µs-ms dynamics. The limited spread of changes in ps-ns dynamics suggests a distinct relationship between motions on the µs-ms and ps-ns timescales in CheY. The allosteric mechanism utilized by CheY highlights the diversity of roles dynamics play in protein function.

Segmental motions, not a two-state concerted switch, underlie allostery in CheY.

The switch between an inactive and active conformation is an important transition for signaling proteins, yet the mechanisms underlying such switches are not clearly understood. Escherichia coli CheY, a response regulator protein from the two-component signal transduction system that regulates bacterial chemotaxis, is an ideal protein for the study of allosteric mechanisms. By using 15N CPMG relaxation dispersion experiments, we monitored the inherent dynamic switching of unphosphorylated CheY. We show that CheY does not undergo a two-state concerted switch between the inactive and active conformations. Interestingly, partial saturation of Mg2+ enhances the intrinsic allosteric motions. Taken together with chemical shift perturbations, these data indicate that the µs-ms timescale motions underlying CheY allostery are segmental in nature. We propose an expanded allosteric network of residues, including W58, that undergo asynchronous, local switching between inactive and active-like conformations as the primary basis for the allosteric mechanism.

Detection of native-state nonadditivity in double mutant cycles via hydrogen exchange.

Proteins have evolved to exploit long-range structural and dynamic effects as a means of regulating function. Understanding communication between sites in proteins is therefore vital to our comprehension of such phenomena as allostery, catalysis, and ligand binding/ejection. Double mutant cycle analysis has long been used to determine the existence of communication between pairs of sites, proximal or distal, in proteins. Typically, nonadditivity (or "thermodynamic coupling") is measured from global transitions in concert with a single probe. Here, we have applied the atomic resolution of NMR in tandem with native-state hydrogen exchange (HX) to probe the structure/energy landscape for information transduction between a large number of distal sites in a protein. Considering the event of amide proton exchange as an energetically quantifiable structural perturbation, m n-dimensional cycles can be constructed from mutation of n-1 residues, where m is the number of residues for which HX data is available. Thus, efficient mapping of a large number of couplings is made possible. We have applied this technique to one additive and two nonadditive double mutant cycles in a model system, eglin c. We find heterogeneity of HX-monitored couplings for each cycle, yet averaging results in strong agreement with traditionally measured values. Furthermore, long-range couplings observed at locally exchanging residues indicate that the basis for communication can occur within the native state ensemble, a conclusion not apparent from traditional measurements. We propose that higher-order couplings can be obtained and show that such couplings provide a mechanistic basis for understanding lower-order couplings via "spheres of perturbation". The method is presented as an additional tool for identifying a large number of couplings with greater coverage of the protein of interest.

Regulation of PKR by HCV IRES RNA: importance of domain II and NS5A.

Protein kinase R (PKR) is an essential component of the innate immune response. In the presence of double-stranded RNA (dsRNA), PKR is autophosphorylated, which enables it to phosphorylate its substrate, eukaryotic initiation factor 2alpha, leading to translation cessation. Typical activators of PKR are long dsRNAs produced during viral infection, although certain other RNAs can also activate. A recent study indicated that full-length internal ribosome entry site (IRES), present in the 5'-untranslated region of hepatitis C virus (HCV) RNA, inhibits PKR, while another showed that it activates. We show here that both activation and inhibition by full-length IRES are possible. The HCV IRES has a complex secondary structure comprising four domains. While it has been demonstrated that domains III-IV activate PKR, we report here that domain II of the IRES also potently activates. Structure mapping and mutational analysis of domain II indicate that while the double-stranded regions of the RNA are important for activation, loop regions contribute as well. Structural comparison reveals that domain II has multiple, non-Watson-Crick features that mimic A-form dsRNA. The canonical and noncanonical features of domain II cumulate to a total of approximately 33 unbranched base pairs, the minimum length of dsRNA required for PKR activation. These results provide further insight into the structural basis of PKR activation by a diverse array of RNA structural motifs that deviate from the long helical stretches found in traditional PKR activators. Activation of PKR by domain II of the HCV IRES has implications for the innate immune response when the other domains of the IRES may be inaccessible. We also study the ability of the HCV nonstructural protein 5A (NS5A) to bind various domains of the IRES and alter activation. A model is presented for how domain II of the IRES and NS5A operate to control host and viral translation during HCV infection.

A highly efficient short hairpin RNA potently down-regulates CCR5 expression in systemic lymphoid organs in the hu-BLT mouse model.

Inhibiting the expression of the HIV-1 coreceptor CCR5 holds great promise for controlling HIV-1 infection in patients. Here we report stable knockdown of human CCR5 by a short hairpin RNA (shRNA) in a humanized bone marrow/liver/thymus (BLT) mouse model. We delivered a potent shRNA against CCR5 into human fetal liver-derived CD34(+) hematopoietic progenitor/stem cells (HPSCs) by lentiviral vector transduction. We transplanted vector-transduced HPSCs solidified with Matrigel and a thymus segment under the mouse kidney capsule. Vector-transduced autologous CD34(+) cells were subsequently injected in the irradiated mouse, intended to create systemic reconstitution. CCR5 expression was down-regulated in human T cells and monocytes/macrophages in systemic lymphoid tissues, including gut-associated lymphoid tissue, the major site of HIV-1 replication. The shRNA-mediated CCR5 knockdown had no apparent adverse effects on T-cell development as assessed by polyclonal T-cell receptor Vbeta family development and naive/memory T-cell differentiation. CCR5 knockdown in the secondary transplanted mice suggested the potential of long-term hematopoietic reconstitution by the shRNA-transduced HPSCs. CCR5 tropic HIV-1 infection was effectively inhibited in mouse-derived human splenocytes ex vivo. These results demonstrate that lentiviral vector delivery of shRNA into human HPSCs could stably down-regulate CCR5 in systemic lymphoid organs in vivo.