Mimicry games: NPC-like MX2 condensates trap viruses.

Recent findings suggest that HIV-1 capsids mimic nuclear transport receptors to engage FG-nucleoporins for entry into host nuclei. In this issue of Cell Host & Microbe, Moschonas et al. report that MX2 forms cytoplasmic condensates comprising FG-nucleoporins resembling nuclear pore complexes to capture viral capsids and hinder their nuclear transport.

MX2 forms nucleoporin-comprising cytoplasmic biomolecular condensates that lure viral capsids.

Human myxovirus resistance 2 (MX2) can restrict HIV-1 and herpesviruses at a post-entry step through a process requiring an interaction between MX2 and the viral capsids. The involvement of other host cell factors, however, remains poorly understood. Here, we mapped the proximity interactome of MX2, revealing strong enrichment of phenylalanine-glycine (FG)-rich proteins related to the nuclear pore complex as well as proteins that are part of cytoplasmic ribonucleoprotein granules. MX2 interacted with these proteins to form multiprotein cytoplasmic biomolecular condensates that were essential for its anti-HIV-1 and anti-herpes simplex virus 1 (HSV-1) activity. MX2 condensate formation required the disordered N-terminal region and MX2 dimerization. Incoming HIV-1 and HSV-1 capsids associated with MX2 at these dynamic cytoplasmic biomolecular condensates, preventing nuclear entry of their viral genomes. Thus, MX2 forms cytoplasmic condensates that likely act as nuclear pore decoys, trapping capsids and inducing premature viral genome release to interfere with nuclear targeting of HIV-1 and HSV-1.

Expression of MxA in esophageal cancer cell lines can influence sensitivity to chemotherapeutic agents but this does not require apoptosis.

Esophageal cancer is a poor prognosis cancer characterized by intrinsic or acquired resistance to chemotherapeutic agents. The primary determinants of treatment failure are unknown. Expression of an anti-viral protein, myxovirus resistance protein A (MxA) is de-regulated in many cancers, including esophageal cancer, and its activity has been linked to apoptosis. This study has assessed whether MxA expression can influence the response of esophageal cancer cells to the chemotherapeutic agents 5-fluorouracil (5-FU) or oxaliplatin. MxA protein was differentially expressed in a panel of five esophageal cancer cell lines. KYSE450 and KYSE140 cells did not express MxA and were apoptosis incompetent. FLO-1, KYSE270, and OE21 cells expressed MxA, were more drug-sensitive and were apoptosis competent. MxA was artificially overexpressed in cell lines with no endogenous expression (KYSE450 and KYSE140). This increased the resistance of KYSE450 but not KYSE140 cells. Both cell lines remained apoptosis incompetent. We then evaluated siRNA knockdown of MxA in FLO-1 cells and CRISPR knockout in OE21 cells. Knockdown of MxA significantly increased drug sensitivity and caspase-3 activation in FLO-1 cells. OE21-MX1KO cells were also more drug-sensitive, but in contrast to FLO-1 cells, caspase-3 activation was reduced. Collectively these data indicate that MxA can promote resistance to chemotherapy, but this does not always correspond with effects on apoptosis. Effects on apoptosis are cell line specific, suggesting that other co-operating pathways determine the overall impact of MxA. Importantly, in cancer cells that overexpress the protein, drug sensitivity can be improved by interfering with MxA.

[Myxovirus resistance protein A (MxA) induces the expression of interferon-stimulated genes in HepG2 cells by enhancing the activity of the interferon-stimulated response element (ISRE)].

