A HST1-like gene controls tiller angle through regulating endogenous auxin in common wheat.

Tiller angle is one of the most important agronomic traits and one key factor for wheat ideal plant architecture, which can both increase photosynthetic efficiency and greatly enhance grain yield. Here, a deacetylase HST1-like (TaHST1L) gene controlling wheat tiller angle was identified by the combination of a genome-wide association study (GWAS) and bulked segregant analysis (BSA). Ethyl methane sulfonate (EMS)-mutagenized tetraploid wheat lines with the premature stop codon of TaHST1L exhibited significantly smaller tiller angles than the wild type. TaHST1L-overexpressing (OE) plants exhibited significantly larger tiller angles and increased tiller numbers in both winter and spring wheat, while TaHST1L-silenced RNAi plants displayed significantly smaller tiller angles and decreased tiller numbers. Moreover, TaHST1L strongly interacted with TaIAA17 and inhibited its expression at the protein level, and thus possibly improved the content of endogenous auxin in the basal tissue of tillers. The transcriptomics and metabolomics results indicated that TaHST1L might change plant architecture by mediating auxin signal transduction and regulating endogenous auxin levels. In addition, a 242-bp insertion/deletion (InDel) in the TaHST1L-A1 promoter altered transcriptional activity and TaHST1L-A1b allele with the 242-bp insertion widened the tiller angle of TaHST1L-OE transgenic rice plants. Wheat varieties with TaHST1L-A1b allele possessed the increased tiller angle and grain yield. Further analysis in wheat and its progenitors indicated that the 242-bp InDel possibly originated from wild emmer and was strongly domesticated in the current varieties. Therefore, TaHST1L involved in the auxin signalling pathway showed the big potential to improve wheat yield by controlling plant architecture.

Identification of TaGL1-B1 gene controlling grain length through regulation of jasmonic acid in common wheat.

Grain length is one of the most important factors in determining wheat yield. Here, a stable QTL for grain length was mapped on chromosome 1B in a F10 recombinant inbred lines (RIL) population, and the gene TaGL1-B1 encoding carotenoid isomerase was identified in a secondary large population through multiple strategies. The genome-wide association study (GWAS) in 243 wheat accessions revealed that the marker for TaGL1-B1 was the most significant among all chromosomes. EMS mutants of TaGL1 possessed significantly reduced grain length, whereas TaGL1-B1-overexpressed lines possessed significantly increased grain length. Moreover, TaGL1-B1 strongly interacted with TaPAP6. TaPAP6-overexpressed lines had significantly increased grain length. Transcriptome analysis suggested that TaPAP6 was possibly involved in the accumulation of JA (jasmonic acid). Consistently, JA content was significantly increased in the TaGL1-B1 and TaPAP6 overexpression lines. Additionally, the role of TaGL1-B1 in regulating carotenoids was verified through QTL mapping, GWAS, EMS mutants and overexpression lines. Notably, overexpression of TaGL1-B1 significantly increased wheat yield in multiple locations. Taken together, overexpression of TaGL1-B1 enhanced grain length, probably through interaction with TaPAP6 to cause the accumulation of JA that improved carotenoid content and photosynthesis, thereby resulted in increased wheat yield. This study provided valuable genes controlling grain length to improve yield and a potential insight into the molecular mechanism of modulating JA-mediated grain size in wheat.

Rb-E2F-HDAC Repressor Complexes Control Interferon-Induced Repression of Adenovirus To Promote Persistent Infection.

