Intracellular nucleic acid delivery by the supercharged dengue virus capsid protein

© 2013 Freire et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.Supercharged proteins are a recently identified class of proteins that have the ability to efficiently deliver functional macromolecules into mammalian cells. They were first developed as bioengineering products, but were later found in the human proteome. In this work, we show that this class of proteins with unusually high net positive charge is frequently found among viral structural proteins, more specifically among capsid proteins. In particular, the capsid proteins of viruses from the Flaviviridae family have all a very high net charge to molecular weight ratio (> +1.07/kDa), thus qualifying as supercharged proteins. This ubiquity raises the hypothesis that supercharged viral capsid proteins may have biological roles that arise from an intrinsic ability to penetrate cells. Dengue virus capsid protein was selected for a detailed experimental analysis. We showed that this protein is able to deliver functional nucleic acids into mammalian cells. The same result was obtained with two isolated domains of this protein, one of them being able to translocate lipid bilayers independently of endocytic routes. Nucleic acids such as siRNA and plasmids were delivered fully functional into cells. The results raise the possibility that the ability to penetrate cells is part of the native biological functions of some viral capsid proteins.This work was supported by Fundação para a Ciência e Tecnologia – Ministério da Educação e Ciência (FCT-MEC, Portugal) [PTDC/QUI-BIQ/112929/2009], by the European Union [projects FP7-PEOPLE IRSES (MEMPEPACROSS) and FP7-HEALTH-F3-2008-223414 (LEISHDRUG)], by the Spanish Ministry of Economy and Competitiveness (SAF2011-24899), the Generalitat de Catalunya (2009 SGR 492), by the Brazilian Conselho Nacional de Desenvolvimento Científico e Tecnoloógico (CNPq), Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ), and the National Institute of Science and Technology in Dengue (INCT-Dengue). JMF also acknowledges FCT-MEC for Ph.D. fellowship SFRH/BD/70423/2010

Pregnant women co-infected with HIV and Zika: Outcomes and birth defects in infants according to maternal symptomatology.

BACKGROUND:Zika virus (ZIKV) was first isolated in Uganda in 1947. In Brazil, the first reported case of ZIKV infection was in May 2015. Additionally, dengue (DENV) is endemic and there has been a recent outbreak of chikungunya (CHIKV). Since the clinical manifestations of different arboviral infections (AI) can be similar, definitive diagnosis requires laboratory testing. OBJECTIVES:To determine the prevalence of ZIKV, DENV, and CHIKV infections in a Brazilian cohort of HIV-infected pregnant women, to assess clinical/immunological characteristics and pregnancy outcomes of women with evidence of recent AI. STUDY DESIGN:Laboratory diagnosis of ZIKV, DENV and CHIKV infections utilized serological assays, RT-PCR and PRNT. The tests were performed at the first visit, 34-36 weeks of gestation and at any time if a woman had symptoms suggestive of AI. Mann-Whitney tests were used for comparison of medians, Chi-square or Fisher's to compare proportions; p< 0.05 was considered statistically significant. Poisson regression was used to analyze risk factors for central nervous system (CNS) malformations in the infant according to maternal symptomatology. RESULTS:Of 219 HIV-infected pregnant women enrolled, 92% were DENV IgG+; 47(22%) had laboratory evidence of recent AI. Of these, 34 (72%) were ZIKV+, nine (19%) CHIKV+, and two (4%) DENV+. Symptoms consistent with AI were observed in 23 (10%) women, of whom 10 (43%) were ZIKV+, eight (35%) CHIKV+. No CNS abnormalities were observed among infants of DENV+ or CHIKV+ women; four infants with CNS abnormalities were born to ZIKV+ women (three symptomatic). Infants born to ZIKV+ women had a higher risk of CNS malformations if the mother was symptomatic (RR = 7.20), albeit not statistically significant (p = 0.066). CONCLUSIONS:Among HIV-infected pregnant women with laboratory evidence of a recent AI, 72% were ZIKV-infected. In this cohort, CNS malformations occurred among infants born to both symptomatic and asymptomatic pregnant women with Zika infection

Viral structural proteins as supercharged proteins.

