Rapid Reversible Osmoregulation of Cytoplasmic Biomolecular Condensates of Human Interferon-α-Induced Antiviral MxA GTPase.

We previously discovered that exogenously expressed GFP-tagged cytoplasmic human myxovirus resistance protein (MxA), a major antiviral effector of Type I and III interferons (IFNs) against several RNA- and DNA-containing viruses, existed in the cytoplasm in phase-separated membraneless biomolecular condensates of varying sizes and shapes with osmotically regulated disassembly and reassembly. In this study we investigated whether cytoplasmic IFN-α-induced endogenous human MxA structures were also biomolecular condensates, displayed hypotonic osmoregulation and the mechanisms involved. Both IFN-α-induced endogenous MxA and exogenously expressed GFP-MxA formed cytoplasmic condensates in A549 lung and Huh7 hepatoma cells which rapidly disassembled within 1-2 min when cells were exposed to 1,6-hexanediol or to hypotonic buffer (~40-50 mOsm). Both reassembled into new structures within 1-2 min of shifting cells to isotonic culture medium (~330 mOsm). Strikingly, MxA condensates in cells continuously exposed to culture medium of moderate hypotonicity (in the range one-fourth, one-third or one-half isotonicity; range 90-175 mOsm) first rapidly disassembled within 1-3 min, and then, in most cells, spontaneously reassembled 7-15 min later into new structures. This spontaneous reassembly was inhibited by 2-deoxyglucose (thus, was ATP-dependent) and by dynasore (thus, required membrane internalization). Indeed, condensate reassembly was preceded by crowding of the cytosolic space by large vacuole-like dilations (VLDs) derived from internalized plasma membrane. Remarkably, the antiviral activity of GFP-MxA against vesicular stomatitis virus survived hypoosmolar disassembly and subsequent reassembly. The data highlight the exquisite osmosensitivity of MxA condensates, and the preservation of antiviral activity in the face of hypotonic stress.

Evaluation of MX1 Gene Promoter Methylation in Different Severities of COVID-19 Considering Patient Gender.

BACKGROUND: Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), led to a pandemic in March 2020. During a viral infection, it has been reported that epigenetic changes occur for both sides: Infected cells elicit an antiviral environmental response, which induces and initiates certain pathways for proper response to the virus, while the virus silences the expression of vital genes in the anti-viral host cell. In this study, we aimed to examine the methylation level of the MX1 gene promoter in different stages in COVID-19 patients compared to the control group. METHODS: In total, 470 COVID-19 patients with a positive polymerase chain reaction (PCR) test (235 women and 235 men) were recruited into the study as the test group. Patients were divided based on the World Health Organization (WHO) classification into three groups: moderate, severe, and critical. Moreover, 100 healthy individuals (50 women and 50 men) were selected as the control group. Peripheral white blood cells were collected and PCR was performed using two types of primers designed for methylated and unmethylated states of the MX1 gene. The PCR products were then loaded on agarose gel and the band intensities were calculated by ImageJ software. RESULTS: The results showed a decrease in the methylation of the MX1 gene promoter in moderate and severe groups and an increase in the MX1 gene promoter methylation in the critical group. In addition, the level of methylation was higher in men than in women. CONCLUSIONS: Increased methylation of the MX1 gene in the critical group may indicate the role of SARS-CoV-2 in reducing the expression levels of this antiviral gene and thus promoting virus replication and disease progression.

Association of human myxovirus resistance protein A with severity of COVID-19.

BACKGROUND: In this retrospective cohort study, we explored the correlation of blood human myxovirus resistance protein A (MxA) level with severity of disease in hospitalized COVID-19 patients. METHODS: All 304 patients admitted for COVID-19 in our hospital until 30th of April 2021 were included in this study. MxA was measured from peripheral blood samples in 268 cases. Patients were divided into groups based on their level of MxA on admission. We studied baseline characteristics and severity of disease on admission based on clinical parameters and inflammatory biomarker levels in each group. Severity of disease during hospitalization was determined by the applied level of respiratory support, by the usage of corticosteroids and by the duration of hospitalization. RESULTS: Higher MxA levels on admission were associated with a shorter duration of symptoms before admission, and with more severe disease. Adjusted Odds Ratios for any respiratory support were 9.92 (95%CI 2.11-46.58; p = 0.004) in patients with MxA between 400 µg/L and 799 µg/L (p = 0.004) and 20.08 (95%CI 4.51-89.44; p < 0.001) in patients with MxA ≥ 800 µg/L in comparison with patients with initial MxA < 400 µg/L. The usage of corticosteroids was significantly higher in the high-MxA group (77%) in comparison with the intermediate-MxA group (62%, p = 0.013) and low-MxA group (47%, p < 0.001). CONCLUSIONS: Higher initial levels of MxA were associated with more severe COVID-19. MxA may be a helpful additional biomarker to predict the severity of the disease.

