Evasion of innate and adaptive immunity by flaviviruses

After a virus infects an animal, antiviral responses are generated that attempt to prevent dissemination. Interferons, antibody, complement, T and natural killer cells all contribute to the control and eradication of viral infections. Most flaviviruses, with the exception of some of the encephalitic viruses, cause acute disease and do not establish persistent infection. The outcome of flavivirus infection in an animal is determined by a balance between the speed of viral replication and spread, and the immune system response. Although many of the mechanistic details require further elucidation, flaviviruses have evolved specific tactics to evade the innate and adaptive immune response. A more thorough understanding of these principles could lead to improved models for viral pathogenesis and to strategies for the development of novel antiviral agents.     

Inhibition of bacterial RNase P by aminoglycoside-arginine conjugates

The potential of RNAs and RNA-protein (RNP) complexes as drug targets is currently being explored in various investigations. For example, a hexa-arginine derivative of neomycin (NeoR) and a tri-arginine derivative of gentamicin (R3G) were recently shown to disrupt essential RNP interactions between the trans-activator protein (Tat) and the Tat-responsive RNA (trans-activating region) in the human immunodeficiency virus (HIV) and also inhibit HIV replication in cell culture. Based on certain structural similarities, we postulated that NeoR and R3G might also be effective in disrupting RNP interactions and thereby inhibiting bacterial RNase P, an essential RNP complex involved in tRNA maturation. Our results indicate that indeed both NeoR and R3G inhibit RNase P activity from evolutionarily divergent pathogenic bacteria and do so more effectively than they inhibit partially purified human RNase P activity.     

Neutrophil-bead collision assay: pharmacologically induced changes in membrane mechanics regulate the PSGL-1/P-selectin adhesion lifetime

Visualization of flowing neutrophils colliding with adherent 1-mum-diameter beads presenting P-selectin allowed the simultaneous measurement of collision efficiency (epsilon), membrane tethering fraction (f), membrane tether growth dynamics, and PSGL-1/P-selectin binding lifetime. For 1391 collisions analyzed over venous wall shear rates from 25 to 200 s(-1), epsilon decreased from 0.17 to 0.004, whereas f increased from 0.15 to 0.70, and the average projected membrane tether length, L(tether)(m), increased from 0.35 mum to approximately 2.0 mum over this shear range. At all shear rates tested, adhesive collisions lacking membrane tethers had average bond lifetimes less than those observed for collisions with tethers. For adhesive collisions that failed to form membrane tethers, the regressed Bell parameters (consistent with single bond Monte Carlo simulation) were zero-stress off-rate, k(off)(0) = 0.56 s(-1) and reactive compliance, r = 0.10 nm, similar to published atomic force microscopy (AFM) measurements. For all adhesion events (+/- tethers), the bond lifetime distributions were more similar to those obtained by rolling assay and best simulated by Monte Carlo with the above Bell parameters and an average of 1.48 bonds (n = 1 bond (67%), n = 2 (22%), and n = 3-5 (11%)). For collisions at 100 s(-1), pretreatment of neutrophils with actin depolymerizing agents, latrunculin or cytochalasin D, had no effect on epsilon, but increased L(tether)(m) by 1.74- or 2.65-fold and prolonged the average tether lifetime by 1.41- or 1.65-fold, respectively. Jasplakinolide, an actin polymerizing agent known to cause blebbing, yielded results similar to the depolymerizing agents. Conversely, cholesterol-depletion with methyl-beta-cyclodextrin or formaldehyde fixation had no effect on epsilon, but reduced L(tether)(m) by 66% or 97% and reduced the average tether lifetime by 30% or 42%, respectively. The neutrophil-bead collision assay combines advantages of atomic force microscopy (small contact zone), aggregometry (discrete interactions), micropipette manipulation (tether visualization), and rolling assays (physiologic flow loading). Membrane tether growth can be enhanced or reduced pharmacologically with consequent effects on PSGL-1/P-selectin lifetimes.     

Phosphorylation of cold shock domain/Y-box proteins by ERK2 and GSK3beta and repression of the human VEGF promoter

The hypoxia responsive region (HRR) of the VEGF promoter plays a key role in regulating VEGF expression. We found that the cold shock domain (Y-box) repressor proteins, dbpA and dbpB/YB-1, bind distinct strands of the human VEGF HRR. We find both dbpA and dbpB are phosphorylated by ERK2 and GSK3beta in vitro, and the binding of dbpB to single-strand VEGF HRR DNA is regulated by this phosphorylation. These findings suggest the ERK/MAPK and PI3K pathways may regulate VEGF expression in part through regulating the action of these repressor proteins.     

Structure-activity relationships of aminoglycoside-arginine conjugates that bind HIV-1 RNAs as determined by fluorescence and NMR spectroscopy

We present here a new set of aminoglycoside-arginine conjugates (AACs) that are either site-specific or per-arginine conjugates of paromomycin, neamine, and neomycin B as well as their structure-activity relationships. Their binding constants (KD) for TAR and RRE RNAs, measured by fluorescence anisotropy, revealed dependence on the number and location of arginines in the different aminoglycoside conjugates. The binding affinity of the per-arginine aminoglycosides to TAR is higher than to RRE, and hexa-arginine neomycin B is the most potent binder (KD=5 and 23 nM, respectively). The 2D TOCSY NMR spectrum of the TAR monoarginine-neomycin complex reveals binding at the bulge region of TAR.     

