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The type I interferon (IFN) system plays an important role in controlling herpesvirus infections, but it is unclear which IFN-mediated effectors interfere with herpesvirus replication. Here we report that human myxovirus resistance protein B (MxB, also designated Mx2) is a potent human herpesvirus restriction factor in the context of IFN. We demonstrate that ectopic MxB expression restricts a range of herpesviruses from the Alphaherpesvirinae and Gammaherpesvirinae , including herpes simplex virus 1 and 2 (HSV-1 and HSV-2), and Kaposi’s sarcoma-associated herpesvirus (KSHV). MxB restriction of HSV-1 and HSV-2 requires GTPase function, in contrast to restriction of lentiviruses. MxB inhibits the delivery of incoming HSV-1 DNA to the nucleus and the appearance of empty capsids, but not the capsid delivery to the cytoplasm or tegument dissociation from the capsid. Our study identifies MxB as a potent pan-herpesvirus restriction factor which blocks the uncoating of viral DNA from the incoming viral capsid. MxB is an interferon-induced GTPase that inhibits HIV replication. Here, Crameri et al. show that MxB restricts replication of herpesviruses by inhibiting delivery of incoming viral DNA into the nucleus, and this antiviral activity depends on MxB’s GTPase activity.

Interferon (IFN) inhibits HIV replication by inducing antiviral effectors. To comprehensively identify IFN-induced HIV restriction factors, we assembled a CRISPR sgRNA library of Interferon Stimulated Genes (ISGs) into a modified lentiviral vector that allows for packaging of sgRNA-encoding genomes in trans into budding HIV-1 particles. We observed that knockout of Zinc Antiviral Protein (ZAP) improved the performance of the screen due to ZAP-mediated inhibition of the vector. A small panel of IFN-induced HIV restriction factors, including MxB, IFITM1, Tetherin/BST2 and TRIM5alpha together explain the inhibitory effects of IFN on the CXCR4-tropic HIV-1 strain, HIV-1LAI, in THP-1 cells. A second screen with a CCR5-tropic primary strain, HIV-1Q23.BG505, described an overlapping, but non-identical, panel of restriction factors. Further, this screen also identifies HIV dependency factors. The ability of IFN-induced restriction factors to inhibit HIV strains to replicate in human cells suggests that these human restriction factors are incompletely antagonized. Editorial note: This article has been through an editorial process in which the authors decide how to respond to the issues raised during peer review. The Reviewing Editor's assessment is that all the issues have been addressed (see decision letter ).

Small fiber neuropathy (SFN) affects small-diameter sensory and autonomic nerve fibers, leading to chronic pain and autonomic dysfunction. While SFN can be associated with diabetes and autoimmune diseases, a significant proportion of cases are idiopathic. Although immune-mediated mechanisms are being recognized increasingly in SFN, their precise role remains unclear. This study investigates the presence of autoantibodies against interferon-induced GTP-binding protein MX (MX1) in SFN patients and explores their potential pathogenic role. A total of 59 patients with skin biopsy-confirmed SFN and 20 healthy controls were recruited. Serum samples were analyzed for the presence of anti-MX1 autoantibodies using enzyme-linked immunosorbent assay (ELISA). Immunohistochemistry was performed on rat sciatic nerves to assess the localization of patient IgG to unmyelinated nerve fibers, and immunocytochemistry and flow cytometry confirmed specific binding to MX1. Functional characterization of MX1 was conducted using whole-cell patch-clamp recordings in dorsal root ganglion (DRG) neurons overexpressing MX1. Additionally, protein interactions between MX1 and transient receptor potential cation channel subfamily C member 6 (TRPC6) were assessed using co-immunoprecipitation and surface biotinylation assays. Anti-MX1 autoantibody levels were significantly elevated in SFN patients compared to controls ( p  = 0.0278), particularly in the autoimmune SFN subgroup. Patient sera exhibited IgG binding to unmyelinated nerve fibers, with idiopathic and autoimmune SFN cases showing similar staining patterns, suggesting a similar immune-mediated mechanism. Immunocytochemistry showed binding to HEK293-MX1 cells and flow cytometry revealed higher MX1/WT fluorescence intensity ratios in patient sera, further confirming specific immune recognition of MX1. Patch-clamp recordings demonstrated that MX1 overexpression in DRG neurons led to significant membrane depolarization and increased action potential firing frequency ( p  < 0.0001), indicating heightened neuronal excitability. However, MX1 did not directly interact with TRPC6 or alter its function, suggesting an alternative pathway for its effects. The addition of anti-MX1 IgG did not further modify DRG electrophysiology, implying that the autoimmune component may contribute to SFN pathogenesis through indirect mechanisms. Our findings support the hypothesis that MX1 influences neuronal excitability and plays a role in SFN pathophysiology. Future studies should validate these findings in larger cohorts and explore potential therapeutic strategies targeting MX1-associated pathways in SFN.