Interferons (IFNs) are cytokines that induce a global change in the cell to establish antiviral immunity. We previously demonstrated that human adenovirus (HAdV) exploits IFN-induced viral repression to persist in infected cells. Although this in vitro persistence model has been described, the mechanism behind how persistent HAdV infection is established is not well understood. In this study, we demonstrate that IFN signaling is essential for viral repression and promoting persistent infection. Cyclin-dependent kinase 4 (CDK4), an antagonist of retinoblastoma (Rb) family proteins, was shown to disrupt the viral repression induced by IFNs. Consistent with this result, knockout of the Rb family proteins pRb, p107, and/or p130 drastically reduced the effect of IFNs on viral replication. The pRb protein specifically contributed the greatest effect to IFN inhibition of viral replication. Interestingly, IFNs did not impact pRb through direct changes in protein or phosphorylation levels. Cells treated with IFNs continued to cycle normally, consistent with observations that persistently infected cells remain for long periods of time in the host and in our in vitro persistent infection model. Finally, we observed that histone deacetylase (HDAC) inhibitors activated productive viral replication in persistently infected cells in the presence of IFN. Thus, HDACs, specifically class I HDACs, which are commonly associated with Rb family proteins, play a major role in the maintenance of persistent HAdV infection in vitro. This study uncovers the critical role of pRb and class I HDACs in the IFN-induced formation of a repressor complex that promotes persistent HAdV infections. IMPORTANCE Adenoviruses are ubiquitous viruses infecting more than 90% of the human population. HAdVs cause persistent infections that may lead to serious complications in immunocompromised patients. Therefore, exploring how HAdVs establish persistent infections is critical for understanding viral reactivation in immunosuppressed individuals. The mechanism underlying HAdV persistence has not been fully explored. Here, we provide insight into the contributions of the host cell to IFN-mediated persistent HAdV infection. We found that HAdV-C5 productive infection is inhibited by an Rb-E2F-HDAC repressor complex. Treatment with HDAC inhibitors converted a persistent infection to a lytic infection. Our results suggest that this process involves the noncanonical regulation of Rb-E2F signaling. This study provides insight into a highly prevalent human pathogen, bringing a new level of complexity and understanding to the replicative cycle.

The adenovirus major core protein VII is dispensable for virion assembly but is essential for lytic infection.

The Adenovirus (Ad) genome within the capsid is tightly associated with a virus-encoded, histone-like core protein-protein VII. Two other Ad core proteins, V and X/µ, also are located within the virion and are loosely associated with viral DNA. Core protein VII remains associated with the Ad genome during the early phase of infection. It is not known if naked Ad DNA is packaged into the capsid, as with dsDNA bacteriophage and herpesviruses, followed by the encapsidation of viral core proteins, or if a unique packaging mechanism exists with Ad where a DNA-protein complex is simultaneously packaged into the virion. The latter model would require an entirely new molecular mechanism for packaging compared to known viral packaging motors. We characterized a virus with a conditional knockout of core protein VII. Remarkably, virus particles were assembled efficiently in the absence of protein VII. No changes in protein composition were evident with VII-virus particles, including the abundance of core protein V, but changes in the proteolytic processing of some capsid proteins were evident. Virus particles that lack protein VII enter the cell, but incoming virions did not escape efficiently from endosomes. This greatly diminished all subsequent aspects of the infectious cycle. These results reveal that the Ad major core protein VII is not required to condense viral DNA within the capsid, but rather plays an unexpected role during virus maturation and the early stages of infection. These results establish a new paradigm pertaining to the Ad assembly mechanism and reveal a new and important role of protein VII in early stages of infection.

E2F/Rb Family Proteins Mediate Interferon Induced Repression of Adenovirus Immediate Early Transcription to Promote Persistent Viral Infection.