Objective To explore the effects of Myxovirus resistance protein A (MxA) on the Janus kinase/Signal transducer and activator of transcription (JAK/STAT) pathway in HepG2 cells. Methods HepG2 cells were transfected with the pcDNA3.1-Flag-MxA construct, and subsequent localization and expression of the MxA protein were detected through immunofluorescence cytochemistry. The presence of MxA protein was further confirmed by using Western blot analysis. Following transfection with MxA small interfering RNA (si-MxA) and subsequent treatment with alpha interferon (IFN-α), real-time fluorescent quantitative PCR was employed to measure the mRNA levels of myxovirus resistance protein A (MxA), protein kinase R (PKR), and oligoadenylate synthase (OAS). Western blot analysis was used to detect the protein expression of MxA, PKR, OAS, signal transducer and activator of transcription 1 (STAT1), phosphorylated STAT1 (pSTAT1), STAT2, phosphorylated STAT2 (p-STAT2) and interferon regulatory factor 9 (IRF9). Additionally, pcDNA3.1-Flag-MxA and pISRE-TA-luc were co-transfected into HepG2 and HepG2.2.15 cells, respectively, to assess the activity of the interferon-stimulated response element (ISRE) by using a luciferase activity assay. Results MxA protein was expressed in both the cytoplasm and nucleus of HepG2 cells, with higher expression levels in the cytoplasm than in the nucleus. Knocking down MxA expression in HepG2 cells did not affect the expression of STAT1, p-STAT1, STAT2, p-STAT2, and IRF9 proteins induced by IFN-α, but significantly reduced the expression of antiviral proteins PKR and OAS. Overexpression of MxA in HepG2 cells enhanced ISRE activity and increased the expression of PKR and OAS proteins, but this effect was inhibited in HepG2.2.15 cells. Conclusion MxA induces the expression of antiviral proteins by enhancing the activity of the JAK/STAT signaling pathway ISRE.

Determination of systemic inflammation response index (SIRI), systemic inflammatory index (SII), HMGB1, Mx1 and TNF levels in neonatal calf diarrhea with systemic inflammatory response syndrome.

The objective of this study was to examine the values of MX dynamin-like GTPase 1 (Mx1), high mobility group box-1 (HMGB1), systemic inflammatory response index (SIRI), systemic inflammatory index (SII), tumor necrosis factor (TNF), and other hematological indices in calves with systemic inflammatory response syndrome (SIRS). The study material was divided into two groups: the SIRS group (comprising 13 calves) and the control group (comprising 10 calves). The independent samples t-test and Mann-Whitney U test were employed for normally distributed and non-normally distributed data, respectively. The relationship between the two groups was determined using Spearman correlation coefficient analysis. Significant differences were identified between the SIRS group and the control group with regard to white blood cell (WBC; P < 0.05), neutrophil (NEU; P < 0.01), and neutrophil-to-lymphocyte ratio (NLR; P < 0.001) values, in addition to SIRI (P < 0.05), SII (P < 0.01) values. Furthermore, HMGB1 (P < 0.001), Mx1 (P < 0.05), and TNF values (P < 0.001) demonstrated notable disparities between the two groups. As a result of this study, it was concluded that there were significant increases in inflammatory hematological indices, as well as in the levels of HMGB1, Mx1, and TNF, in calves with SIRS.

Characterization of Myxovirus resistance (Mx) gene from Chinese seabass Lateolabrax maculatus: Insights into the evolution and function of Mx genes.

Chinese seabass (Lateolabrax maculatus) stands out as one of the most sought-after and economically significant species in aquaculture within China. Diseases of L. maculatus occur frequently due to the degradation of the germplasm, the aggravation of environmental pollution of water, and the reproduction of pathogenic microorganisms, inflicting considerable economic losses on the Chinese seabass industry. The Myxovirus resistance (Mx) gene plays pivotal roles in the antiviral immune response ranging from mammals to fish. However, the function of the Mx gene in L. maculatus is still unknown. Firstly, the origin and evolutionary history of Mx proteins was elucidated in this study. Subsequently, an Mx gene from L. maculatus (designed as LmMxA gene) was identified, and its functions in combating antiviral and antibacterial threats were investigated. Remarkably, our findings suggested that while Mx group genes were present in chordates, DYN group genes were present in everything from single-celled animals to humans. Furthermore, our investigation revealed that the LmMxA mRNA level increased in the kidney, spleen and liver subsequent to Vibrio anguillarum and poly(I:C) challenged. Immunofluorescence analysis indicated that LmMxA is predominantly localization in the nucleus and the cytoplasm. Notably, the expression of MAVS, IFN1 and Mx1 increased when LmMxA was overexpression within the EPC cells. Moreover, through assessment via cytopathic effect (CPE), virus titer, and antibacterial activity, it becomes evident that LmMxA exerts a dual role in bolstering both antiviral and antibacterial immune responses. These compelling findings laid the foundation for further exploring the mechanism of LmMxA in response to innate immunity of L. maculatus.