The dynamics of autophagy visualized in live cells: from autophagosome formation to fusion with endo/lysosomes

Autophagy has been implicated in a range of disorders and hence is of major interest. However, imaging autophagy in real time has been hampered by lack of suitable markers. We have compared the potential of monodansylcadaverine, widely used as an autophagosomal marker, and the Atg8 homologue LC3, to follow autophagy by fluorescence microscopy whilst labelling late endosomes and lysosomes simultaneously using EGFP-CD63. Monodansylcadaverine labelled only acidic CD63-positive compartments in response to a range of autophagic inducers in various live or post-fixed cells, staining being identical in atg5(+/+) and atg5(-/-) MEFs in which autophagosome formation is disabled. Monodansylcadaverine staining was essentially indistinguishable from that of LysoTracker Red, LAMP-1 or LAMP-2. In contrast, 60-90% of EGFP-LC3-positive punctate organelles did not colocalise with LAMP-1/LAMP-2/CD63 and were monodansylcadaverine-negative while EGFP-LC3 puncta that did colocalise with LAMP-1/LAMP-2/CD63 were also monodansylcadaverine-positive. Hence monodansylcadaverine is no different from other markers of acidic compartments and it cannot be used to follow autophagosome formation. In contrast, fusion of mRFP-LC3-labelled autophagosomes with EGFP-CD63-positive endosomes and lysosomes and sequestration of dsRed-labelled mitochondria by EGFP-LC3- and EGFP-CD63-positive compartments could be visualized in real time. Moreover, transition of EGFP-LC3-I (45 kDa) to EGFP-LC3-II (43 kDa)-traced by immunoblotting and verified by [(3)H]ethanolamine labelling-revealed novel insights into the dynamics of autophagosome homeostasis, including the rapid activation of autophagy by the apoptotic inducer staurosporine prior to apoptosis proper. Use of fluorescent LC3 and a counter-fluorescent endosomal/lysosomal protein clearly allows the entire autophagic process to be followed by live cell imaging with high fidelity.     

Recombinant modified vaccinia virus Ankara expressing a soluble form of glycoprotein B causes durable immunity and neutralizing antibodies against multiple strains of human cytomegalovirus

Human cytomegalovirus (CMV) is a viral pathogen that infects both genders, who remain asymptomatic unless they receive immunosuppressive drugs or acquire infections that cause reactivation of latent virus. CMV infection also causes serious birth defects following primary maternal infection during gestation. A safe and effective vaccine to limit disease in this population continues to be elusive. A well-studied antigen is glycoprotein B (gB), which is the principal target of neutralizing antibodies (NAb) towards CMV in humans and has been implicated as the viral partner in the receptor-mediated infection by CMV in a variety of cell types. Antibody-mediated virus neutralization has been proposed as a mechanism by which host immunity could modify primary infection. Towards this goal, an attenuated poxvirus, modified vaccinia virus Ankara (MVA), has been constructed to express soluble CMV gB (gB680-MVA) to induce CMV NAb. Very high levels of gB-specific CMV NAb were produced after two doses of the viral vaccine. NAb were durable within a twofold range for up to 6 months. Neutralization titers developed in immunized mice are equivalent to titers found clinically after natural infection. This viral vaccine, expressing gB derived from CMV strain AD169, induced antibodies that neutralized CMV strains of three different genotypes. Remarkably, preexisting MVA and vaccinia virus (poxvirus) immunity did not interfere with subsequent immunizations of gB680-MVA. The safety characteristics of MVA, combined with the robust immune response to CMV gB, suggest that this approach could be rapidly translated into the clinic.     

FIE and CURLY LEAF polycomb proteins interact in the regulation of homeobox gene expression during sporophyte development

The Arabidopsis FERTILIZATION-INDEPENDENT ENDOSPERM (FIE) polycomb group (PcG) protein, a WD40 homologue of Drosophila extra sex comb (ESC), regulates endosperm and embryo development and represses flowering during embryo and seedling development. As fie alleles are not transmitted maternally, homozygous mutant plants cannot be obtained. To study FIE function during the entire plant life cycle, we used Arabidopsis FIE co-suppressed plants. Low FIE level in these plants produced dramatic morphological aberrations, including loss of apical dominance, curled leaves, early flowering and homeotic conversion of leaves, flower organs and ovules into carpel-like structures. These morphological aberrations are similar to those exhibited by plants overexpressing AGAMOUS (AG) or CURLY LEAF (clf) mutants. Furthermore, the aberrant leaf morphology of FIE-silenced and clf plants correlates with de-repression of the class I KNOTTED-like homeobox (KNOX) genes including KNOTTED-like from Arabidopsis thaliana 2 (KNAT2) and SHOOTMERISTEMLESS (STM), whereas BREVIPEDICELLUS (BP) was upregulated in FIE-silenced plants, but not in the clf mutant. Thus, FIE is essential for the control of shoot and leaf development. Yeast two-hybrid and pull-down assays demonstrate that FIE interacts with CLF. Collectively, the morphological characteristics, together with the molecular and biochemical data presented in this work, strongly suggest that in plants, as in mammals and insects, PcG proteins control expression of homeobox genes. Our findings demonstrate that the versatility of the plant FIE function, which is derived from association with different SET (SU (VAR)3-9, E (Z), Trithorax) domain PcG proteins, results in differential regulation of gene expression throughout the plant life cycle.