Interferons (IFNs) are cytokines that have pleiotropic effects and play important roles in innate and adaptive immunity. IFNs have broad antiviral properties and function by different mechanisms. IFNs fail to inhibit wild-type Adenovirus (Ad) replication in established cancer cell lines. In this study, we analyzed the effects of IFNs on Ad replication in normal human cells. Our data demonstrate that both IFNα and IFNγ blocked wild-type Ad5 replication in primary human bronchial epithelial cells (NHBEC) and TERT-immortalized normal human diploid fibroblasts (HDF-TERT). IFNs inhibited the replication of divergent adenoviruses. The inhibition of Ad5 replication by IFNα and IFNγ is the consequence of repression of transcription of the E1A immediate early gene product. Both IFNα and IFNγ impede the association of the transactivator GABP with the E1A enhancer region during the early phase of infection. The repression of E1A expression by IFNs requires a conserved E2F binding site in the E1A enhancer, and IFNs increased the enrichment of the E2F-associated pocket proteins, Rb and p107, at the E1A enhancer in vivo. PD0332991 (Pabociclib), a specific CDK4/6 inhibitor, dephosphoryles pocket proteins to promote their interaction with E2Fs and inhibited wild-type Ad5 replication dependent on the conserved E2F binding site. Consistent with this result, expression of the small E1A oncoprotein, which abrogates E2F/pocket protein interactions, rescued Ad replication in the presence of IFNα or IFNγ. Finally, we established a persistent Ad infection model in vitro and demonstrated that IFNγ suppresses productive Ad replication in a manner dependent on the E2F binding site in the E1A enhancer. This is the first study that probes the molecular basis of persistent adenovirus infection and reveals a novel mechanism by which adenoviruses utilize IFN signaling to suppress lytic virus replication and to promote persistent infection.

To increase the incorporation efficiency of genetically encoding Nε-acetyllysine in recombinant protein.

Reversible Nε-lysine (Nε-Lys) acetylation is a dynamic post-translational modification. Genetic incorporation of Nε-acetyllysine (Nε-AcK) into the specific site of a protein is a powerful method for producing recombinant protein with acetylation and studying the functional role of protein acetylation. Because of the universal existence of deacetylase such as CobB in vivo, the acetyl group of Nε-AcK may be removed from recombinant protein. So in the process of incorporating acetyl lysine into protein, nicotinamide (NAM), a lysine deacetylase (KDAC) inhibitor, is needed to inhibit the KDAC activity and protect the acetyl group of Nε-acetyllysine incorporated from removal in vivo. In this study, we knocked out cobB gene from an E. coli strain and used it as host for incorporating of Nε-AcK into recombinant protein by expanding genetic code. Comparing with the addition of KDAC inhibitor, this is a novel method to protect the acetyl group of Nε-acetyllysine from deacetylation by using cobB knockout strain. Thus, this method provides a better way for protecting recombinant acetylated protein from deacetylation. By knocking out cobB gene from the strain of E. coli, we successfully produced homogeneous acetylated protein by expanding genetic code with a high effectiveness. This method will also provide a good reference for genetically introducing other varieties of modified amino acids into protein.

Resveratrol Exerts Antioxidant Effects by Activating SIRT2 To Deacetylate Prx1.

Resveratrol is a promising chemical agent that treats multiple aging-related diseases and improves life span. While reactive oxygen species undoubtedly play ubiquitous roles in the aging process and resveratrol has been shown to be an effective antioxidant, the mechanism through which resveratrol acts against oxidative stress remains unknown. Here we show that resveratrol activates SIRT2 to deacetylate Prx1, leading to an increased H2O2 reduction activity and a decreased cellular H2O2 concentration. Knockdown of SIRT2 or Prx1 by RNA interference abrogates resveratrol's ability to reduce the H2O2 level in HepG2 cells. Using purified SIRT2 and a Prx1 mutant harboring acetyllysine at position 27 (Prx1-27AcK), we show that resveratrol enhances SIRT2's activity to deacetylate Prx1-27AcK, resulting in a significantly increased H2O2 reducing activity. Thus, SIRT2 and Prx1 are targets for modulating intracellular redox status in the therapeutic strategies for the treatment of aging-related disorders.

Non-viral expression of chimeric antigen receptors with multiplex gene editing in primary T cells.

