Top-Down and Bottom-Up Proteomics Methods to Study RNA Virus Biology

RNA viruses cause a wide range of human diseases that are associated with high mortality and morbidity. In the past decades, the rise of genetic-based screening methods and high-throughput sequencing approaches allowed the uncovering of unique and elusive aspects of RNA virus replication and pathogenesis at an unprecedented scale. However, viruses often hijack critical host functions or trigger pathological dysfunctions, perturbing cellular proteostasis, macromolecular complex organization or stoichiometry, and post-translational modifications. Such effects require the monitoring of proteins and proteoforms both on a global scale and at the structural level. Mass spectrometry (MS) has recently emerged as an important component of the RNA virus biology toolbox, with its potential to shed light on critical aspects of virus–host perturbations and streamline the identification of antiviral targets. Moreover, multiple novel MS tools are available to study the structure of large protein complexes, providing detailed information on the exact stoichiometry of cellular and viral protein complexes and critical mechanistic insights into their functions. Here, we review top-down and bottom-up mass spectrometry-based approaches in RNA virus biology with a special focus on the most recent developments in characterizing host responses, and their translational implications to identify novel tractable antiviral targets

Preventive effects of folic acid on Zika virus-associated poor pregnancy outcomes in immunocompromised mice.

Zika virus (ZIKV) infection may lead to congenital microcephaly and pregnancy loss in pregnant women. In the context of pregnancy, folic acid (FA) supplementation may reduce the risk of abnormal pregnancy outcomes. Intriguingly, FA may have a beneficial effect on the adverse pregnancy outcomes associated with ZIKV infection. Here, we show that FA inhibits ZIKV replication in human umbilical vein endothelial cells (HUVECs) and a cell culture model of blood-placental barrier (BPB). The inhibitory effect of FA against ZIKV infection is associated with FRα-AMPK signaling. Furthermore, treatment with FA reduces pathological features in the placenta, number of fetal resorptions, and stillbirths in two mouse models of in utero ZIKV transmission. Mice with FA treatment showed lower viral burden and better prognostic profiles in the placenta including reduced inflammatory response, and enhanced integrity of BPB. Overall, our findings suggest the preventive role of FA supplementation in ZIKV-associated abnormal pregnancy and warrant nutritional surveillance to evaluate maternal FA status in areas with active ZIKV transmission

Ebselen alleviates testicular pathology in mice with Zika virus infection and prevents its sexual transmission

<div><p>Despite the low case fatality, Zika virus (ZIKV) infection has been associated with microcephaly in infants and Guillain-Barré syndrome. Antiviral and vaccine developments against ZIKV are still ongoing; therefore, in the meantime, preventing the disease transmission is critical. Primarily transmitted by Aedes species mosquitoes, ZIKV also can be sexually transmitted. We used AG129 mice lacking interferon-α/β and -γ receptors to study the testicular pathogenesis and sexual transmission of ZIKV. Infection of ZIKV progressively damaged mouse testes, increased testicular oxidative stress as indicated by the levels of reactive oxygen species, nitric oxide, glutathione peroxidase 4, spermatogenesis-associated-18 homolog in sperm and pro-inflammatory cytokines including IL-1β, IL-6, and G-CSF. We then evaluated the potential role of the antioxidant ebselen (EBS) in alleviating the testicular pathology with ZIKV infection. EBS treatment significantly reduced ZIKV-induced testicular oxidative stress, leucocyte infiltration and production of pro-inflammatory response. Furthermore, it improved testicular pathology and prevented the sexual transmission of ZIKV in a male-to-female mouse sperm transfer model. EBS is currently in clinical trials for various diseases. ZIKV infection could be on the list for potential use of EBS, for alleviating the testicular pathogenesis with ZIKV infection and preventing its sexual transmission.</p></div

ZIKV infection increases testicular oxidative stress.