<p><b>A)</b> Plot of formal net charge to Molecular Weight (M_W) ratio <i>vs.</i> formal net charge of viral structural proteins (Capsid, Membrane and Envelope) annotated at Viralzone (<a href="http://viralzone.expasy.org" target="_blank">http://viralzone.expasy.org</a>). The yellow background region of the plot is the area with formal net charge/M_W exceeding +0.75/kDa, i.e. the region comprising the supercharged proteins <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081450#pone.0081450-Thompson1" target="_blank">[2]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081450#pone.0081450-McNaughton1" target="_blank">[12]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081450#pone.0081450-Cronican2" target="_blank">[14]</a>. A viral protein net charge/M_W trend line is plotted as a guide to the eye to facilitate the identification of the most significant supercharged proteins. Capsid proteins from flaviviruses (yellow solid circles) clearly form a subgroup of supercharged proteins. <b>B)</b> Linear sequence of DENV C protein monomer and structural representation of the dimer (PDB ID: 1R6R). Hydrophobic (MBS) and cationic (RBS) conserved regions are highlighted in yellow and green, respectively. The surface charge distribution of DENV C protein is represented: basic residues (blue), hydrophobic (red) and all other residues (grey) are shown. Tridimensional representation obtained by the PyMol software <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081450#pone.0081450-DeLano1" target="_blank">[33]</a>.</p

Quantification of DENV C protein-mediated translocation of ssDNA, GFP-encoding plasmid and TLR3 siRNA into cells.

<p><b>A)</b> Flow cytometry fluorescence distribution histograms of trypsinized BHK-21 cells incubated with ssDNA-Alexa488 in the presence or in the absence (control) of DENV C protein. The percentage of BHK-21 cells positive for DENV C protein-mediated ssDNA internalization is indicated. <b>B)</b> Real time-flow cytometry of DENV C-mediated ssDNA intracellular delivery in BHK-21 cells at 4°C (black) or 37°C (green). The average fluorescence intensity signal of ssDNA is plotted over time. <b>C)</b> DENV C-mediated ssDNA delivery was also carried out on astrocytes at 37°C and 4°C, and imaged by confocal microscopy. Nuclear staining with Hoechst 33342 at 1 mg.mL⁻¹– blue. <b>D)</b> Confocal imaging of BHK-21 cells cultured for one day and then transfected with pEGFP-C3 using DENV C protein or Lipofectamine (control – no transfecting agent). Positive GFP expression (green) was imaged 48 h after transfection. Nuclear staining with Hoechst 33342 at 1 mg.mL⁻¹– blue, as in C). The right panel shows GFP fluorescence excitation (dashed lines) and emission (solid lines) spectra in BHK-21cells suspensions after Lipofectamine- (red) and DENV C-mediated (green) transfections (control – black). <b>E)</b> Percentage of GFP-positive cells, quantified by flow cytometry after transfection with Lipofectamine (red), Tat (black and white) or DENV C protein (green). Flow cytometry histograms of BHK-21 cells after transfection with Lipofectamine (red) or DENV C protein (green) are shown on the right. The fraction of GFP-positive cells is indicated. Statistical analysis was carried out using the two-tailed <i>t-</i>test (n.s. – non-significant; *** – p<0.0003)<b>. F)</b> TLR3 gene silencing assay using siRNA sc-36685 in BMEC. Transfection was performed using Lipofectamine (red), DENV C protein (green), pepM (blue) or pepR (orange). TLR3 gene silencing was analyzed by real-time PCR 48 h after transfection. Results reflect TLR3 expression changes in the presence and absence of the TLR3 siRNA for each transfection agent tested. Relative expression values <i>vs.</i> control (absence of siRNA) were analyzed by the two-tailed <i>t</i>-test (***– p<0.0007; * – p<0.025). Relative expression values <i>vs</i>. Lipofectamine were also analyzed (n.s. – non-significant; # – p<0.04). The resulting data are the mean of three experiments with standard deviation.</p

DENV C protein and DENV C protein-derived peptides pepR and pepM interaction with lipid vesicles.

