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.

Polymorphisms of IFN signaling genes and FOXP4 influence the severity of COVID-19.

BACKGROUND: The clinical manifestations of COVID-19 range from asymptomatic, mild to moderate, severe, and critical disease. Host genetic variants were recognized to affect the disease severity. However, the genetic landscape differs among various populations. Therefore, we explored the variants associated with COVID-19 severity in the Guangdong population. METHODS: A total of 314 subjects were selected, of which the severe and critical COVID-19 patients were defined as "cases", and the mild and moderate patients were defined as "control". Twenty-two variants in interferon-related genes and FOXP4 were genotyped using the MassARRAY technology platform. RESULTS: IFN signaling gene MX1 rs17000900 CA + AA genotype was correlated with a reduced risk of severe COVID-19 in males (P = 0.001, OR = 0.050, 95%CI = 0.008-0.316). The AT haplotype comprised of MX1 rs17000900 and rs2071430 was more likely to protect against COVID-19 severity (P = 6.3E-03). FOXP4 rs1886814 CC genotype (P = 0.001, OR = 3.747, 95%CI = 1.746-8.043) and rs2894439 GA + AA genotype (P = 0.001, OR = 5.703, 95% CI = 2.045-15.903) were correlated with increased risk of severe COVID-19. Haplotype CA comprised of rs1886814 and rs2894439 was found to be correlated with adverse outcomes (P = 7.0E-04). FOXP4 rs1886814 CC (P = 0.0004) and rs2894439 GA + AA carriers had higher neutralizing antibody titers (P = 0.0018). The CA + AA genotype of MX1 rs17000900 tended to be correlated with lower neutralizing antibody titers than CC genotype (P = 0.0663), but the difference was not statistically significant. CONCLUSION: Our study found a possible association between MX1 and FOXP4 polymorphisms and the severity of COVID-19. Distinguishing high-risk patients who develop severe COVID-19 will provide clues for early intervention and individual treatment strategies.

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.

MX1 and UBE2L6 are potential metaflammation gene targets in both diabetes and atherosclerosis.

Background: The coexistence of diabetes mellitus (DM) and atherosclerosis (AS) is widespread, although the explicit metabolism and metabolism-associated molecular patterns (MAMPs) responsible for the correlation are still unclear. Methods: Twenty-four genetically wild-type male Ba-Ma mini pigs were randomly divided into five groups distinguished by different combinations of 90 mg/kg streptozotocin (STZ) intravenous injection and high-cholesterol/lipid (HC) or high-lipid (HL) diet feeding for 9 months in total. Pigs in the STZ+HC and STZ+HL groups were injected with STZ first and then fed the HC or HL diet for 9 months. In contrast, pigs in the HC+STZ and HL+STZ groups were fed the HC or HL diet for 9 months and injected with STZ at 3 months. The controls were only fed a regular diet for 9 months. The blood glucose and abdominal aortic plaque observed through oil red O staining were used as evaluation indicators for successful modelling of DM and AS. A microarray gene expression analysis of all subjects was performed. Results: Atherosclerotic lesions were observed only in the HC+STZ and STZ+HC groups. A total of 103 differentially expressed genes (DEGs) were identified as common between them. The most significantly enriched pathways of 103 common DEGs were influenza A, hepatitis C, and measles. The global and internal protein-protein interaction (PPI) networks of the 103 common DEGs consisted of 648 and 14 nodes, respectively. The top 10 hub proteins, namely, ISG15, IRG6, IRF7, IFIT3, MX1, UBE2L6, DDX58, IFIT2, USP18, and IFI44L, drive aspects of DM and AS. MX1 and UBE2L6 were the intersection of internal and global PPI networks. The expression of MX1 and UBE2L6 was 507.22 ± 342.56 and 96.99 ± 49.92 in the HC+STZ group, respectively, which was significantly higher than others and may be linked to the severity of hyperglycaemia-related atherosclerosis. Further PPI network analysis of calcium/micronutrients, including MX1 and UBE2L6, consisted of 58 and 18 nodes, respectively. The most significantly enriched KEGG pathways were glutathione metabolism, pyrimidine metabolism, purine metabolism, and metabolic pathways. Conclusions: The global and internal PPI network of the 103 common DEGs consisted of 648 and 14 nodes, respectively. The intersection of the nodes of internal and global PPI networks was MX1 and UBE2L6, suggesting their key role in the comorbidity mechanism of DM and AS. This inference was partly verified by the overexpression of MX1 and UBE2L6 in the HC+STZ group but not others. Further calcium- and micronutrient-related enriched KEGG pathway analysis supported that MX1 and UBE2L6 may affect the inflammatory response through micronutrient metabolic pathways, conceptually named metaflammation. Collectively, MX1 and UBE2L6 may be potential common biomarkers for DM and AS that may reveal metaflammatory aspects of the pathological process, although proper validation is still needed to determine their contribution to the detailed mechanism.