Efficient engineering of T cells to express exogenous tumor-targeting receptors such as chimeric antigen receptors (CARs) or T-cell receptors (TCRs) is a key requirement of effective adoptive cell therapy for cancer. Genome editing technologies, such as CRISPR/Cas9, can further alter the functional characteristics of therapeutic T cells through the knockout of genes of interest while knocking in synthetic receptors that can recognize cancer cells. Performing multiple rounds of gene transfer with precise genome editing, termed multiplexing, remains a key challenge, especially for non-viral delivery platforms. Here, we demonstrate the efficient production of primary human T cells incorporating the knockout of three clinically relevant genes (B2M, TRAC, and PD1) along with the non-viral transfection of a CAR targeting disialoganglioside GD2. Multiplexed knockout results in high on-target deletion for all three genes, with low off-target editing and chromosome alterations. Incorporating non-viral delivery to knock in a GD2-CAR resulted in a TRAC-B2M-PD1-deficient GD2 CAR T-cell product with a central memory cell phenotype and high cytotoxicity against GD2-expressing neuroblastoma target cells. Multiplexed gene-editing with non-viral delivery by CRISPR/Cas9 is feasible and safe, with a high potential for rapid and efficient manufacturing of highly potent allogeneic CAR T-cell products.

Tiled microarray identification of novel viral transcript structures and distinct transcriptional profiles during two modes of productive murine gammaherpesvirus 68 infection.

We applied a custom tiled microarray to examine murine gammaherpesvirus 68 (MHV68) polyadenylated transcript expression in a time course of de novo infection of fibroblast cells and following phorbol ester-mediated reactivation from a latently infected B cell line. During de novo infection, all open reading frames (ORFs) were transcribed and clustered into four major temporal groups that were overlapping yet distinct from clusters based on the phorbol ester-stimulated B cell reactivation time course. High-density transcript analysis at 2-h intervals during de novo infection mapped gene boundaries with a 20-nucleotide resolution, including a previously undefined ORF73 transcript and the MHV68 ORF63 homolog of Kaposi's sarcoma-associated herpesvirus vNLRP1. ORF6 transcript initiation was mapped by tiled array and confirmed by 5' rapid amplification of cDNA ends. The ∼1.3-kb region upstream of ORF6 was responsive to lytic infection and MHV68 RTA, identifying a novel RTA-responsive promoter. Transcription in intergenic regions consistent with the previously defined expressed genomic regions was detected during both types of productive infection. We conclude that the MHV68 transcriptome is dynamic and distinct during de novo fibroblast infection and upon phorbol ester-stimulated B cell reactivation, highlighting the need to evaluate further transcript structure and the context-dependent molecular events that govern viral gene expression during chronic infection.

Detecting Melanocortin 1 Receptor Gene's SNPs by CRISPR/enAsCas12a.

Beyond its powerful genome-editing capabilities, the CRISPR/Cas system has opened up a new era of molecular diagnostics due to its highly specific base recognition and trans-cleavage activity. However, most CRISPR/Cas detection systems are mainly used to detect nucleic acids of bacteria or viruses, while the application of single nucleotide polymorphism (SNP) detection is limited. The MC1R SNPs were investigated by CRISPR/enAsCas12a and are not limited to the protospacer adjacent motif (PAM) sequence in vitro. Specifically, we optimized the reaction conditions, which proved that the enAsCas12a has a preference for divalent magnesium ion (Mg2+) and can effectively distinguish the genes with a single base difference in the presence of Mg2+, and the Melanocortin l receptor (MC1R) gene with three kinds of SNP sites (T305C, T363C, and G727A) was quantitatively detected. Since the enAsCas12a is not limited by PAM sequence in vitro, the method shown here can extend this extraordinary CRISPR/enAsCas12a detection system to other SNP targets, thus providing a general SNP detection toolbox.

Synthetic immune checkpoint engagers protect HLA-deficient iPSCs and derivatives from innate immune cell cytotoxicity.

