Development of a novel NS1 competitive enzyme-linked immunosorbent assay for the early detection of Zika virus infection

Zika virus (ZIKV) is a flavivirus that has emerged as a global health threat after the 2015 outbreak in the Americas, where devastating congenital defects were documented. There are currently no vaccines to prevent ZIKV infections nor commercially available clinical diagnostic tests demonstrated to identify ZIKV without cross-reactive interference of related flaviviruses. Early diagnosis is critical when treating symptomatic patients and in preventing ZIKV transmission. In this context, the development of sensitive and accurate diagnostic methods are urgently needed for the detection of ZIKV acute infection. The aim of this study consisted of obtaining monoclonal antibodies (mAbs) against denatured monomeric ZIKV Nonstructural protein 1 (ZNS1), a useful diagnostic marker for flavivirus early detection, in order to develop a highly specific and sensitive ZNS1 indirect competitive ELISA (icELISA). The production of hybridomas secreting ZNS1 mAbs was carried out through immunizations with denatured monomeric ZNS1. We selected 1F5 and 6E2 hybridoma clones, which recognized the heat-denatured ZNS1 hexameric form by indirect ELISA. Cross-reaction studies indicated that these mAbs specifically bind to a ZNS1 linear epitope, and that they do not cross-react with the NS1 protein from other related flaviviruses. The 1F5 mAb enabled the development of a sensitive and reproducible icELISA to detect and quantify small amounts of ZNS1 disease marker in heat-denatured human sera. Here, we establish a reliable 1F5 based-icELISA that constitutes a promising diagnostic tool for control strategies and the prevention of ZIKV propagation.Fil: Roldan, Julieta Suyay. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. - Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Cassola, Alejandro Carlos. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. - Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Castillo, Daniela Susana. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. - Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Biotecnológicas; Argentin

Optimization of recombinant Zika virus NS1 protein secretion from HEK293 cells

Sensitive, accurate and cost-effective diagnostic tests are urgently needed to detect Zika virus (ZIKV) infection. Nonstructural 1 (NS1) glycoprotein is an excellent diagnostic marker since it is released in a hexameric conformation from infected cells into the patient's bloodstream early in the course of the infection. We established a stable rZNS1-His-expression system in HEK293 cells through lentiviral transduction. A novel optimization approach to enhance rZNS1-His protein secretion in the mammalian expression system was accomplished through 50 nM rapamycin incubation followed by serum-free media incubation for 9 days, reaching protein yields of ∼10 mg/l of culture medium. Purified rZNS1-His hexamer was recognized by anti-NS1 antibodies in ZIKV patient's serum, and showed the ability to induce a humoral response in immunized mice. The obtained recombinant protein is a reliable biological tool that can potentially be applied in the development of diagnostic tests to detect ZIKV in infected patients during the acute phase.Fil: Roldan, Julieta Suyay. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. - Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Cassola, Alejandro Carlos. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. - Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Castillo, Daniela Susana. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. - Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Biotecnológicas; Argentin

Regulation of RNA binding proteins in trypanosomatid protozoan parasites

Posttranscriptional mechanisms have a critical role in the overall outcome of gene expression. These mechanisms are especially relevant in protozoa from the genus Trypanosoma, which is composed by death threatening parasites affecting people in Sub-saharan Africa or in the Americas. In these parasites the classic view of regulation of transcription initiation to modulate the products of a given gene cannot be applied. This is due to the presence of transcription start sites that give rise to long polycistronic units that need to be processed costranscriptionally by trans-splicing and polyadenylation to give mature monocistronic mRNAs. Posttranscriptional mechanisms such as mRNA degradation and translational repression are responsible for the final synthesis of the required protein products. In this context, RNA-binding proteins (RBPs) in trypanosomes have a relevant role as modulators of mRNA abundance and translational repression by associating to the 3’ untranslated regions in mRNA. Many different RBPs have been proposed to modulate cohorts of mRNAs in trypanosomes. However, the current understanding of their functions lacks a dynamic view on the different steps at which these RBPs are regulated. Here, we discuss different evidences to propose regulatory events for different RBPs in these parasites. These events vary from regulated developmental expression, to biogenesis of cytoplasmic ribonucleoprotein complexes in the nucleus, and condensation of RBPs and mRNA into large cytoplasmic granules. Finally, we discuss how newly identified posttranslational modifications of RBPs and mRNA metabolism-related proteins could have an enormous impact on the modulation of mRNA abundance. To understand these modifications is especially relevant in these parasites due to the fact that the enzymes involved could be interesting targets for drug therapy.Fil: Romaniuk, María Albertina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús). Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús); ArgentinaFil: Cervini Bohm, Gabriela Marta . Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús). Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús); ArgentinaFil: Cassola, Alejandro Carlos. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús). Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús); Argentin