Type I interferon gene expression signature as a marker to predict response to cyclophosphamide based treatment in proliferative lupus nephritis.

OBJECTIVES: To assess the longitudinal effect of cyclophosphamide (CYC) treatment on type-I interferon (IFN) signature in proliferative lupus nephritis (LN) and its role in predicting treatment response. METHODS: Fifty-four biopsy proven proliferative LN patients scheduled to receive high-dose (HD) or low-dose (LD) CYC were recruited and followed up for six months. At six months, patients were classified as clinical responders (CR) or non-responders (NR) to treatment, using the EULAR/EDTA criteria. An IFN-gene based score (IGS) was developed from the mean log-transformed gene expression of MX1, OAS1, IFIT1, OASL, IFIT4, LY6E, IRF7 at baseline, three and six months. Longitudinal changes of IGS within and between groups were assessed and ΔIGS, which is the difference in IGS between baseline and three months was calculated. Independent predictors of non-response were identified and an ROC analysis was performed to evaluate their utility to predict NR. RESULTS: There was a dynamic change in IGS within the HD, LD, CR, and NR groups. Compared to baseline, there was a significant decrease in IGS at three months in HD and LD groups (HD group: 2.01 to 1.14, p = .001; LD group = 2.01 to 0.81, p < .001), followed by a significant increase from three to six months in LD group (LD: 0.81 to 1.51, p = .03; HD: 1.14 to 1.54, p = .300). A decrease in IGS from baseline to three months was seen in both CR (2.13 to 0.79, p < .001) and NR groups (1.83 to 1.27, p = .046), and a significant increase from three to six months was observed only in the CR group (CR: 0.79 to 1.57, p = .006; NR: 1.27 to 1.46, p = 1). ΔIGS (baseline to three months) was higher in CR compared to NR group (-1.339 vs -0.563, p = .017). ROC analysis showed that the model comprising of 0.81 fold decrease in IGS from baseline to three months, endocapillary hypercellularity and interstitial inflammation on renal histopathology predicted non-response with a sensitivity of 83.3% and specificity of 71.4%. CONCLUSION: In proliferative LN, treated with HD or LD-CYC, combined model comprising of decrease in IGS score by 0.81 fold from baseline to three months, along with important histopathological features such as endocapillary hypercellularity and interstitial inflammation had better predictive capability for non-response.

Pathogenic role and diagnostic utility of interferon-α in histiocytic necrotizing lymphadenitis.

PURPOSE: Histiocytic necrotizing lymphadenitis (HNL) is an inflammatory disease of unknown etiology clinically characterized by painful lymphadenopathy. This study aimed to investigate the role of interferon (IFN)-α in the pathogenesis of HNL and the clinical significance of serum IFN-α levels for the diagnosis and monitoring of HNL disease activity. METHODS: This study enrolled 47 patients with HNL and 43 patients with other inflammatory diseases that require HNL differentiation including malignant lymphoma (ML), bacterial lymphadenitis, and Kawasaki disease. Expression of IFN-stimulated genes (ISGs) and MX1 in the lymph nodes was measured by real-time quantitative reverse transcription polymerase chain reaction and immunofluorescence staining, respectively. Enzyme-linked immunosorbent assay was used to quantify serum cytokine levels. The results were compared with the clinical features and disease course of HNL. RESULTS: Patients with HNL had a significantly elevated ISG expression in the lymph nodes compared with those with ML. MX1 and CD123, a specific marker of plasmacytoid dendritic cells (pDCs), were colocalized. In patients with HNL, serum IFN-α levels were significantly elevated and positively correlated with disease activity. The serum IFN-α level cutoff value for differentiating HNL from other diseases was 11.5 pg/mL. CONCLUSION: IFN-α overproduction from pDCs may play a critical role in HNL pathogenesis. The serum IFN-α level may be a valuable biomarker for the diagnosis and monitoring of disease activity in patients with HNL.