Integration of ATAC-seq and RNA-seq identifies MX1-mediated AP-1 transcriptional regulation as a therapeutic target for Down syndrome.

BACKGROUND: Growing evidence has suggested that Type I Interferon (I-IFN) plays a potential role in the pathogenesis of Down Syndrome (DS). This work investigates the underlying function of MX1, an effector gene of I-IFN, in DS-associated transcriptional regulation and phenotypic modulation. METHODS: We performed assay for transposase-accessible chromatin with high-throughout sequencing (ATAC-seq) to explore the difference of chromatin accessibility between DS derived amniocytes (DSACs) and controls. We then combined the annotated differentially expressed genes (DEGs) and enriched transcriptional factors (TFs) targeting the promoter region from ATAC-seq results with the DEGs in RNA-seq, to identify key genes and pathways involved in alterations of biological processes and pathways in DS. RESULTS: Binding motif analysis showed a significant increase in chromatin accessibility of genes related to neural cell function, among others, in DSACs, which is primarily regulated by members of the activator protein-1 (AP-1) transcriptional factor family. Further studies indicated that MX Dynamin Like GTPase 1 (MX1), defined as one of the key effector genes of I-IFN, is a critical upstream regulator. Its overexpression induced expression of AP-1 TFs and mediated inflammatory response, thus leading to decreased cellular viability of DS cells. Moreover, treatment with specific AP-1 inhibitor T-5224 improved DS-associated phenotypes in DSACs. CONCLUSIONS: This study demonstrates that MX1-mediated AP-1 activation is partially responsible for cellular dysfunction of DS. T-5224 effectively ameliorated DS-associated phenotypes in DSACs, suggesting it as a potential treatment option for DS patients.

A single polymorphic residue in humans underlies species-specific restriction of HSV-1 by the antiviral protein MxB.

IMPORTANCE: Herpesviruses present a major global disease burden. Understanding the host cell mechanisms that block viral infections, as well as how viruses can evolve to counteract these host defenses, is critically important for understanding viral disease pathogenesis. This study reveals that the major human variant of the antiviral protein myxovirus resistance protein B (MxB) inhibits the human pathogen herpes simplex virus (HSV-1), whereas a minor human variant and orthologous MxB genes from even closely related primates do not. Thus, in contrast to the many antagonistic virus-host interactions in which the virus is successful in thwarting the host's defense systems, here the human gene appears to be at least temporarily winning at this interface of the primate-herpesvirus evolutionary arms race. Our findings further show that a polymorphism at amino acid 83 in a small fraction of the human population is sufficient to abrogate MxB's ability to inhibit HSV-1, which could have important implications for human susceptibility to HSV-1 pathogenesis.

Anti-Schmallenberg Virus Activities of Type I/III Interferons-Induced Mx1 GTPases from Different Mammalian Species.

Mx proteins are key factors of the innate intracellular defense mechanisms that act against viruses induced by type I/III interferons. The family Peribunyaviridae includes many viruses of veterinary importance, either because infection results in clinical disease or because animals serve as reservoirs for arthropod vectors. According to the evolutionary arms race hypothesis, evolutionary pressures should have led to the selection of the most appropriate Mx1 antiviral isoforms to resist these infections. Although human, mouse, bat, rat, and cotton rat Mx isoforms have been shown to inhibit different members of the Peribunyaviridae, the possible antiviral function of the Mx isoforms from domestic animals against bunyaviral infections has, to our knowledge, never been studied. Herein, we investigated the anti-Schmallenberg virus activity of bovine, canine, equine, and porcine Mx1 proteins. We concluded that Mx1 has a strong, dose-dependent anti-Schmallenberg activity in these four mammalian species.

The Antiviral Activity of Equine Mx1 against Thogoto Virus Is Determined by the Molecular Structure of Its Viral Specificity Region.