Immune rejection of allogeneic cell therapeutics remains a major problem for immuno-oncology and regenerative medicine. Allogeneic cell products so far have inferior persistence and efficacy when compared with autologous alternatives. Engineering of hypoimmune cells may greatly improve their therapeutic benefit. We present a new class of agonistic immune checkpoint engagers that protect human leukocyte antigen (HLA)-depleted induced pluripotent stem cell-derived endothelial cells (iECs) from innate immune cells. Engagers with agonistic functionality to their inhibitory receptors TIM3 and SIRPα effectively protect engineered iECs from natural killer (NK) cell and macrophage killing. The SIRPα engager can be combined with truncated CD64 to generate fully immune evasive iECs capable of escaping allogeneic cellular and immunoglobulin G (IgG) antibody-mediated rejection. Synthetic immune checkpoint engagers have high target specificity and lack retrograde signaling in the engineered cells. This modular design allows for the exploitation of more inhibitory immune pathways for immune evasion and could contribute to the advancement of allogeneic cell therapeutics.

Cap Is the Protease of the Porcine Circovirus 2.

Circoviruses are the smallest single-stranded DNA viruses that infect mammalian species, avian species, fish, and insects. The infections of circoviruses are known to be associated with a series of fatal diseases, but the protease of circovirus still remains unknown. In this research, we identified viral capsid protein (Cap) as the protease of porcine circovirus type 2 (PCV2), to our knowledge the first circoviruses protease to be reported. First, we found that the expression of host proteins is affected due to PCV2 infection in the porcine kidney (PK-15) cells. Then, by proteomic analysis, 253 host proteins that were down regulated were identified due to direct or indirect effects of PCV2. Further, Cap expression, but not other ORFs of PCV2, significantly reduced both JMJD6 (bifunctional arginine demethylase and lysyl-hydroxylase) and CCT5 (the chaperonin containing TCP1 subunit 5) in PK-15 cells. Finally, the results in vitro hydrolysis assays demonstrated that Cap could directly degraded either JMJD6 or CCT5 with different catalytic efficiency. In summary, our study expands repertoire of PCV2 Cap and promotes the development of inhibitors toward the anti-PCV2.

Human IFIT3 Protein Induces Interferon Signaling and Inhibits Adenovirus Immediate Early Gene Expression.

Interferons (IFNs) are one of the hallmarks of host antiviral immunity. IFNs exert their antiviral activities through the induction of IFN-stimulated genes (ISGs) and antiviral proteins; however, the mechanism by which ISGs inhibit adenovirus (Ad) replication is not clearly understood. IFNs repress Ad immediate early gene expression and, consequently, all subsequent aspects of the viral life cycle. In this study, we found that IFN-induced protein with tetratricopeptide repeats 3, IFIT3 (ISG60), restricts Ad replication. IFIT3 repressed Ad E1A immediate early gene expression but did not alter Ad genome entry into the nucleus. Expression of IFIT3 led to phosphorylation of TBK1, IRF3, and STAT1; increased expression of IFNß and ISGs; and required IFIT1 and IFIT2 partner proteins. During RNA virus infections, it is known that IFIT3 stimulates IFN production through mitochondrial antiviral signaling (MAVS)-mediated activation of TBK1 which synergizes activation of IRF3 and NF-κB. MAVS or TBK1 depletion in cells expressing IFIT3 blocked IFN signaling and reversed the Ad replication restriction. In addition, STING depletion phenocopied the effect suggesting that IFIT3 activates the STING pathway with cross talk to the MAVS pathway. This occurs independently of viral pathogen-associated molecular patterns (PAMPs). These results demonstrate that the expression of a single ISG, IFIT3, activates IFN signaling and establishes a cellular antiviral state independent of viral PAMPs. IMPORTANCE IFITs belong to a family of IFN-induced proteins that have broad antiviral functions, primarily studied with RNA viruses leaving a gap of knowledge on the effects of these proteins on DNA viruses. In this study we show that IFIT3, with its partner proteins IFIT1 and IFIT2, specifically restricts replication of human Ad, a DNA virus, by stimulating IFNß production via the STING and MAVS pathways. This effect enhanced the IFN response and is independent of viral PAMPs. These results reveal a novel mechanism of activation of IFN signaling to enhance cellular antiviral responses.