Analyses of the Mx family members in lumpfish: Molecular characterization, phylogeny, and gene expression analyses.

Members of the myxovirus resistance (Mx) protein family play an essential role in antiviral immunity. They are Dynamin-like GTPases, induced by interferons. In the current study, we have characterized two predicted MX genes (MX1 and MX2) from lumpfish (Cyclopterus lumpus L.), having 12 and 13 exons, respectively. Mx2 has two isoforms (Mx2-X1 and Mx2-X2) which differ in exon 1. The lumpfish Mx proteins contain an N-terminal Dynamin-like GTPase domain, the middle domain (MD) and GTPase effector domain (GED) characteristic for Mx proteins. Phylogenetic analyses grouped all the lumpfish Mx sequences in group 1, and synteny analyses showed that both genes were localized at chromosome 5 in proximity to the genes Tohc7, Atxn7 and Psmd6. In vitro stimulation experiment showed that both MX1 and MX2-X2 were highly upregulated upon exposure to poly(I:C), but not bacteria, 24 h post exposure, indicating their role in antiviral immunity.

The SAMHD1-MX2 axis restricts HIV-1 infection at postviral DNA synthesis.

HIV-1 replication is tightly regulated in host cells, and various restriction factors have important roles in inhibiting viral replication. SAMHD1, a well-known restriction factor, suppresses HIV-1 replication by hydrolyzing intracellular dNTPs, thereby limiting the synthesis of viral cDNA in quiescent cells. In this study, we revealed an additional and distinct mechanism of SAMHD1 inhibition during the postviral cDNA synthesis stage. Using immunoprecipitation and mass spectrometry analysis, we demonstrated the interaction between SAMHD1 and MX2/MxB, an interferon-induced antiviral factor that inhibits HIV-1 cDNA nuclear import. The disruption of endogenous MX2 expression significantly weakened the ability of SAMHD1 to inhibit HIV-1. The crucial region within SAMHD1 that binds to MX2 has been identified. Notably, we found that SAMHD1 can act as a sensor that recognizes and binds to the incoming HIV-1 core, subsequently delivering it to the molecular trap formed by MX2, thereby blocking the nuclear entry of the HIV-1 core structure. SAMHD1 mutants unable to recognize the HIV-1 core showed a substantial decrease in antiviral activity. Certain mutations in HIV-1 capsids confer resistance to MX2 inhibition while maintaining susceptibility to suppression by the SAMHD1-MX2 axis. Overall, our study identifies an intriguing antiviral pattern wherein two distinct restriction factors, SAMHD1 and MX2, collaborate to establish an alternative mechanism deviating from their actions. These findings provide valuable insight into the complex immune defense networks against exogenous viral infections and have implications for the development of targeted anti-HIV therapeutics. IMPORTANCE: In contrast to most restriction factors that directly bind to viral components to exert their antiviral effects, SAMHD1, the only known deoxynucleotide triphosphate (dNTP) hydrolase in eukaryotes, indirectly inhibits viral replication in quiescent cells by reducing the pool of dNTP substrates available for viral cDNA synthesis. Our study provides a novel perspective on the antiviral functions of SAMHD1. In addition to its role in dNTP hydrolysis, SAMHD1 cooperates with MX2 to inhibit HIV-1 nuclear import. In this process, SAMHD1 acts as a sensor for incoming HIV-1 cores, detecting and binding to them, before subsequently delivering the complex to the molecular trap formed by MX2, thereby immobilizing the virus. This study not only reveals a new antiviral pathway for SAMHD1 but also identifies a unique collaboration and interaction between two distinct restriction factors, establishing a novel line of defense against HIV-1 infection, which challenges the traditional view of restriction factors acting independently. Overall, our findings further indicate the intricate complexity of the host immune defense network and provide potential targets for promoting host antiviral immune defense.