Mammalian myxovirus resistance (Mx) proteins are interferon-induced, large dynamin-like GTPases with a broad antiviral spectrum. Here, we analyzed the antiviral activity of selected mammalian Mx1 proteins against Thogoto virus (THOV). Of those, equine Mx1 (eqMx1) showed antiviral activity comparable to that of the human MX1 gene product, designated huMxA, whereas most Mx1 proteins were antivirally inactive. We previously demonstrated that the flexible loop L4 protruding from the stalk domain of huMxA, and especially the phenylalanine at position 561 (F561), determines its antiviral specificity against THOV (P. S. Mitchell, C. Patzina, M. Emerman, O. Haller, et al., Cell Host Microbe 12:598-604, 2012, https://doi.org/10.1016/j.chom.2012.09.005). However, despite the similar antiviral activity against THOV, the loop L4 sequence of eqMx1 substantially differs from the one of huMxA. Mutational analysis of eqMx1 L4 identified a tryptophan (W562) and the adjacent glycine (G563) as critical antiviral determinants against THOV, whereas the neighboring residues could be exchanged for nonpolar alanines without affecting the antiviral activity. Further mutational analyses revealed that a single bulky residue at position 562 and the adjacent tiny residue G563 were sufficient for antiviral activity. Moreover, this minimal set of L4 amino acids transferred anti-THOV activity to the otherwise inactive bovine Mx1 (boMx1) protein. Taken together, our data suggest a fairly simple architecture of the antiviral loop L4 that could serve as a mutational hot spot in an evolutionary arms race between Mx-escaping viral variants and their hosts. IMPORTANCE Most mammals encode two paralogs of the interferon-induced Mx proteins: Mx1, with antiviral activity largely against RNA viruses, like orthomyxoviruses and bunyaviruses; and Mx2, which is antivirally active against HIV-1 and herpesviruses. The human Mx1 protein, also called huMxA, is the best-characterized example of mammalian Mx1 proteins and was recently shown to prevent zoonotic virus transmissions. To evaluate the antiviral activity of other mammalian Mx1 proteins, we used Thogoto virus, a tick-transmitted orthomyxovirus, which is efficiently blocked by huMxA. Interestingly, we detected antiviral activity only with equine Mx1 (eqMx1) but not with other nonprimate Mx1 proteins. Detailed functional analysis of eqMx1 identified amino acid residues in the unstructured loop L4 of the stalk domain critical for antiviral activity. The structural insights of the present study explain the unique position of eqMx1 antiviral activity within the collection of nonhuman mammalian Mx1 proteins.

An evolutionarily conserved N-terminal leucine is essential for MX1 GTPase antiviral activity against different families of RNA viruses.

Myxovirus resistance protein 1 (MX1) and MX2 are homologous, dynamin-like large GTPases, induced upon interferon exposure. Human MX1 (HsMX1) is known to inhibit many viruses, including influenza A virus, by likely acting at various steps of their life cycles. Despite decades of studies, the mechanism(s) of action with which MX1 proteins manage to inhibit target viruses is not fully understood. MX1 proteins are mechano-enzymes and share a similar organization to dynamin, with a GTPase domain and a carboxy-terminal stalk domain, connected by a bundle signaling element. These three elements are known to be essential for antiviral activity. HsMX1 has two unstructured regions, the L4 loop, also essential for antiviral activity, and a short amino (N)-terminal region, which greatly varies between MX1 proteins of different species. The role of this N-terminal domain in antiviral activity is not known. Herein, using mutagenesis, imaging, and biochemical approaches, we demonstrate that the N-terminal domain of HsMX1 is essential for antiviral activity against influenza A virus, Vesicular Stomatitis Virus, and La Crosse virus. Furthermore, we pinpoint a highly conserved leucine within this region, which is absolutely crucial for human, mouse, and bat MX1 protein antiviral activity. Importantly, mutation of this leucine does not compromise GTPase activity or oligomerization capabilities but does modify MX1 protein subcellular localization. The discovery of this essential and highly conserved residue defines this region as key for antiviral activity and may reveal insights as to the mechanism(s) of action of MX1 proteins.

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.

Increased Polymerase Activity of Zoonotic H7N9 Allows Partial Escape from MxA.

