Inactivating a cellular intrinsic immune defense mediated by Daxx is the mechanism through which the human cytomegalovirus pp71 protein stimulates viral immediate-early gene expression

Human cytomegalovirus (HCMV) masterfully evades adaptive and innate immune responses, allowing infection to be maintained and periodically reactivated for the life of the host. Here we show that cells also possess an intrinsic immune defense against HCMV that is disarmed by the virus. In HCMV-infected cells, the promyelocytic leukemia nuclear body (PML-NB) protein Daxx silences viral immediate-early gene expression through the action of a histone deacetylase. However, this antiviral tactic is efficiently neutralized by the viral pp71 protein, which is incorporated into virions, delivered to cells upon infection, and mediates the proteasomal degradation of Daxx. This work demonstrates the mechanism through which pp71 activates viral immediate-early gene expression in HCMV-infected cells. Furthermore, it provides insight into how a PML-NB protein institutes an intrinsic immune defense against a DNA virus and how HCMV pp71 inactivates this defense.     

Differential regulation of host cellular genes by HIV-1 viral protein R (Vpr): cDNA microarray analysis using isogenic virus

HIV-1 Vpr is a protein with multiple functions. It has been suggested that such pleiotropic effects by a viral protein may be mediated by its association with viral and cellular proteins or through modulation of expression of specific cellular genes. To address this, we used cDNA microarray techniques to analyze the regulation of a panel of host cellular genes by HIV-1 Vpr using isogenic HIV-1 either with or without Vpr expression. Results indicate that Vpr downregulated the expression of genes involved in cell cycle/proliferation regulation, DNA repair, tumor antigens, and immune activation factors, and upregulated many ribosomal and structural proteins. These results for the first time reveal the involvement of several potential cellular genes, which may be useful, both for understanding Vpr functions and for the development of therapeutics targeting the Vpr molecule.     

Binding free energy differences in a TCR-peptide-MHC complex induced by a peptide mutation: a simulation analysis

Recognition by the T-cell receptor (TCR) of immunogenic peptides presented by class I major histocompatibility complexes (MHCs) is the determining event in the specific cellular immune response against virus-infected cells or tumor cells. It is of great interest, therefore, to elucidate the molecular principles upon which the selectivity of a TCR is based. These principles can in turn be used to design therapeutic approaches, such as peptide-based immunotherapies of cancer. In this study, free energy simulation methods are used to analyze the binding free energy difference of a particular TCR (A6) for a wild-type peptide (Tax) and a mutant peptide (Tax P6A), both presented in HLA A2. The computed free energy difference is 2.9 kcal/mol, in good agreement with the experimental value. This makes possible the use of the simulation results for obtaining an understanding of the origin of the free energy difference which was not available from the experimental results. A free energy component analysis makes possible the decomposition of the free energy difference between the binding of the wild-type and mutant peptide into its components. Of particular interest is the fact that better solvation of the mutant peptide when bound to the MHC molecule is an important contribution to the greater affinity of the TCR for the latter. The results make possible identification of the residues of the TCR which are important for the selectivity. This provides an understanding of the molecular principles that govern the recognition. The possibility of using free energy simulations in designing peptide derivatives for cancer immunotherapy is briefly discussed.     

Immunosuppression induced by expression of a viral RNase enhances susceptibility of Plutella xylostella to microbial pesticides