Altered expression of human myxovirus resistance protein A in amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder. The etiology of sporadic ALS (sALS) has not yet been clarified. An increasing body of evidence suggests the involvement of viral infections and interferons (IFNs). Human myxovirus resistance protein A (MxA) is an IFN-induced dynamin-like GTPase that acts as a potent antiviral factor. This study examined MxA expression in ALS patient spinal cords using immunohistochemistry. Thirty-two cases of sALS (pathologically proven ALS-TDP), 10 non-ALS, other neurological disease control cases were examined. In most ALS cases, MxA cytoplasmic condensates were observed in the remaining spinal anterior horn neurons. The ALS group had a significantly higher rate of MxA-highly expressing neurons than the non-ALS group. Colocalization of MxA cytoplasmic condensate and transactive response DNA-binding protein 43 kDa (TDP-43)-positive inclusions was rarely observed. Because MxA has antiviral activity induced by IFNs, our results suggest that IFNs are involved in the pathogenesis of ALS in spinal cord anterior horn neurons. Our study also suggests that monitoring viral infections and IFN activation in patients with ALS may be critically important.

[Diagnosis of anti-melanoma differentiation-associated gene 5 antibody-positive dermatomyositis led by sarcoplasmic myxovirus resistance protein A expression on muscle pathology].

A 44-year-old woman with autism spectrum disorder developed bulbar symptoms and generalized muscle weakness 7 months before referral. Six months before, she was administered glucocorticoid for liver involvement. During the course, while she presented alopecia, skin ulcers, and poikiloderma, hyperCKemia was observed only twice. Due to complications including cardiac involvement and hearing loss as well, we suspected mitochondrial disease and performed a muscle biopsy. The muscle pathology showed sarcoplasmic myxovirus resistance A (MxA) expression with scattered pattern. Since anti-melanoma differentiation-associated gene 5 (MDA5) antibody was detected, we diagnosed the patient with anti-MDA5 antibody-positive dermatomyositis (DM). We reinforced immunosuppressive therapy, and her clinical symptoms and liver involvement were improved. When we diagnose a case of anti-MDA5 antibody-positive DM who is difficult to make clinical diagnosis, it may be valuable to evaluate sarcoplasmic MxA expression on muscle pathology.

Induction and antiviral activity of ferret myxovirus resistance (Mx) protein 1 against influenza A viruses.

Myxovirus resistance (Mx) proteins are products of interferon stimulated genes (ISGs) and Mx proteins of different species have been reported to mediate antiviral activity against a number of viruses, including influenza A viruses (IAV). Ferrets are widely considered to represent the 'gold standard' small animal model for studying pathogenesis and immunity to human IAV infections, however little is known regarding the antiviral activity of ferret Mx proteins. Herein, we report induction of ferret (f)Mx1/2 in a ferret lung cell line and in airway tissues from IAV-infected ferrets, noting that fMx1 was induced to higher levels that fMx2 both in vitro and in vivo. Overexpression confirmed cytoplasmic expression of fMx1 as well as its ability to inhibit infection and replication of IAV, noting that this antiviral effect of fMx1was modest when compared to cells overexpressing either human MxA or mouse Mx1. Together, these studies provide the first insights regarding the role of fMx1 in cell innate antiviral immunity to influenza viruses. Understanding similarities and differences in the antiviral activities of human and ferret ISGs provides critical context for evaluating results when studying human IAV infections in the ferret model.

Dissemination of influenza B virus to the lower respiratory tract of mice is restricted by the interferon response.