Translational repression by an RNA-binding protein promotes differentiation to infective forms in Trypanosoma cruzi.

Trypanosomes, protozoan parasites of medical importance, essentially rely on post-transcriptional mechanisms to regulate gene expression in insect vectors and vertebrate hosts. RNA binding proteins (RBPs) that associate to the 3'-UTR of mature mRNAs are thought to orchestrate master developmental programs for these processes to happen. Yet, the molecular mechanisms by which differentiation occurs remain largely unexplored in these human pathogens. Here, we show that ectopic inducible expression of the RBP TcUBP1 promotes the beginning of the differentiation process from non-infective epimastigotes to infective metacyclic trypomastigotes in Trypanosoma cruzi. In early-log epimastigotes TcUBP1 promoted a drop-like phenotype, which is characterized by the presence of metacyclogenesis hallmarks, namely repositioning of the kinetoplast, the expression of an infective-stage virulence factor such as trans-sialidase, increased resistance to lysis by human complement and growth arrest. Furthermore, TcUBP1-ectopic expression in non-infective late-log epimastigotes promoted full development into metacyclic trypomastigotes. TcUBP1-derived metacyclic trypomastigotes were infective in cultured cells, and developed normally into amastigotes in the cytoplasm. By artificial in vivo tethering of TcUBP1 to the 3' untranslated region of a reporter mRNA we were able to determine that translation of the reporter was reduced by 8-fold, while its mRNA abundance was not significantly compromised. Inducible ectopic expression of TcUBP1 confirmed its role as a translational repressor, revealing significant reduction in the translation rate of multiple proteins, a reduction of polysomes, and promoting the formation of mRNA granules. Expression of TcUBP1 truncated forms revealed the requirement of both N and C-terminal glutamine-rich low complexity sequences for the development of the drop-like phenotype in early-log epimastigotes. We propose that a rise in TcUBP1 levels, in synchrony with nutritional deficiency, can promote the differentiation of T. cruzi epimastigotes into infective metacyclic trypomastigotes

Irreversible growth arrest in parasites ectopically expressing TcUBP1.

<p>(A and B) Growth curves of induced and non-induced epimastigotes expressing TcUBP1-GFP or GFP. Values represent mean ± SD of 5 independent replicates. (C) Growth curves of wt epimastigotes in the presence or absence of CHX. Values represent mean ± SD of 3 independent replicates. (D) The percentage of dead cells was determined by flow cytometry by incubating parasites with propidium iodide at 5 and 10 days after Tet addition. Values represent mean ± SD of 4 independent replicates. (E) Growth curves of induced and non-induced epimastigotes expressing TcUBP1-GFP or GFP. At day 5 of culture, Tet was removed by washing and fresh medium was added. Values represent mean ± SD of 4 independent replicates. (F) Kinetoplast repositioning was evaluated by microscopy. Cells were stained with DAPI. (G) Staining of TS-SAPA by immunofluorescence in induced early-log epimastigotes expressing TcUBP1-GFP, and after Tet wash. DNA was stained with DAPI, shown in cyan. Images are representative of 3 independent experiments.</p

Induced ectopic expression of TcUBP1-GFP in early-log phase epimastigotes promotes the development of metacyclic trypomastigotes features.