Abstract Polydnaviruses are a group of insect DNA viruses and are characterized in their segmented genome that is located in the chromosome(s) of host wasps. A polydnavirus, Cotesia plutellae bracovirus (CpBV), encodes a viral ribonuclease (RNase) T2 in a specific segment #3 (CpBV‐S3). This study tested its effect on gene expression associated with host immune responses in the diamondback moth, Plutella xylostella. Micro‐injection of CpBV‐S3 into nonparasitized larvae induced expression of its two encoded genes, CpBV‐ORF301 (=CpBV‐RNase T2) and CpBV‐ORF302. In response to a bacterial challenge, four antimicrobial peptide genes (hemolin, gloverin, cecropin and lysozyme) and six phenoloxidase (PO)–associated genes (proPO‐activating proteinase, PO, serine proteinase homolog and serpins 1–3) were up‐regulated in their expressions. However, the transient expression of CpBV‐S3 suppressed the expressions of cecropin, PO and serpin 1. Double‐stranded RNA specific to the viral RNase T2 could specifically knockdown the viral gene expression and restored the three gene expressions suppressed in the larvae injected with CpBV‐S3. The inhibitory activity of the viral RNase T2 on the target genes was further proven by the suppression of PO activation in response to bacterial challenge in the larvae injected with CpBV‐S3. This immunosuppression by the expression of the viral RNase T2 resulted in significant increase of pathogen susceptibility of P. xylostella against Bacillus thuringiensis or baculovirus infection.     

Administration of a maple syrup extract to mitigate their hepatic inflammation induced by a high-fat diet: a transcriptome analysis

Effects of the administration of maple syrup extract (MSX) on hepatic gene expression were investigated in mice fed a high-fat diet. Gene annotation enrichment analysis based on gene ontology revealed some changes in the expression of genes related to lipid metabolism and the immune response in MSX-fed mice. Detailed analysis of these data indicated that MSX ingestion mitigates hepatic inflammation.     

A circular channel crucible oscillating viscometer: Detection of DNA damage induced in vivo by exceedingly small doses of dimethylnitrosamine

A new oscillating crucible viscometer, having a U-shaped circular channel, is described. The damping coefficient δ is lowered by an increase of the viscosity η. The instrument described here allows the solution to come in contact with inert plastic only. At all steps of its preparation and during viscosity measurements, giant DNA from rat liver nuclei was maintained at shear stresses around 10^?4 dynes cm^?2. Viscosity was studied as a function of surface tension, DNA concentration and shear stress. It was found that under our experimental conditions it was possible to obtain meaningful values for reduced viscosity, η_red, practically identical to intrinsic viscosity [η]. Rat liver nuclei are incubated in an alkaline lysing solution (pH 12.5; 22 °C): they are lysed immediately and the released DNA starts to uncoil. The viscosity of solutions of this giant DNA increases very slowly with time, reaching a maximum only after about ten hours. The process was accelerated by single-stranded breaks arising from methylation of DNA in vivo with dimethylnitrosamine. It was found that the time of DNA disentanglement was sensitive to an exceedingly small number of breaks. We think that we were able to measure molecular weights around the length of the single strand of an average chromosome (M_n 5 × 10¹⁰). An empirical relation between molecular weight and reduced viscosity after complete disentanglement was also established, as a linear log-log plot, covering a molecular weight range between 10⁸ and 2.5 × 10¹⁰. It is suggested that the viscosimetric evaluation of DNA disentanglement is probably the most sensitive method for studying DNA damage induced “in vivo” by chemical carcinogens.     

A plant kinase plays roles in defense response against geminivirus by phosphorylation of a viral pathogenesis protein

The plant SNF1-related kinase (SnRK1) is the α-subunit of the SnRK1 heterotrimeric compleses. Although SnRK1 is widely known as a key regulator of plant response to various physiological processes including nutrient- and energy-sensing, regulation of global metabolism, and control of cell cycle, development, as well as abiotics stress, less is known about the function of SnRK1 during pathogen infection. Our previous work has demonstrated that a tomato SNF1-related kinase (SlSnRK1) can interact with and phosphorylate βC1, a pathogenesis protein encoded by tomato yellow leaf curl China betasatellite. Our results also showed that the plant SnRK1 can affect genimivirus infection in plant and reduce viral DNA accumulation. Phosphorylation of βC1 protein negatively impacts its function as a pathogenicity determinant. Here we provide more information on interaction between βC1 and SlSnRK1 and propose a mechanistic model for the SlSnRK1-mediated defense responses against geminiviruses and the potential role of SnRK1 in plant resistance to geminivirus.     