The global burden of disease caused by influenza B virus (IBV) is substantial; however, IBVs remain overlooked. Understanding host-pathogen interactions and establishing physiologically relevant models of infection are important for the development and assessment of therapeutics and vaccines against IBV. In this study, we assessed an upper respiratory tract (URT)-restricted model of mouse IBV infection, comparing it to the conventional administration of the virus to the total respiratory tract (TRT). We found that URT infections caused by different strains of IBV disseminate to the trachea but resulted in limited dissemination of IBV to the lungs. Infection of the URT did not result in weight loss or systemic inflammation even at high inoculum doses and despite robust viral replication in the nose. Dissemination of IBV to the lungs was enhanced in mice lacking functional type I IFN receptor (IFNAR2), but not IFNγ. Conversely, in mice expressing the IFN-inducible gene Mx1, we found reduced IBV replication in the lungs and reduced dissemination of IBV from the URT to the lungs. Inoculation of IBV in both the URT and TRT resulted in seroconversion against IBV. However, priming at the TRT conferred superior protection from a heterologous lethal IBV challenge compared to URT priming, as determined by improved survival rates and reduced viral replication throughout the respiratory tract. Overall, our study establishes a URT-restricted IBV infection model, highlights the critical role of IFNs in limiting dissemination of IBV to the lungs, and also demonstrates that the lack of viral replication in the lungs may impact protection from subsequent infections. IMPORTANCE: Our study investigated how influenza B virus (IBV) spreads from the nose to the lungs of mice and the impact this has on disease and protection from re-infection. We found that when applied to the nose only, IBV does not spread very efficiently to the lungs in a process controlled by the interferon response. Priming immunity at the nose only resulted in less protection from re-infection than priming immunity at both the nose and lungs. These insights can guide the development of potential therapies targeting the interferon response as well as of intranasal vaccines against IBV.

Genetic insertion of mouse Myxovirus-resistance gene 1 increases innate resistance against both high and low pathogenic avian influenza virus by significantly decreasing replication in chicken DF1 cell line.

Avian influenza virus (AIV) is a constant threat to animal health with recent global outbreaks resulting in the death of hundreds of millions of birds with spillover into mammals. Myxovirus-resistance (Mx) proteins are key mediators of the antiviral response that block virus replication. Mouse (Mu) Mx (Mx1) is a strong antiviral protein that interacts with the viral nucleoprotein to inhibit polymerase function. The ability of avian Mx1 to inhibit AIV is unclear. In these studies, Mu Mx1 was stably introduced into chicken DF1 cells to enhance the immune response against AIV. Following infection, titers of AIV were significantly decreased in cells expressing Mu Mx1. In addition, considerably less cytopathic effect (CPE) and matrix protein staining was observed in gene-edited cells expressing Mu Mx1, suggesting Mu Mx1 is broadly effective against multiple AIV subtypes. This work provides foundational studies for use of gene-editing to enhance innate disease resistance against AIV.

Increased Interferon Signaling in Vaginal Tissue of Patients With Primary Sjögren Syndrome.

OBJECTIVE: Vaginal dryness is an important factor influencing sexual function in women with primary Sjögren syndrome (pSS). Previous studies showed a higher degree of inflammation in vaginal biopsies from patients with pSS compared to non-pSS controls. However, the molecular pathways that drive this inflammation remain unclear. Therefore, the aim of this study was to investigate inflammatory pathway activity in the vaginal tissue of patients with pSS. METHODS: Vaginal biopsies of 8 premenopausal patients with pSS experiencing vaginal dryness and 7 age-matched non-pSS controls were included. Expression of genes involved in inflammation and tissue homeostasis was measured using NanoString technology and validated using TaqMan Real-Time PCR. Vaginal tissue sections were stained by immunohistochemistry for myxovirus resistance protein 1 (MxA) and CD123 (plasmacytoid dendritic cells [pDCs]). RESULTS: The most enriched pathway in vaginal biopsies from patients with pSS compared to non-pSS controls was the interferon (IFN) signaling pathway (P < 0.01). Pathway scores for Janus kinase and signal transducer and activator of transcription (JAK-STAT) and Notch signaling were also higher (P < 0.01 for both pathways). Conversely, transforming growth factor-ß signaling and angiogenesis pathway scores were lower in pSS (P = 0.02 and P = 0.04, respectively). Differences in IFN signaling between patients with pSS and non-pSS controls were confirmed by PCR and MxA tissue staining. No CD123+ pDCs were detected in vaginal biopsies. IFN-stimulated gene expression levels correlated positively with CD45+ cell numbers in vaginal biopsies and serum anti-SSA/Ro positivity. CONCLUSION: Upregulation of IFN signaling in vaginal tissue of women with pSS, along with its association with tissue pathology, suggests that IFNs contribute to inflammation of the vaginal wall and potentially also to clinical symptomatology (ie, vaginal dryness).