<p>(A) Schematic experimental design. Mice were subcutaneously infected in the footpad with 5x10⁴ PFU/mouse of ZIKV. Mice were intraperitoneally treated with the antioxidant ebselen (EBS; 10 mg/kg body weight/mouse/day) or solvent control on day 1–2 (group: 3-dpi), day 1–5 (group: 6-dpi), or day 1–6 (group: 9-dpi) after infection. Sperm was collected on 3, 6, and 9 dpi. (B) Intracellular reactive oxygen species (ROS) assay. ROS levels in sperms were measured by use of the OxiSelect intracellular ROS indicator, with H2O2-treated sperms as a positive control. Relative fluorescence intensity was determined by use of a fluorescence plate reader. (C) Relative fluorescence intensity of GPx4 to CellTracker. (D) Relative fluorescence intensity of SPATA18 to CellTracker. Data are mean ± SD (n = 6 mice or 5 random confocal fields/group). *P<0.05 and **P<0.01 by Kruskal-Wallis, Bonferroni post-hoc test.</p

EBS reduces testicular inflammatory response and leucocyte infiltration.

<p>AG129 mice subcutaneously infected in the footpad with 5x10⁴ PFU/mouse of ZIKV were intraperitoneally treated with EBS (10 mg/kg body weight/mouse/day) or solvent control on day 1–6 after infection. Testes and sperm were collected on 9 dpi. (A) Cytokine profile of seminal fluid on 9 dpi. Cytokine levels were evaluated by ELISA array. Data are mean ± standard deviation (n = 5 mice/group). A two-tailed 0.01 significance level; a (Mock vs. ZIKV) and b (ZIKV vs. ZIKV+EBS) by Kruskal-Wallis, Bonferroni Post Hoc Test. (B-C) Confocal microscopy of testes sections immunostained for CD45 (green, B), IL-1β (red, C), and Hoechst for nuclei (blue). Scale bar: 100 μm.</p

Antioxidant EBS prevents sexual transmission of ZIKV.

<p>(A) Schematic experimental design of sexual transmission. AG129 mice subcutaneously infected in the footpad with 5x10⁴ PFU/mouse of ZIKV were intraperitoneally treated with EBS (10 mg/kg body weight/mouse/day) or solvent control on day 1–5 (group: 6-dpi), or day 1–6 (group: 9-dpi) after infection. Sperm and sera were collected on 6 and 9 dpi. Sperm (50 μl) was used for vaginal inoculation into female mice (1 male to 1 female mouse). (B) Plaque-forming assay of viral load in sperm and sera. (C) Survival of female mice receiving sperm. (D) Relative quantitative analysis of ZIKV RNA in brain, ovaries and fallopian tubes (OV+FT), and spleen of recipient female mice. Data are mean ± SD (n = 6 mice/group). *P<0.05 and **P<0.01 by Mann-Whitney U test. Survival curves of female mice were compared by Log-rank test (**P = 0.0005).</p

Antioxidant EBS alleviates ZIKV-associated testicular pathology.

<p>AG129 mice subcutaneously infected in the footpad with 5x10⁴ PFU/mouse of ZIKV were intraperitoneally treated with EBS (10 mg/kg body weight/mouse/day) or solvent control on 1–6 dpi. Testes and semen were collected on 9 dpi. (A) Histology of testes sections stained with haematoxylin and eosin. Arrows indicate lumen (green), sperm (red), blood capillary (blue), interstitial cell (yellow), and degeneration of SNT (black). Scale bar: 200 μm. (B) Confocal microscopy of testes sections immunostained for TRA98 (green, germ cells), ETV5 (red, blood—testis barrier), and Hoechst for nuclei (blue). Differential interference contrast (DIC). Scale bar: 100 μm. (C) Total number of sperm (cell/ml). (D) Proportion of sperm with normal and abnormal morphology. Data are mean ± SD (n = 6 mice/group). **P<0.01 by Kruskal-Wallis, Bonferroni post-hoc test. (E) Confocal microscopy of sperm morphology with CellTracker staining for cytoplasm (red) and Hoechst for nuclei (blue). Scale bar: 100 μm. Arrows indicate sperm with abnormal morphology.</p

The effects of EBS treatment on viral replication in vitro and ZIKV-induced lethality <i>in vivo</i>.