Inkjet-Printed Quantum Dot Fluorescent Security Labels with Triple-Level Optical Encryption.

Optical security labels play a significant role in protecting both our wealth and health. However, simultaneously meeting the requirements including low-cost fabrication, easy detection, and high-level security is still challenging for security labels. Here, we design an unclonable anti-counterfeiting system with triple-level security by using the inkjet printing technique, which can be authenticated by naked eyes, a portable microscope, and a fluorescence microscope. These labels are achieved by printing microscale quantum dot (QD) ink droplets on premodified substrates with random-distributed glass microspheres. Due to the unique capillary action induced by the glass microspheres, QDs in the ink droplets are deposited around the microspheres, forming microscale multicircular patterns. Multiple pinning of QDs at the three-phase contact lines appears during the evaporation of the droplet, resulting in the formation of a nanoscale labyrinthine pattern around the microspheres. The nanoscale labyrinth pattern and the microscale multicircular microsphere array, together with the printed macroscopic image, constitute a triple-level progressive anti-counterfeiting system. Moreover, the system is compatible with an artificial intelligence-based identification strategy that allows rapid identification and verification of the unclonable security labels.

Unclonable Perovskite Fluorescent Dots with Fingerprint Pattern for Multilevel Anticounterfeiting.

Anticounterfeiting techniques based on physical unclonable functions exhibit great potential in security protection of extensive commodities from daily necessities to high-end products. Herein, we propose a facile strategy to fabricate an unclonable super micro fingerprint (SMFP) array by introducing in situ grown perovskite crystals for multilevel anticounterfeiting labels. The unclonable features are formed on the basis of the differential transportation of a microscale perovskite precursor droplet during the inkjet printing process, coupled with random crystallization and Ostwald ripening of perovskite crystals originating from their ion crystal property. Furthermore, the unclonable patterns can be readily tailored by tuning in situ crystallization conditions of the perovskite. Three-dimensional height information on the perovskite patterns are introduced into a security label and further transformed into structural color, significantly enhancing the capacity of anticounterfeiting labels. The SMFPs are characterized with tunable multilevel anticounterfeiting properties, including macroscale patterns, microscale unclonable pattern, fluorescent two-dimensional pattens, and colorful three-dimensional information.

Tonic 4-1BB Costimulation in Chimeric Antigen Receptors Impedes T Cell Survival and Is Vector-Dependent.

Antigen-independent tonic signaling by chimeric antigen receptors (CARs) can increase differentiation and exhaustion of T cells, limiting their potency. Incorporating 4-1BB costimulation in CARs may enable T cells to resist this functional exhaustion; however, the potential ramifications of tonic 4-1BB signaling in CAR T cells remain unclear. Here, we found that tonic CAR-derived 4-1BB signaling can produce toxicity in T cells via continuous TRAF2-dependent activation of the nuclear factor κB (NF-κB) pathway and augmented FAS-dependent cell death. This mechanism was amplified in a non-self-inactivating gammaretroviral vector through positive feedback on the long terminal repeat (LTR) promoter, further enhancing CAR expression and tonic signaling. Attenuating CAR expression by substitution with a self-inactivating lentiviral vector minimized tonic signaling and improved T cell expansion and anti-tumor function. These studies illuminate the interaction between tonic CAR signaling and the chosen expression platform and identify inhibitory properties of the 4-1BB costimulatory domain that have direct implications for rational CAR design.

The Yersinia Virulence Factor YopM Hijacks Host Kinases to Inhibit Type III Effector-Triggered Activation of the Pyrin Inflammasome.

