Expression of Measles Virus Nucleoprotein Induces Apoptosis and Modulates Diverse Functional Proteins in Cultured Mammalian Cells

Background: Measles virus nucleoprotein (N) encapsidates the viral RNA, protects it from endonucleases and forms a virus specific template for transcription and replication. It is the most abundant protein during viral infection. Its C-terminal domain is intrinsically disordered imparting it the flexibility to interact with several cellular and viral partners. Principal Findings: In this study, we demonstrate that expression of N within mammalian cells resulted in morphological transitions, nuclear condensation, DNA fragmentation and activation of Caspase 3 eventuating into apoptosis. The rapid generation of intracellular reactive oxygen species (ROS) was involved in the mechanism of cell death. Addition of ascorbic acid (AA) or inhibitor of caspase-3 in the extracellular medium partially reversed N induced apoptosis. We also studied the protein profile of cells expressing N protein. MS analysis revealed the differential expression of 25 proteins out of which 11 proteins were up regulated while 14 show signs of down regulation upon N expression. 2DE results were validated by real time and semi quantitative RT-PCR analysis. Conclusion: These results show the pro-apoptotic effects of N indicating its possible development as an apoptogenic tool. Our 2DE results present prima facie evidence that the MV nucleoprotein interacts with or causes differential expression of a wide range of cellular factors. At this stage it is not clear as to what the adaptive response of the host cell is and what reflects a strategic modulation exerted by the virus

Inhibition of apoptosis by AA and Z-VAD-FMK.

<p>(a) Percentage of apoptosis in control N expressing cells analyzed by flow cytometry after PI staining. Apoptotic cells were estimated by the percentage of cells in the sub-G1 peak. Each bar represents the mean ± SD of three independent experiments. (b) Nuclear staining with hoechst 33258. N transfected MCF7 cells showed apoptotic morphology; DNA condensation and nuclear fragmentation whereas rest of the cells remained uniformly stained with round and unpunctuated nucleus. White arrows indicate nuclear condensation and fragmentation. The figures represent one of the three independent experiments. (c) Single Cell Gel Electrophoresis assessment of N toxicity in human breast cancer cells (MCF7). Cells were harvested for comet tail formation assays under alkaline conditions. Comet images were captured using fluorescence microscopy, and tail moment was analyzed in 50–60 randomly chosen comets using Comet assay IV software. Representative comet images observed are shown. Histograms represent changes in the comet tail moments between control and treated cells. N transfected cells show a long tail indicating DNA damage. Each experiment was done in triplicate. Data is represented as means ±SD.</p

List of oligonucleotides used in this study.

<p>List of oligonucleotides used in this study.</p

N induces ROS production and caspase 3 activation in MCF7 and 293T cells.

<p>(a) N and P expression in MCF-7 cells. 24 h after transfection, total RNA was extracted from the cells and RT-PCR was performed as described in methods. Gel pictures showing expression of N and P. (b) Analysis of ROS generation by DCF fluorescence as described in Methods. Representative fluorescent images and corresponding mean fluorescence intensity. (c) Determination of caspase 3 activation in MCF7 cells by FITC fluorescence as described in Methods. Results are expressed as the fold increase in fluorescence and are given as the mean ± SD for three experiments.</p

Typical 2D gel analysis of N transfected cells compared with control cells.

<p>Cells were transfected with either pCA or pCA-N and 24 h post transfection total cellular protein was extracted. Protein extracts were subjected to separation by 2DE prior to staining and visualization as described under methods. (a) Representative 2D gels of control and treated samples. Encircled proteins were consistently seen to vary in intensity in multiple experiments. Those spots were picked for sequencing. (b) The MALDI-TOF-MS mass spectrum of a spot, identified as the Poly(rC) binding protein 1 according to the matched peaks is shown.</p

N induces cell death in MCF7 cells.

<p>(a) Immunoblot analysis of N expression in MCF7 cells. (b) Representative fluorescent images of cells co transfected with GFP and N expression vectors showing 70–80% transfection efficiency. (c) Morphological features of MCF7 cells expressing N as seen by light microscopy. (d) Flow cytometric profile of representative cell populations 24 h post transfection, fixed and PI stained for cell cycle analysis as described in methods. The data is representative of three independent experiments.</p

Validation of 2DE by real time and semi quantitative RT-PCR.

<p>24 h after transfection, total RNA was extracted from the cells and real time and semi quantitative RT-PCR was performed as described in methods. (a) Gel pictures showing differential expression of PARK7 and PHB. Fold change in expression was analyzed by software ImageJ. Data was normalized with beta actin as the control house keeping gene. (b) Relative quantification of GAPDH and MAP2K4 mRNA in N transfected and control MCF7 cells. The change in gene expression is expressed as fold change in relation to control. Results are presented as the mean ± SD from 3 different experiments.</p

Decoding the rare diagnostic enigma of pythium insidiosum keratitis – A narrative review

Pythium insidiosum, an oomycete, is frequently referred to as a “parafungus” due to its striking similarity to fungal keratitis. Over the past 10 years, there has been a significant rise in Pythium insidiosum keratitis (PIK) cases, notably in Asia and India. This trend is potentially attributable to heightened research interest in this microorganism and advancements in diagnostic and therapeutic techniques. Clinically, its features, such as subepithelial infiltration, fluffy stromal infiltrates resembling cotton wool, satellite lesions, corneal perforation, endoexudates, and anterior chamber hypopyon, mirror those of fungal infections. However, hallmark clinical features like reticular dots, tentacular extensions, peripheral indentation, and prompt limbal progression distinguish Pythium from other pathogens and necessitate vigilant clinical scrutiny. From a morphological and microbiological perspective, Pythium closely mirrors fungi when subjected to routine smear tests, often revealing perpendicular or obtuse septate or non-septate branching hyphae. Culturing on nutritional media like blood agar, chocolate agar, and so on remains the diagnostic gold standard, with the organism presenting as cream-white colonies that form zoospores. This can be further verified through the leaf incarnation technique. Culture and polymerase chain reaction (PCR) are the gold standard diagnostic modalities for PIK. Historically, due to constraints in diagnostic techniques and the slow growth of cultures, there has been a trend toward employing advanced molecular diagnostic tools, including PCR, confocal microscopy, enzyme-linked immunosorbent assay, and immunodiffusion. When corneal scraping tests (10% KOH, Gram stain) depict fungal-like hyphae, antifungal treatments are often initiated even before culture results are ready. However, recent molecular research recommends the use of antibacterials, specifically 0.2% linezolid and 1% azithromycin, as the primary therapeutic agents. In cases that do not respond to treatment, early therapeutic keratoplasty is advised. This narrative review aims to provide a comprehensive overview of the epidemiology, clinical manifestations, laboratory and molecular diagnostic procedures, and therapeutic approaches for PIK