<p>(A) Immunofluorescence of infective metacyclic trypomastigotes showing staining with anti-TS-SAPA antibody, and non-infective epimastigotes showing no staining. (B) Western blot of epimastigotes (E) and metacyclic trypomastigotes (MT) protein extracts with the anti-TS-SAPA antibody. An extract from cell-derived trypomastigotes (T) served as a positive control. Tubulin immunoblot was used as loading control. (C) Staining of TS-SAPA by immunofluorescence in induced (10 days) or uninduced early-log epimastigotes expressing TcUBP1-GFP or GFP. DNA was stained with DAPI. Images are representative of 3 independent experiments. (D) Western blot of uninduced or induced epimastigote protein extracts ectopically expressing TcUBP1-GFP. As a positive control for the anti-TS-SAPA antibody we used a protein extract from cell-derived trypomastigotes (T). Tubulin blot served as a loading control. Image is representative of 4 independent experiments. Scale bars, 5 μm. (E) Survival of epimastigotes to lysis by human complement. TcUBP1-GFP uninduced or induced parasites were incubated in PBS supplemented with 10% fresh human serum for the defined time course. Microscopic analysis was performed at each time point without fixation, only motile parasites were considered alive. **P< 0.01 by Two-way Anova- test.</p

TcUBP1-GFP ectopic expression in epimastigotes.

<p>(A) Wt epimastigotes were incubated in TAU-3AAG medium and TcUBP1 expression was assessed by Western blot for four days. Tubulin blot served as loading control. Values represent mean ± SD of 3 independent replicates. TcUBP1/Tubulin before TAU incubation was considered as 1. A representative Western blot experiment is shown. **P< 0.01 by Anova-Dunnett test. (B) Levels of TcUBP1-GFP induced expression 48 hours after the addition of different concentrations of Tet determined by Immunoblot (IB), and compared to TcUBP1 endogenous levels using an anti-TcUBP1 antibody. The ratio between the sum (Σ) of TcUBP1 forms (endogenous and ectopic) and Tubulin before Tet addition was considered as 1. (C) Levels of GFP induced expression at different Tet concentrations detected with an anti-GFP antibody and compared to Tubulin levels. (D) Percentage of GFP positive epimastigotes before and after induction with Tet determined by flow cytometry. Values represent mean ± SD of 6 independent replicates. (E) Immunoblot from a time course analysis of TcUBP1-GFP induced expression using 0.05 μg/ml Tet, together with endogenous TcUBP1. Tubulin was used as a loading control. The ratio between the sum of TcUBP1 forms (endogenous and ectopic) and Tubulin before Tet addition was considered as 1. (F) Fluorescence microscopy of epimastigotes expressing TcUBP1-GFP, or GFP, after 5 days of induction. Magnifications highlight a red line connecting the base of the flagellum, the nucleus and the kinetoplast, evidencing the repositioning of the kinetoplast. DNA was stained with DAPI, shown in cyan. Images are representative of 6 independent experiments. Scale bars, 5 μm. (G) Parasites schemes represent approximate forms obtained from induced TcUBP1-GFP or GFP cultures. In these parasites, the angle formed by the flagellum, the nucleus and the kinetoplast (FNK angle) was measured. The mean FNK angle is shown with black lines, and the kinetoplast is placed in this mean position. The gray spectrum represents SD above and below the mean FNK angle. ****P<0.0001 by T test (TcUBP1 N = 64, GFP N = 58). (H) Pie chart representing the mean percentages of cells with orthogonal or normal kinetoplast position in parasites induced for the expression of TcUBP1-GFP or GFP after 5 days. Values were obtained from 4 independent experiments. (I) Time course quantitation of parasites displaying drop-like phenotype in induced TcUBP1-GFP and GFP cultures.</p

Tethering of <i>T</i>. <i>cruzi</i> RBPs to reporter mRNA.