The immune-evasive proline-283 substitution in influenza nucleoprotein increases aggregation propensity without altering the native structure.

Nucleoprotein (NP) is a key structural protein of influenza ribonucleoprotein complexes and is central to viral RNA packing and trafficking. NP also determines the sensitivity of influenza to myxovirus resistance protein 1 (MxA), an innate immunity factor that restricts influenza replication. A few critical MxA-resistant mutations have been identified in NP, including the highly conserved proline-283 substitution. This essential proline-283 substitution impairs influenza growth, a fitness defect that becomes particularly prominent at febrile temperature (39°C) when host chaperones are depleted. Here, we biophysically characterize proline-283 NP and serine-283 NP to test whether the fitness defect is caused by the proline-283 substitution introducing folding defects. We show that the proline-283 substitution changes the folding pathway of NP, making NP more aggregation prone during folding, but does not alter the native structure of the protein. These findings suggest that influenza has evolved to hijack host chaperones to promote the folding of otherwise biophysically incompetent viral proteins that enable innate immune system escape.

Oral Antiviral Defense: Saliva- and Beverage-like Hypotonicity Dynamically Regulate Formation of Membraneless Biomolecular Condensates of Antiviral Human MxA in Oral Epithelial Cells.

The oral mucosa represents a defensive barrier between the external environment and the rest of the body. Oral mucosal cells are constantly bathed in hypotonic saliva (normally one-third tonicity compared to plasma) and are repeatedly exposed to environmental stresses of tonicity, temperature, and pH by the drinks we imbibe (e.g., hypotonic: water, tea, and coffee; hypertonic: assorted fruit juices, and red wines). In the mouth, the broad-spectrum antiviral mediator MxA (a dynamin-family large GTPase) is constitutively expressed in healthy periodontal tissues and induced by Type III interferons (e.g., IFN-λ1/IL-29). Endogenously induced human MxA and exogenously expressed human GFP-MxA formed membraneless biomolecular condensates in the cytoplasm of oral carcinoma cells (OECM1 cell line). These condensates likely represent storage granules in equilibrium with antivirally active dispersed MxA. Remarkably, cytoplasmic MxA condensates were exquisitely sensitive sensors of hypotonicity-the condensates in oral epithelium disassembled within 1-2 min of exposure of cells to saliva-like one-third hypotonicity, and spontaneously reassembled in the next 4-7 min. Water, tea, and coffee enhanced this disassembly. Fluorescence changes in OECM1 cells preloaded with calcein-AM (a reporter of cytosolic "macromolecular crowding") confirmed that this process involved macromolecular uncrowding and subsequent recrowding secondary to changes in cell volume. However, hypertonicity had little effect on MxA condensates. The spontaneous reassembly of GFP-MxA condensates in oral epithelial cells, even under continuous saliva-like hypotonicity, was slowed by the protein-phosphatase-inhibitor cyclosporin A (CsA) and by the K-channel-blocker tetraethylammonium chloride (TEA); this is suggestive of the involvement of the volume-sensitive WNK kinase-protein phosphatase (PTP)-K-Cl cotransporter (KCC) pathway in the regulated volume decrease (RVD) during condensate reassembly in oral cells. The present study identifies a novel subcellular consequence of hypotonic stress in oral epithelial cells, in terms of the rapid and dynamic changes in the structure of one class of phase-separated biomolecular condensates in the cytoplasm-the antiviral MxA condensates. More generally, the data raise the possibility that hypotonicity-driven stresses likely affect other intracellular functions involving liquid-liquid phase separation (LLPS) in cells of the oral mucosa.

