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.

The Mechanism of the Zika Virus Crossing the Placental Barrier and the Blood-Brain Barrier.

Zika virus (ZIKV) infection causes severe neurological symptoms in adults and fetal microcephaly and the virus is detected in the brain of microcephaly and meningoencephalitis patient. However, the mechanism of ZIKV crossing the physiological barrier to the central nervous systems (CNS) remains elusive. The placental barrier and the blood brain barrier (BBB) protect the fetus from pathogens and ensure healthy brain development during pregnancy. In this study, we used human placenta trophoblasts cells (JEG-3) and human brain-derived endothelial cells (hCMEC/D3) as in vitro models of the physiological barriers. Results showed that ZIKV could infect JEG-3 cells effectively and reduce the amounts of ZO-1 and occludin between adjacent cells by the proteasomal degradation pathway, suggesting that the permeability of the barrier differentially changed in response to ZIKV infection, allowing the virus particle to cross the host barrier. In contrast, ZIKV could infect hCMEC/D3 cells without disrupting the BBB barrier permeability and tight junction protein expression. Although no disruption to the BBB was observed during ZIKV infection, ZIKV particles were released on the basal side of the BBB model and infected underlying cells. In addition, we observed that fluorescence-labeled ZIKV particles could cross the in vitro placenta barrier and BBB model by transcytosis and the action of transcytosis could be blocked by either low temperature or pharmacological inhibitors of endocytosis. In summary, the ZIKV uses a cell-type specific paracellular pathway to cross the placenta monolayer barrier by disrupting cellular tight junction. In addition, the ZIKV can also cross both the placenta barrier and the BBB by transcytosis. Our study provided new insights into on the mechanism of the cellular barrier penetration of ZIKV particles.

Benzenesulfonamide Derivatives as Calcium/Calmodulin-Dependent Protein Kinase Inhibitors and Antiviral Agents against Dengue and Zika Virus Infections.

Emerging and resurging mosquito-borne flaviviruses are an important public health challenge. The increased prevalence of dengue virus (DENV) infection has had a significant socioeconomic impact on epidemic countries. The recent outbreak of Zika virus (ZIKV) has created an international public health emergency because ZIKV infection has been linked to congenital defects and Guillain-Barré syndrome. To develop potentially prophylactic antiviral drugs for combating these acute infectious diseases, we have targeted the host calcium/calmodulin-dependent kinase II (CaMKII) for inhibition. By using CaMKII structure-guided inhibitor design, we generated four families of benzenesulfonamide (BSA) derivatives for SAR analysis. Among these substances, N-(4-cycloheptyl-4-oxobutyl)-4-methoxy-N-phenylbenzenesulfonamide (9) showed superior properties as a lead CaMKII inhibitor and antiviral agent. BSA 9 inhibited CaMKII activity with an IC50 value of 0.79 µM and displayed EC50 values of 1.52 µM and 1.91 µM against DENV and ZIKV infections of human neuronal BE(2)C cells, respectively. Notably, 9 significantly reduced the viremia level and increased animal survival time in mouse-challenge models.

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

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/ß 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-1ß, 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.

Japanese Encephalitis Virus Exploits Dopamine D2 Receptor-phospholipase C to Target Dopaminergic Human Neuronal Cells.

Despite the availability of vaccines for Japanese encephalitis virus (JEV), the re-emerging virus remains a clinically important pathogen that causes acute encephalitis and permanent neuropsychiatric sequels. JEV highly targets dopaminergic neuron-rich brain regions including the thalamus and midbrain. The molecular mechanism contributing to the high susceptibility of these particular brain regions remains largely unclear. This study addressed whether this tissue tropism of JEV is associated with signaling of dopaminergic neurons. Three pieces of evidence indicate that JEV exploits dopamine signaling to facilitate its infection: (1) JEV infection modulates dopamine level; (2) a selective dopamine D2 receptor (D2R) agonist enhances JEV infection; and (3) stimulation of D2R activates phospholipase C (PLC) to enhance the surface expression of JEV binding/entry molecules, integrin ß3 and vimentin. Overall, JEV may exploit dopamine-mediated neuronal communication to increase the susceptibility of D2R-expressing cells to JEV infection. This study identifies a potential underlying mechanism of viral invasiveness in the dopaminergic brain regions and suggests antiviral strategies against viral infection by targeting D2R-PLC signaling.

Repurposing of prochlorperazine for use against dengue virus infection.

The increasing prevalence of dengue virus (DENV) infection presents serious disease and economic burdens in countries where dengue epidemics are occurring. Despite the clinical importance, no DENV vaccine or anti-DENV drug is available. In this study, we found that prochlorperazine (PCZ), a dopamine D2 receptor (D2R) antagonist approved to treat nausea, vomiting, and headache in humans has potent in vitro and in vivo antiviral activity against DENV infection. PCZ can block DENV infection by targeting viral binding and viral entry through D2R- and clathrin-associated mechanisms, respectively. Administration of PCZ immediately or 6 hours after DENV infection in a Stat1-deficient mouse model completely protected against or delayed lethality. Overall, PCZ showed a previously unknown antiviral effect against DENV infection, and D2R may play a role in the DENV life cycle. Prophylactic and/or therapeutic treatment with PCZ might reduce viral replication and relieve the clinical symptoms of patients with dengue.