In vivo control of soluble guanylate cyclase activation by nitric oxide: a kinetic analysis

Free nitric oxide (NO) activates soluble guanylate cyclase (sGC), an enzyme, within both pulmonary and vascular smooth muscle. sGC catalyzes the cyclization of guanosine 5'-triphosphate to guanosine 3',5'-cyclic monophosphate (cGMP). Binding rates of NO to the ferrous heme(s) of sGC have been measured in vitro. However, a missing link in our understanding of the control mechanism of sGC by NO is a comprehensive in vivo kinetic analysis. Available literature data suggests that NO dissociation from the heme center of sGC is accelerated by its interaction with one or more cofactors in vivo. We present a working model for sGC activation and NO consumption in vivo. Our model predicts that NO influences the cGMP formation rate over a concentration range of approximately 5-100 nM (apparent Michaelis constant approximately 23 nM), with Hill coefficients between 1.1 and 1.5. The apparent reaction order for NO consumption by sGC is dependent on NO concentration, and varies between 0 and 1.5. Finally, the activation of sGC (half-life approximately 1-2 s) is much more rapid than deactivation (approximately 50 s). We conclude that control of sGC in vivo is most likely ultra-sensitive, and that activation in vivo occurs at lower NO concentrations than previously reported.     

Human cytomegalovirus pp71: a new viral tool to probe the mechanisms of cell cycle progression and oncogenesis controlled by the retinoblastoma family of tumor suppressors

The DNA tumor virus oncogenes (adenovirus E1A, simian virus 40 (SV40) large T antigen, and papillomavirus E7) have been instrumental in illuminating the molecules and mechanisms of cell cycle progression and carcinogenesis. However, since these multifunctional proteins target so many important cellular regulators, it is sometimes difficult to establish the functional importance of any individual interaction. Perhaps a herpesvirus protein, newly defined as a cell cycle regulator, can help address these issues. Like the DNA tumor virus proteins, the human cytomegalovirus (HCMV) pp71 protein contains a retinoblastoma protein (Rb) binding motif (LxCxD), and stimulates DNA synthesis in quiescent cells. Unlike E1A, T antigen, and E7, pp71 expression does not induce apoptosis, nor does it cooperate to transform primary cells. Determining how pp71 induces cell cycle progression without invoking apoptosis or leading to cellular transformation may help in defining the signals that ultimately lead to these processes.     

Induction of humoral and cellular immune responses in mice by a recombinant fowlpox virus expressing the E2 protein of bovine viral diarrhea virus

A recombinant fowlpox virus (rFPV/E2) expressing the E2 protein of bovine viral diarrhea virus (BVDV) was constructed and characterized. Mice were immunized with recombinant virus and both humoral and cellular immune responses were studied. rFPV/E2 induced BVDV-specific antibodies which were detected by ELISA. In addition, mouse sera were shown to neutralize BVDV. A cytokine ELISA assay revealed that mice vaccinated with rFPV/E2 induced 7-fold more interferon-gamma than parental fowlpox virus.     

In vivo topological analysis of Ste2, a yeast plasma membrane protein, by using beta-lactamase gene fusions.

Gene fusions were constructed between Ste2, the receptor for the Saccharomyces cerevisiae alpha-factor, and beta la, the secreted form of beta-lactamase encoded by the bla gene of pBR322. The Ste2 and beta la components were linked by a processing fragment (P) from the yeast killer preprotoxin containing a C-terminal lysine-arginine site for cleavage by the Golgi-associated Kex2 protease. Ste2 is predicted to have a rhodopsinlike topology, with an external N terminus and seven transmembrane segments. Fusions to three of the four Ste2 domains predicted to be external resulted in beta la secretion from yeast cells. A fusion at a site just preceding the first transmembrane segment was an exception; the product was cell associated, indicating that the first 44 residues of Ste2 are insufficient to direct secretion of beta la; translocation of this domain presumably requires the downstream transmembrane segment. Expression of fusions located in two domains predicted to be cytoplasmic failed to result in beta la secretion. Following insertion of the preprotoxin signal peptide (S) between the Ste2 and P components of these cytoplasmic fusions, secretion of beta la activity occurred, which is consistent with inversion of the orientation of the beta la reporter. Conversely, insertion of S between Ste2 and P in an external fusion sharply reduced beta la secretion. Complementary information about both cytoplasmic and external domains of Ste2 was therefore provided, and most aspects of the predicted topology were confirmed. The steady-state levels of beta la detected were low, presumably because of efficient degradation of the fusions in the secretory pathway; levels, however, were easily detectable. This method should be valuable in the analysis of in vivo topologies of both homologous and foreign plasma membrane proteins expressed in yeast cells.     