GTPase activity of porcine Mx1 plays a dominant role in inhibiting the N-Nsp9 interaction and thus inhibiting PRRSV replication.

Porcine Mx1 is a type of interferon-induced GTPase that inhibits the replication of certain RNA viruses. However, the antiviral effects and the underlying mechanism of porcine Mx1 for porcine reproductive and respiratory syndrome virus (PRRSV) remain unknown. In this study, we demonstrated that porcine Mx1 could significantly inhibit PRRSV replication in MARC-145 cells. By Mx1 segment analysis, it was indicated that the GTPase domain (68-341aa) was the functional area to inhibit PRRSV replication and that Mx1 interacted with the PRRSV-N protein through the GTPase domain (68-341aa) in the cytoplasm. Amino acid residues K295 and K299 in the G domain of Mx1 were the key sites for Mx1-N interaction while mutant proteins Mx1(K295A) and Mx1(K299A) still partially inhibited PRRSV replication. Furthermore, we found that the GTPase activity of Mx1 was dominant for Mx1 to inhibit PRRSV replication but was not essential for Mx1-N interaction. Finally, mechanistic studies demonstrated that the GTPase activity of Mx1 played a dominant role in inhibiting the N-Nsp9 interaction and that the interaction between Mx1 and N partially inhibited the N-Nsp9 interaction. We propose that the complete anti-PRRSV mechanism of porcine Mx1 contains a two-step process: Mx1 binds to the PRRSV-N protein and subsequently disrupts the N-Nsp9 interaction by a process requiring the GTPase activity of Mx1. Taken together, the results of our experiments describe for the first time a novel mechanism by which porcine Mx1 evolves to inhibit PRRSV replication. IMPORTANCE: Mx1 protein is a key mediator of the interferon-induced antiviral response against a wide range of viruses. How porcine Mx1 affects the replication of porcine reproductive and respiratory syndrome virus (PRRSV) and its biological function has not been studied. Here, we show that Mx1 protein inhibits PRRSV replication by interfering with N-Nsp9 interaction. Furthermore, the GTPase activity of porcine Mx1 plays a dominant role and the Mx1-N interaction plays an assistant role in this interference process. This study uncovers a novel mechanism evolved by porcine Mx1 to exert anti-PRRSV activities.

Virus specificity and nucleoporin requirements for MX2 activity are affected by GTPase function and capsid-CypA interactions.

Human myxovirus resistance 2 (MX2/MXB) is an interferon-induced GTPase that inhibits human immunodeficiency virus-1 (HIV-1) infection by preventing nuclear import of the viral preintegration complex. The HIV-1 capsid (CA) is the major viral determinant for sensitivity to MX2, and complex interactions between MX2, CA, nucleoporins (Nups), cyclophilin A (CypA), and other cellular proteins influence the outcome of viral infection. To explore the interactions between MX2, the viral CA, and CypA, we utilized a CRISPR-Cas9/AAV approach to generate CypA knock-out cell lines as well as cells that express CypA from its endogenous locus, but with specific point mutations that would abrogate CA binding but should not affect enzymatic activity or cellular function. We found that infection of CypA knock-out and point mutant cell lines with wild-type HIV-1 and CA mutants recapitulated the phenotypes observed upon cyclosporine A (CsA) addition, indicating that effects of CsA treatment are the direct result of blocking CA-CypA interactions and are therefore independent from potential interactions between CypA and MX2 or other cellular proteins. Notably, abrogation of GTP hydrolysis by MX2 conferred enhanced antiviral activity when CA-CypA interactions were abolished, and this effect was not mediated by the CA-binding residues in the GTPase domain, or by phosphorylation of MX2 at position T151. We additionally found that elimination of GTPase activity also altered the Nup requirements for MX2 activity. Our data demonstrate that the antiviral activity of MX2 is affected by CypA-CA interactions in a virus-specific and GTPase activity-dependent manner. These findings further highlight the importance of the GTPase domain of MX2 in regulation of substrate specificity and interaction with nucleocytoplasmic trafficking pathways.