Pathogenic Yersinia, including Y. pestis, the agent of plague in humans, and Y. pseudotuberculosis, the related enteric pathogen, deliver virulence effectors into host cells via a prototypical type III secretion system to promote pathogenesis. These effectors, termed Yersinia outer proteins (Yops), modulate multiple host signaling responses. Studies in Y. pestis and Y. pseudotuberculosis have shown that YopM suppresses infection-induced inflammasome activation; however, the underlying molecular mechanism is largely unknown. Here we show that YopM specifically restricts the pyrin inflammasome, which is triggered by the RhoA-inactivating enzymatic activities of YopE and YopT, in Y. pseudotuberculosis-infected macrophages. The attenuation of a yopM mutant is fully reversed in pyrin knockout mice, demonstrating that YopM inhibits pyrin to promote virulence. Mechanistically, YopM recruits and activates the host kinases PRK1 and PRK2 to negatively regulate pyrin by phosphorylation. These results show how a virulence factor can hijack host kinases to inhibit effector-triggered pyrin inflammasome activation.

Impact of Adenovirus E4-ORF3 Oligomerization and Protein Localization on Cellular Gene Expression.

The Adenovirus E4-ORF3 protein facilitates virus replication through the relocalization of cellular proteins into nuclear inclusions termed tracks. This sequestration event disrupts antiviral properties associated with target proteins. Relocalization of Mre11-Rad50-Nbs1 proteins prevents the DNA damage response from inhibiting Ad replication. Relocalization of PML and Daxx impedes the interferon-mediated antiviral response. Several E4-ORF3 targets regulate gene expression, linking E4-ORF3 to transcriptional control. Furthermore, E4-ORF3 was shown to promote the formation of heterochromatin, down-regulating p53-dependent gene expression. Here, we characterize how E4-ORF3 alters cellular gene expression. Using an inducible, E4-ORF3-expressing cell line, we performed microarray experiments to highlight cellular gene expression changes influenced by E4-ORF3 expression, identifying over four hundred target genes. Enrichment analysis of these genes suggests that E4-ORF3 influences factors involved in signal transduction and cellular defense, among others. The expression of mutant E4-ORF3 proteins revealed that nuclear track formation is necessary to induce these expression changes. Through the generation of knockdown cells, we demonstrate that the observed expression changes may be independent of Daxx and TRIM33 suggesting that an additional factor(s) may be responsible. The ability of E4-ORF3 to manipulate cellular gene expression through the sequestration of cellular proteins implicates a novel role for E4-ORF3 in transcriptional regulation.

The use of chromatin immunoprecipitation (ChIP) to study the binding of viral proteins to the adenovirus genome in vivo.

The encapsidation of adenovirus (Ad) DNA into virus particles depends on cis-acting sequences located at the left end of the viral genome. Repeated DNA sequences in the packaging domain contribute to viral DNA encapsidation and several viral proteins bind to these repeats when analyzed using in vitro DNA-protein binding assays. In this chapter, we describe a chromatin immunoprecipitation (ChIP) approach to study the binding of viral proteins to packaging sequences in vivo. This assay permits accurate quantification over a wide range of DNA concentrations. The use of formaldehyde cross-linking to stabilize DNA-protein and protein-protein complexes formed in vivo allows the identification of macromolecular complexes found in living cells.

Crystal structure of E. coli arginyl-tRNA synthetase and ligand binding studies revealed key residues in arginine recognition.

The arginyl-tRNA synthetase (ArgRS) catalyzes the esterification reaction between L-arginine and its cognate tRNA(Arg). Previously reported structures of ArgRS shed considerable light on the tRNA recognition mechanism, while the aspect of amino acid binding in ArgRS remains largely unexplored. Here we report the first crystal structure of E. coli ArgRS (eArgRS) complexed with L-arginine, and a series of mutational studies using isothermal titration calorimetry (ITC). Combined with previously reported work on ArgRS, our results elucidated the structural and functional roles of a series of important residues in the active site, which furthered our understanding of this unique enzyme.