<p>(A) EBS cytotoxicity assay. Human microglial CHME3 cells were treated with solvent or the indicated concentrations of EBS for 24 hours. LDH assays was performed to determine cellular cytotoxicity. Data are mean ± SD (n = 5 mice/group or 3 independent experiments). **P < 0.01 by Kruskal-Wallis, Bonferroni post-hoc test. (B-C) Human microglial CHME3 cells were infected with ZIKV (Multiplicity of infection: 0.1) in the presence or absence of EBS for 24 hours. (B) Plaque-forming assay of viral progeny production in culture supernatants. (C) Immunofluorescence microscopy was performed on cells immunostained for ZIKV-E (green) and Hoechst for nuclei (blue). (D-F) AG129 mice were subcutaneously infected in the footpad with 5x10⁴ PFU/mouse of ZIKV. Mice were intraperitoneally treated with EBS (5 or 10 mg/kg body weight/mouse/day) or solvent control on 1–6 dpi marked by arrows. Mice survival was presented as percentage of survival. The median survival time (T₅₀) is presented. Survival curves were compared by the use of Log-rank test (Vehicle control vs. EBS 5 mg, P = 0.1327 and Vehicle control vs. EBS 10 mg, P = 0.0505). (E) Viremia level of mice receiving EBS (10 mg/kg body weight) or solvent control. (F) Testicular distribution of ZIKV in mice receiving EBS (10 mg/kg body weight) or solvent control. Confocal microscopy of testes sections immunostained for ZIKV-E (green) and Hoechst for nuclei (blue). Scale bar: 100 μm.</p

EBS improves oxidative stress, cytokine profile, and testicular pathology of ZIKV-infected C57BL/6 mice.

<p>(A) Schematic experimental design. C57BL/6 mice were pre-treated with purified anti-mouse IFNAR-1 antibody (1 mg/kg body weight/mouse/intraperitoneal) one day prior subcutaneous infection of ZIKV (1x10⁵ PFU/mouse) in the footpad. Mice were intraperitoneally treated with EBS (10 mg/kg body weight/mouse/day) or solvent control on 1–6 dpi. Testes and sperm were collected on 9 dpi. (B-C) Intracellular nitric oxide (NO) and reactive oxygen species (ROS) assays. NO (B) and ROS (C) levels in sperms were measured by use of the OxiSelect intracellular NO and ROS indicator, respectively. H₂O₂-treated sperms as a positive control. Relative fluorescence intensity was determined by use of a fluorescence plate reader. (D) Cytokine profile of seminal fluid on 9 dpi. Cytokine levels were evaluated by ELISA array. (E) Viremia level on the indicated day after infection. Data are mean ± standard deviation (n = 5 mice/group). *P<0.05 and **P<0.01; a (Mock vs. ZIKV) and b (ZIKV vs. ZIKV+EBS) by Kruskal-Wallis, Bonferroni Post Hoc Test. (F) Confocal microscopy of testes sections immunostained for TRA98 (green, germ cells), ZIKV-E (red), and Hoechst for nuclei (blue). Differential interference contrast (DIC). Scale bar: 100 μm. (G) Histological analysis of testis sections stained with haematoxylin and eosin. Arrows indicate lumen (green), sperm (red), blood capillary (blue), interstitial cell (yellow), and degeneration of SNT (black). Scale bar: 200 μm.</p

ZIKV infects and damages testes.

<p>AG129 mice were subcutaneously infected in the footpad with 5x10⁴ plaque-forming units (PFU)/mouse of ZIKV. Testes, sperm, and sera were collected on 3, 6, and 9 days post-infection (dpi). (A) Confocal microscopy of testes sections immunostained for ZIKV-E (green) and Hoechst for nuclei (blue). (B) Confocal microscopy of sperm immunostained for ZIKV-E (green), CellTracker for cytoplasm (red), and Hoechst for nuclei (blue). Differential interference contrast (DIC). Scale bar: 100 μm. (C) Plaque-forming assay of viral load in sperm and sera. (D) Total number of sperm (cells/ml). (E) Proportion of sperm with normal and abnormal morphology. Data are mean ± SD (n = 6 mice/group). *P<0.05 and **P<0.01 by Mann-Whitney U test.</p