<p>(A) Scheme of the DNA construct cloned in the pTEX vector. See <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1007059#sec009" target="_blank">Methods</a> section for details. (B) Normalization of reporter mRNA and luciferase activity values. See <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1007059#sec009" target="_blank">Methods</a> section for details. RNA: normalized luciferase mRNA, P: normalized RBP protein, L: normalized luciferase activity, A: reporter mRNA abundance, T: reporter mRNA translation. (C) <i>Trypanosoma cruzi</i> epimastigotes were transfected with the reporter construct in which different RBPs (TcPABP1, TcUBP1, TcRBP4, TcZFP2 and TcZFP3) were artificially tethered to the 3´ UTR of the reporter gene. The results indicate folds over values obtained with GFP as the tethered protein. Folds higher than 2 were considered significative (*). Results are shown for 3 or 4 independent experiments.</p

Translation repression in parasites ectopically expressing TcUBP1.

<p>(A) Wt parasites were incubated with CHX (50 μg/ml) or vehicle and then with puromycin (10 μg/ml) or vehicle for 30 minutes. Puromycin incorporation was determined by Immunoblot (IB) using an anti-puromycin antibody. Protein loading was monitored using an anti-tubulin antibody. Image is representative of 3 independent experiments. Proteins unspecifically detected by the anti-puromycin antibody in the absence of puromycin, are pointed with arrowheads. (B) GFP or TcUBP1-GFP induced or GFP uninduced parasites were cultured for 5 and 10 days. 3 x 10⁷ parasites were incubated with puromycin (10 μg/ml) or vehicle for 30 minutes. Puromycin incorporation was determined by immunoblot using an anti-puromycin antibody. Protein loading was monitored using an anti-tubulin antibody. The ratio between Puromycin fluorescence/Tubulin fluorescence (Puro/Tub) is shown in arbitrary units (AU). Images are representative of 4 independent experiments. Unspecific bands are pointed with arrowheads. (C) Left panel, polysome profiles of induced (green line) and uninduced TcUBP1-GFP (black line), and from induced GFP parasites (filled light blue profile) after 5 days of induction, in the presence of CHX. Right panel, a control polysome profile from wt parasites using CHX is shown (black line), together with the disruption of 80S ribosomes into 60S and 40S ribosomal subunits with EDTA as a specificity control (red line). (D) Percentage of parasites displaying mRNA granules under optimal starvation conditions. (E) Percentage of parasites displaying mRNA granules under suboptimal starvation conditions. (F) Microscopic images of epimastigotes expressing TcUBP1-GFP, or GFP, under suboptimal starvation conditions, together with poly(A) mRNA detection by FISH and staining of DNA with DAPI, shown in cyan. Images are representative of 5 different experiments. (G) Analysis of granule area. (H) Analysis of the number of mRNA granules per parasite. Values represent mean ± SD of 5 independent replicates. *P< 0.05 by Anova-Dunnett test. Scale bar, 5 μm.</p

TcUBP1-GFP ectopic expression in late-log phase epimastigotes promote full metacyclogenesis.

<p>(A) The percentage of metacyclic trypomastigotes was determined in uninduced or induced TcUBP1-GFP or GFP late-log epimastigotes cultures 5 days after addition of Tet. Values represent mean ± SD of 4 independent replicates. (B) TS-SAPA staining was assessed in metacyclic trypomastigotes derived from TcUBP1-GFP induced cultures (5 days). Images are representative of 3 independent experiments. (C) The percentage of metacyclics was determined in uninduced or induced TcUBP1-GFP or GFP late-log epimastigotes incubated in TAU-3AAG medium for 96 hs. (D) Staining for intracellular amastigotes in VERO cells 7 days after incubating with induced and uninduced TcUBP1-GFP cultures. (E) Percentage of infected VERO cells 7 days post-infection with parasites derived from induced or uninduced TcUBP1-GFP or GFP cultures. Values represent mean ± SD of 3 independent replicates. (F) Infected VERO cells with intracellular amastigotes 7 days post infection with TcUBP1-GFP cultures. Amastigotes were stained with mouse anti-<i>T</i>. <i>cruzi</i> before permeabilization (red) and with rabbit anti-<i>T</i>. <i>cruzi</i> after permeabilization (green). DNA was stained with DAPI, shown in cyan. Images are representative of 3 independent experiments. ***P< 0.001 by Anova-Tukey test. Scale bars, 5 μm. (G) Model for TcUBP1-induced morphological differentiation.</p