In vivo analysis of scaffold-associated regions in Drosophila: a synthetic high-affinity SAR binding protein suppresses position effect variegation.

Scaffold-associated regions (SARs) were studied in Drosophila melanogaster by expressing a synthetic, high-affinity SAR-binding protein called MATH (multi-AT-hook), which consists of reiterated AT-hook peptide motifs; each motif is known to recognize a wide variety of short AT-rich sequences. MATH proteins were expressed specifically in the larval eye imaginal discs by means of the tetracycline-regulated transactivation system and tested for their effect on position effect variegation (PEV). MATH20, a highly potent SAR ligand consisting of 20 AT-hooks, was found to suppress whitemottled 4 variegation. This suppression required MATH20 expression at an early larval developmental stage. Our data suggest an involvement of the high AT-rich SARs in higher order chromatin structure and gene expression.     

In vivo DNA gene electro-transfer: a systematic analysis of different electrical parameters

BACKGROUND: Intramuscular plasmid injection followed by electroporation is an efficient method for gene therapy or vaccination. Several protocols have been described that give good transduction levels with several reporter genes. METHODS: In this work we have explored the efficiency of gene delivery upon variation of the different electrical parameters such as pulse length frequency and voltage monitoring both on short- and long-term protein production. RESULTS: Having defined the best performing parameters, we have designed a short electric treatment that gives good levels of plasmid-encoded protein in different species such as mice, rabbits and monkeys.     

In vivo reconstitution of an active siderophore transport system by a binding protein derivative lacking a signal sequence

Transport of iron(III) hydroxamates across the inner membrane ofEscherichia coli depends on a binding protein-dependent transport system composed of the FhuB,C and D proteins. The FhuD protein, which is synthesized as a precursor and exported through the cytoplasmic membrane, represents the periplasmic binding protein of the system, accepting as substrates a number of hydroxamate siderophores and the antibiotic albomycin. A FhuD derivative, carrying an N-terminal His-tag sequence instead of its signal sequence and therefore not exported through the inner membrane, was purified from the cytoplasm. Functional activity, comparable to that of wild-type FhuD, was demonstrated for this His-tag-FhuD in vitro by protease protection experiments in the presence of different substrates, and in vivo by reconstitution of iron transport in afhuD mutant strain. The experimental data demonstrate that the primary sequence of the portion corresponding to the mature FhuD contains all the information required for proper folding of the polypeptide chain into a functional solute-binding protein. Moreover, purification of modified periplasmic proteins from the cytosol may be a useful approach for recovery of many polypeptides which are normally exported across the inner membrane and can cause toxicity problems when overproduced.     

In vivo fragment complementation of a (beta/alpha)(8) barrel protein: generation of variability by recombination

The high representation of the TIM barrel as a scaffold for enzymatic proteins makes it an interesting model for protein engineering. Based on previous reports of folding mechanisms of TIM barrels that suggest an independent folding unit formed by six (beta/alpha) subunits, we interrupted the gene of phosphoribosylanthranilate isomerase (PRAI) from Escherichia coli at three different positions to yield fragments with different combinations of (beta/alpha) subunits. When these constructions were expressed as polycistrons in a TrpF-E. coli strain, complementation of the function only occurred with fragments beta1-alpha4 and beta5-alpha8, demonstrating that (beta/alpha)(4) subunits are stable enough to survive in vivo conditions and to assemble to yield a functional enzyme. The expression of these fragments in a separated plasmid/phagemid system to complement the function gave a slower complementation in the TrpF-E. coli strain; this was overcome by introducing extra secondary elements to the structure that reinforce their interaction.