<p><b>A)</b> Lipid membrane translocation of pepR (red; dashed black) and pepM (green; solid black) in lipid vesicles of POPC:POPG 4∶1 (MLV – black; LUV – colored). <b>B)</b> Lipid membrane translocation of pepR-ssDNA (orange; dashed black) or pepM-ssDNA (blue; solid black) in lipid vesicles of POPC:POPG 4∶1 (MLV – black; LUV – colored). In <b>C, E, G)</b> LUV composed of POPC:POPG 4∶1 (triangles), or POPC:POPE 4∶1 (squares) were titrated with C) DENV C protein (black), E) pepR:ssDNA or G) pepM-ssDNA complexes (red and green, respectively) up to 36 µM, with a 4∶1 peptide:ssDNA molar ratio, at pH 7.4. The percentage of vesicle fusion dependence on concentration is represented. In <b>D, F, H)</b> ζ-potential of LUV composed of POPC (circles) or POPC:POPG (4∶1- triangles; 3∶2 - squares; 2∶3 - stars) titrated with D) DENV C protein, F) pepR or G) pepM up to 6, 32 and 100 µM, respectively, at pH 7.4. ζ = 0 is the point of vesicle surface electroneutrality.</p

DENV C protein-mediated delivery of ssDNA into different cells.

<p>Confocal imaging of BHK-21 cells, astrocytes and HepG2 cells. Cells were cultured for two days and then stained with Hoechst 33342 at 1 mg.mL⁻¹ (blue – nuclei), followed by the addition of ssDNA-Alexa488 at 1 µM (green) and unlabeled DENV C protein (3 µM).</p

Cell delivery of ssDNA mediated by DENV C protein-derived peptides (pepR and pepM).

<p><b>A)</b> Confocal imaging of BHK-21 cells. Cells were cultured for two days and then stained with Hoechst 33342 at 1 mg.mL⁻¹ (blue – nuclei), followed by the addition of 5 µM of either pepR-rhodamineB or pepM-rhodamineB (red) conjugated with 1 µM of ssDNA-Alexa488. <b>B)</b> Flow cytometry quantification of the cellular internalization of ssDNA labeled with Alexa488, by using pepM, pepR, DENV C protein or Tat as delivery vectors, in BHK-21 cells.</p

Characterization of a porin channel in the endosymbiont of the trypanosomatid protozoan Crithidia deanei.

Crithidia deanei is a trypanosomatid protozoan that harbours a symbiotic bacterium. The partners maintain a mutualistic relationship, thus constituting an excellent model for studying metabolic exchanges between the host and the symbiont, the origin of organelles and cellular evolution. According to molecular analysis, symbionts of different trypanosomatid species share high identity and descend from a common ancestor, a β-proteobacterium of the genus Bordetella. The endosymbiont is surrounded by two membranes, like Gram-negative bacteria, but its envelope presents special features, since phosphatidylcholine is a major membrane component and the peptidoglycan layer is highly reduced, as described in other obligate intracellular bacteria. Like the process that generated mitochondria and plastids, the endosymbiosis in trypanosomatids depends on pathways that facilitate the intensive metabolic exchanges between the bacterium and the host protozoan. A search of the annotated symbiont genome database identified one sequence with identity to porin-encoding genes of the genus Bordetella. Considering that the symbiont outer membrane has a great accessibility to cytoplasm host factors, it was important to characterize this single porin-like protein using biochemical, molecular, computational and ultrastructural approaches. Antiserum against the recombinant porin-like molecule revealed that it is mainly located in the symbiont envelope. Secondary structure analysis and comparative modelling predicted the protein 3D structure as an 18-domain β-barrel, which is consistent with porin channels. Electrophysiological measurements showed that the porin displays a slight preference for cations over anions. Taken together, the data presented herein suggest that the C. deanei endosymbiont porin is phylogenetically and structurally similar to those described in Gram-negative bacteria, representing a diffusion channel that might contribute to the exchange of nutrients and metabolic precursors between the symbiont and its host cell.Journal ArticleResearch Support, Non-U.S. Gov'tSCOPUS: ar.jinfo:eu-repo/semantics/publishe