The interferon-induced myxovirus resistance protein A (MxA) is a potent restriction factor that prevents zoonotic infection from influenza A virus (IAV) subtype H7N9. Individuals expressing antivirally inactive MxA variants are highly susceptible to these infections. However, human-adapted IAVs have acquired specific mutations in the viral nucleoprotein (NP) that allow escape from MxA-mediated restriction but that have not been observed in MxA-sensitive, human H7N9 isolates. To date, it is unknown whether H7N9 can adapt to escape MxA-mediated restriction. To study this, we infected Rag2-knockout (Rag2-/-) mice with a defect in T and B cell maturation carrying a human MxA transgene (MxAtg/-Rag2-/-). In these mice, the virus could replicate for several weeks facilitating host adaptation. In MxAtg/-Rag2-/-, but not in Rag2-/- mice, the well-described mammalian adaptation E627K in the viral polymerase subunit PB2 was acquired, but no variants with MxA escape mutations in NP were detected. Utilizing reverse genetics, we could show that acquisition of PB2 E627K allowed partial evasion from MxA restriction in MxAtg/tg mice. However, pretreatment with type I interferon decreased viral replication in these mice, suggesting that PB2 E627K is not a true MxA escape mutation. Based on these results, we speculate that it might be difficult for H7N9 to acquire MxA escape mutations in the viral NP. This is consistent with previous findings showing that MxA escape mutations cause severe attenuation of IAVs of avian origin.

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.

Salmonid alphavirus non-structural protein 2 is a key protein that activates the NF-κB signaling pathway to mediate inflammatory responses.

Salmonid alphavirus (SAV) infection of Atlantic salmon (Salmo salar) and rainbow trout (Oncorhynchus mykiss) causes pancreas disease (PD) with typical inflammatory responses, such as necrosis of the exocrine pancreas, cardiomyopathy and skeletal myopathy. However, the pathogenic mechanism underlying SAV infection is still unclear. Inflammation may cause damage to the body, but it is a defense response against infection by pathogenic microorganisms, of which nuclear factor-kappa B (NF-κB) is the main regulator. This study revealed that SAV can activate NF-κB, of which the viral nonstructural protein Nsp2 is the major activating protein. SAV activates the NF-κB signaling pathway by simultaneously up-regulating TLR3, 7, 8 and then the expression of the signaling molecule myeloid differentiation factor 88 (Myd88) and tumor necrosis factor receptor-associated factor 6 (TRAF6). We found that Nsp2 can induce IκB degradation and p65 phosphorylation and transnucleation, and activate NF-κB downstream inflammatory cytokines. Nsp2 may simultaneously activate NF-κB through TLR3,7,8-dependent signaling pathways. Overexpression of Nsp2 can up-regulate mitochondrial antiviral signaling protein (MAVS) and then promote the expression of IFNa1 and antiviral protein Mx, which inhibits viral replication. This study shows that Nsp2 acts as a key activator protein for the NF-κB signaling pathway, which induces inflammation post-SAV infection. This study systematically analyzes the molecular mechanism of SAV activation of the NF-κB signaling pathway, and provides a theoretical basis for revealing the mechanism of innate immune response and inflammatory injury caused by SAV.

Antitumour Effects of Selected Pyridinium Salts on Sensitive Leukaemia HL60 Cells and Their Multidrug Resistant Topoisomerase II-Defective HL60/MX2 Counterparts.

Multidrug resistance (MDR), having a multifactorial nature, is one of the major clinical problems causing the failure of anticancer therapy. The aim of this study was to examine the antitumour effects of selected pyridinium salts, 1-methyl-3-nitropyridine chloride (MNP) and 3,3,6,6,10-pentamethyl-3,4,6,7-tetrahydro-[1,8(2H,5H)-dion]acridine chloride (MDION), on sensitive leukaemia HL60 cells and resistant topoisomerase II-defective HL60/MX2 cells. Cell growth was determined by the MTT test. Intracellular ROS level was measured with the aid of 2',7'-DCF-DA. The cell cycle distribution was investigated by performing PI staining. DSB formation was examined using the γ-H2AX histone phosphorylation assay. The activity of caspase-3 and caspase-8 was measured with the use of the FLICA test. The assays for examining the lysosome membrane permeabilization were carried out with the aid of LysoTracker Green DND-26. Both studied compounds exerted very similar cytotoxic activities towards sensitive HL60 cells and their MDR counterparts. They modulated the cellular ROS level in a dose-dependent and time-dependent manner and significantly increased the percentage of sensitive HL60 and resistant HL60/MX2 cells with sub-diploid DNA (sub-G1 fraction). However, the induction of DSB formation was not a significant mechanism of action of these pyridinium salts in studied cells. Both examined compounds triggered caspase-3/caspase-8-dependent apoptosis of sensitive HL60 cells and their MDR counterparts. Additionally, the findings of the study indicate that lysosomes may also participate in the programmed death of HL60 as well as HL60/MX2 cells induced by MDION. The data obtained in this work showed that both examined pyridinium salts, MNP and MDION, are able to retain high antileukaemic effects against multidrug resistant topoisomerase II-defective HL60/MX2 cells.