Developing a Virus-Binding Bacterium Expressing Mx Protein on the Bacterial Surface to Prevent Grouper Nervous Necrosis Virus Infection.

Grouper nervous necrosis virus (GNNV) infection causes mass grouper mortality, leading to substantial economic loss in Taiwan. Traditional methods of controlling GNNV infections involve the challenge of controlling disinfectant doses; low doses are ineffective, whereas high doses may cause environmental damage. Identifying potential methods to safely control GNNV infection to prevent viral outbreaks is essential. We engineered a virus-binding bacterium expressing a myxovirus resistance (Mx) protein on its surface for GNNV removal from phosphate-buffered saline (PBS), thus increasing the survival of grouper fin (GF-1) cells. We fused the grouper Mx protein (which recognizes and binds to the coat protein of GNNV) to the C-terminus of outer membrane lipoprotein A (lpp-Mx) and to the N-terminus of a bacterial autotransporter adhesin (Mx-AIDA); these constructs were expressed on the surfaces of Escherichia coli BL21 (BL21/lpp-Mx and BL21/Mx-AIDA). We examined bacterial surface expression capacity and GNNV binding activity through enzyme-linked immunosorbent assay; we also evaluated the GNNV removal efficacy of the bacteria and viral cytotoxicity after bacterial adsorption treatment. Although both constructs were successfully expressed, only BL21/lpp-Mx exhibited GNNV binding activity; BL21/lpp-Mx cells removed GNNV and protected GF-1 cells from GNNV infection more efficiently. Moreover, salinity affected the GNNV removal efficacy of BL21/lpp-Mx. Thus, our GNNV-binding bacterium is an efficient microparticle for removing GNNV from 10‰ brackish water and for preventing GNNV infection in groupers.

Transcriptomic studies revealed pathophysiological impact of COVID-19 to predominant health conditions.

Despite the association of prevalent health conditions with coronavirus disease 2019 (COVID-19) severity, the disease-modifying biomolecules and their pathogenetic mechanisms remain unclear. This study aimed to understand the influences of COVID-19 on different comorbidities and vice versa through network-based gene expression analyses. Using the shared dysregulated genes, we identified key genetic determinants and signaling pathways that may involve in their shared pathogenesis. The COVID-19 showed significant upregulation of 93 genes and downregulation of 15 genes. Interestingly, it shares 28, 17, 6 and 7 genes with diabetes mellitus (DM), lung cancer (LC), myocardial infarction and hypertension, respectively. Importantly, COVID-19 shared three upregulated genes (i.e. MX2, IRF7 and ADAM8) with DM and LC. Conversely, downregulation of two genes (i.e. PPARGC1A and METTL7A) was found in COVID-19 and LC. Besides, most of the shared pathways were related to inflammatory responses. Furthermore, we identified six potential biomarkers and several important regulatory factors, e.g. transcription factors and microRNAs, while notable drug candidates included captopril, rilonacept and canakinumab. Moreover, prognostic analysis suggests concomitant COVID-19 may result in poor outcome of LC patients. This study provides the molecular basis and routes of the COVID-19 progression due to comorbidities. We believe these findings might be useful to further understand the intricate association of these diseases as well as for the therapeutic development.

Interferon-Induced Transmembrane Protein (IFITM3) Is Upregulated Explicitly in SARS-CoV-2 Infected Lung Epithelial Cells.

Current guidelines for COVID-19 management recommend the utilization of various repurposed drugs. Despite ongoing research toward the development of a vaccine against SARS-CoV-2, such a vaccine will not be available in time to contribute to the containment of the ongoing pandemic. Therefore, there is an urgent need to develop a framework for the rapid identification of novel targets for diagnostic and therapeutic interventions. We analyzed publicly available transcriptomic datasets of SARS-CoV infected humans and mammals to identify consistent differentially expressed genes then validated in SARS-CoV-2 infected epithelial cells transcriptomic datasets. Comprehensive toxicogenomic analysis of the identified genes to identify possible interactions with clinically proven drugs was carried out. We identified IFITM3 as an early upregulated gene, and valproic acid was found to enhance its mRNA expression as well as induce its antiviral action. These findings indicate that analysis of publicly available transcriptomic and toxicogenomic data represents a rapid approach for the identification of novel targets and molecules that can modify the action of such targets during the early phases of emerging infections like COVID-19.