MX2 Viral Substrate Breadth and Inhibitory Activity Are Regulated by Protein Phosphorylation.

Human immunodeficiency virus type-1 (HIV-1) infection is potently inhibited by human myxovirus resistance 2 (MX2/MxB), which binds to the viral capsid and blocks the nuclear import of viral DNA. We have recently shown that phosphorylation is a key regulator of MX2 antiviral activity, with phosphorylation of serine residues at positions 14, 17, and 18 repressing MX2 function. Here, we extend the study of MX2 posttranslational modifications and identify serine and threonine phosphorylation in all domains of MX2. By substituting these residues with aspartic acid or alanine, hence mimicking the presence or absence of a phosphate group, respectively, we identified key positions that control MX2 antiviral activity. Aspartic acid substitutions of residues Ser306 or Thr334 and alanine substitutions of Thr343 yielded proteins with substantially reduced antiviral activity, whereas the presence of aspartic acid at positions Ser28, Thr151, or Thr343 resulted in enhanced activity: referred to as hypermorphic mutants. In some cases, these hypermorphic mutations, particularly when paired with other MX2 mutations (e.g., S28D/T151D or T151D/T343A) acquired the capacity to inhibit HIV-1 capsid mutants known to be insensitive to wild-type MX2, such as P90A or T210K, as well as MX2-resistant retroviruses such as equine infectious anemia virus (EIAV) and murine leukemia virus (MLV). This work highlights the complexity and importance of MX2 phosphorylation in the regulation of antiviral activity and in the selection of susceptible viral substrates. IMPORTANCE Productive infection by human immunodeficiency virus type-1 (HIV-1) requires the import of viral replication complexes into the nuclei of infected cells. Myxovirus resistance 2 (MX2/MxB) blocks this step, halting nuclear accumulation of viral DNA and virus replication. We recently demonstrated how phosphorylation of a stretch of three serines in the amino-terminal domain of MX2 inhibits the antiviral activity. Here, we identify additional positions in MX2 whose phosphorylation status reduces or enhances antiviral function (hypomorphic and hypermorphic variants, respectively). Importantly, hypermorphic mutant proteins not only increased inhibitory activity against wild-type HIV-1 but can also exhibit antiviral capabilities against HIV-1 capsid mutant viruses that are resistant to wild-type MX2. Furthermore, some of these proteins were also able to inhibit retroviruses that are insensitive to MX2. Therefore, we propose that phosphorylation comprises a major element of MX2 regulation and substrate determination.

Mouse Mx1 Inhibits Herpes Simplex Virus Type 1 Genomic Replication and Late Gene Expression In Vitro and Prevents Lesion Formation in the Mouse Zosteriform Model.

Myxovirus resistance (Mx) proteins are dynamin-like GTPases that are inducible by interferons (IFNs) following virus infections. Most studies investigating Mx proteins have focused on their activity against influenza A viruses (IAV), although emerging evidence suggests that some Mx proteins may exhibit broader antiviral activity. Herein, we demonstrate that in addition to IAV, overexpression of mouse Mx1 (mMx1), but not mMx2, resulted in potent inhibition of growth of the human alphaherpesviruses herpes simplex virus 1 (HSV-1) and HSV-2, whereas neither inhibited the mouse betaherpesvirus murine cytomegalovirus (MCMV) in vitro. IFN induction of a functional endogenous mMx1 in primary mouse fibroblasts ex vivo was also associated with inhibition of HSV-1 growth. Using an in vitro overexpression approach, we demonstrate that mutations that result in redistribution of mMx1 from the nucleus to the cytoplasm or in loss of its combined GTP binding and GTPase activity also abrogated its ability to inhibit HSV-1 growth. Overexpressed mMx1 did not inhibit early HSV-1 gene expression but was shown to inhibit both replication of the HSV-1 genome as well as subsequent late gene expression. In a mouse model of cutaneous HSV-1 infection, mice expressing a functional endogenous mMx1 showed significant reductions in the severity of skin lesions as well as reduced HSV-1 titers in both the skin and dorsal root ganglia (DRG). Together, these data demonstrate that mMx1 mediates potent antiviral activity against human alphaherpesviruses by blocking replication of the viral genome and subsequent steps in virus replication. Moreover, endogenous mMx1 potently inhibited pathogenesis in the zosteriform mouse model of HSV-1 infection. IMPORTANCE While a number of studies have demonstrated that human Mx proteins can inhibit particular herpesviruses in vitro, we are the first to report the antiviral activity of mouse Mx1 (mMx1) against alphaherpesviruses both in vitro and in vivo. We demonstrate that both overexpressed mMx1 and endogenous mMx1 potently restrict HSV-1 growth in vitro. mMx1-mediated inhibition of HSV-1 was not associated with inhibition of virus entry and/or import of the viral genome into the nucleus, but rather with inhibition of HSV-1 genomic replication as well as subsequent late gene expression. Therefore, inhibition of human alphaherpesviruses by mMx1 occurs by a mechanism that is distinct from that reported for human Mx proteins against herpesviruses. Importantly, we also provide evidence that expression of a functional endogenous mMx1 can limit HSV-1 pathogenesis in a mouse model of infection.

MxB inhibits long interspersed element type 1 retrotransposition.

Long interspersed element type 1 (LINE-1, also L1 for short) is the only autonomously transposable element in the human genome. Its insertion into a new genomic site may disrupt the function of genes, potentially causing genetic diseases. Cells have thus evolved a battery of mechanisms to tightly control LINE-1 activity. Here, we report that a cellular antiviral protein, myxovirus resistance protein B (MxB), restricts the mobilization of LINE-1. This function of MxB requires the nuclear localization signal located at its N-terminus, its GTPase activity and its ability to form oligomers. We further found that MxB associates with LINE-1 protein ORF1p and promotes sequestration of ORF1p to G3BP1-containing cytoplasmic granules. Since knockdown of stress granule marker proteins G3BP1 or TIA1 abolishes MxB inhibition of LINE-1, we conclude that MxB engages stress granule components to effectively sequester LINE-1 proteins within the cytoplasmic granules, thus hindering LINE-1 from accessing the nucleus to complete retrotransposition. Thus, MxB protein provides one mechanism for cells to control the mobility of retroelements.

Fifty Shades of Erns: Innate Immune Evasion by the Viral Endonucleases of All Pestivirus Species.

The genus Pestivirus, family Flaviviridae, includes four historically accepted species, i.e., bovine viral diarrhea virus (BVDV)-1 and -2, classical swine fever virus (CSFV), and border disease virus (BDV). A large number of new pestivirus species were identified in recent years. A common feature of most members is the presence of two unique proteins, Npro and Erns, that pestiviruses evolved to regulate the host's innate immune response. In addition to its function as a structural envelope glycoprotein, Erns is also released in the extracellular space, where it is endocytosed by neighboring cells. As an endoribonuclease, Erns is able to cleave viral ss- and dsRNAs, thus preventing the stimulation of the host's interferon (IFN) response. Here, we characterize the basic features of soluble Erns of a large variety of classified and unassigned pestiviruses that have not yet been described. Its ability to form homodimers, its RNase activity, and the ability to inhibit dsRNA-induced IFN synthesis were investigated. Overall, we found large differences between the various Erns proteins that cannot be predicted solely based on their primary amino acid sequences, and that might be the consequence of different virus-host co-evolution histories. This provides valuable information to delineate the structure-function relationship of pestiviral endoribonucleases.

Rapid, efficient and activation-neutral gene editing of polyclonal primary human resting CD4+ T cells allows complex functional analyses.

CD4+ T cells are central mediators of adaptive and innate immune responses and constitute a major reservoir for human immunodeficiency virus (HIV) in vivo. Detailed investigations of resting human CD4+ T cells have been precluded by the absence of efficient approaches for genetic manipulation limiting our understanding of HIV replication and restricting efforts to find a cure. Here we report a method for rapid, efficient, activation-neutral gene editing of resting, polyclonal human CD4+ T cells using optimized cell cultivation and nucleofection conditions of Cas9-guide RNA ribonucleoprotein complexes. Up to six genes, including HIV dependency and restriction factors, were knocked out individually or simultaneously and functionally characterized. Moreover, we demonstrate the knock in of double-stranded DNA donor templates into different endogenous loci, enabling the study of the physiological interplay of cellular and viral components at single-cell resolution. Together, this technique allows improved molecular and functional characterizations of HIV biology and general immune functions in resting CD4+ T cells.