Antagonistic interactions drive host-virus evolutionary arms races, which often manifest as recurrent amino acid changes (i.e., positive selection) at their protein-protein interaction interfaces. Here, we investigated whether combinatorial mutagenesis of positions under positive selection in a host antiviral protein could enhance its restrictive properties. We tested approximately 700 variants of human MxA, generated by combinatorial mutagenesis, for their ability to restrict Thogotovirus (THOV). We identified MxA super-restrictors with increased binding to the THOV nucleoprotein (NP) target protein and 10-fold higher anti-THOV restriction relative to wild-type human MxA, the most potent naturally occurring anti-THOV restrictor identified. Our findings reveal a means to elicit super-restrictor antiviral proteins by leveraging signatures of positive selection. Although some MxA super-restrictors of THOV were impaired in their restriction of H5N1 influenza A virus (IAV), other super-restrictor variants increased THOV restriction without impairment of IAV restriction. Thus, broadly acting antiviral proteins such as MxA mitigate breadth-versus-specificity trade-offs that could otherwise constrain their adaptive landscape.

Dynamin-like GTPase myxovirus resistance protein 1 (Mx1) is an intracellular anti-viral protein following the activation of type I and type III interferon signaling. Mx1 inhibits viral replication by blocking the transcription of viral RNA, and a deficiency in this protein enhances susceptibility to influenza infection. Thus, Mx1 could be another efficient target of anti-influenza therapy. To test our hypothesis, we fused poly-arginine cell-penetrating peptides to the C terminus of Mx1 (Mx1-9R) and examined the anti-viral activity of Mx1-9R in vitro and in vivo. Madin-Darby Canine Kidney epithelial cells internalized the Mx1-9R within 12 h. Pre-exposure Mx1-9R treatment inhibited viral replication and viral RNA expression in infected cells. Further, intranasal administration of Mx1-9R improved the survival of mice infected with the PR8 influenza viral strain. These data support the consideration of Mx1-9R as a novel therapeutic agent against mucosal influenza virus infection.

The innate-immune restriction factor MxA inhibits influenza replication by targeting the viral nucleoprotein (NP). Human influenza virus is more resistant than avian influenza virus to inhibition by human MxA, and prior work has compared human and avian viral strains to identify amino-acid differences in NP that affect sensitivity to MxA. However, this strategy is limited to identifying sites in NP where mutations that affect MxA sensitivity have fixed during the small number of documented zoonotic transmissions of influenza to humans. Here we use an unbiased deep mutational scanning approach to quantify how all single amino-acid mutations to NP affect MxA sensitivity in the context of replication-competent virus. We both identify new sites in NP where mutations affect MxA resistance and re-identify mutations known to have increased MxA resistance during historical adaptations of influenza to humans. Most of the sites where mutations have the greatest effect are almost completely conserved across all influenza A viruses, and the amino acids at these sites confer relatively high resistance to MxA. These sites cluster in regions of NP that appear to be important for its recognition by MxA. Overall, our work systematically identifies the sites in influenza nucleoprotein where mutations affect sensitivity to MxA. We also demonstrate a powerful new strategy for identifying regions of viral proteins that affect inhibition by host factors.

•I.m. injection of IFNb and IFNc plasmids induced antiviral genes systemically.•IFNa1 plasmid induced antiviral genes only at the muscle injection site.•Systemic induction of antiviral genes by IFNc plasmid lasted at least 8 weeks.•IFNc plasmid protected salmon against ISAV infection 8 weeks after injection.•IFNa1 and IFNb plasmids induced no or little protection against ISAV infection. In this work we have tested the in vivo antiviral activity of type I interferons (IFNs) in Atlantic salmon by injecting presmolts intramuscularly (i.m.) with plasmids encoding IFNa1, IFNb or IFNc under the control of a CMV promoter, and measured expression of antiviral genes in organs and protection against infection with infectious salmon anemia virus (ISAV) infection. All three IFN plasmids induced expression of antiviral genes (Mx, Viperin, ISG15 and IFIT5) at the muscle injection site while the control plasmid had little effect. Only IFNb and IFNc plasmids induced expression of antiviral genes in head kidney, liver and heart. This suggests that IFNb and IFNc are distributed systemically while IFNa1 is active only at the injection site. Injection of IFNc plasmid was found to induce expression of antiviral genes and receptors for virus RNA (RIG-I, TLR3 and TLR7) in head kidney from 1 to at least 8 weeks. Immunoblotting showed increased expression of ISG15 and Mx protein in liver with time during this time period. Challenge of presmolts with ISAV 8 weeks after injection of IFN plasmids, showed strong protection of the IFNc plasmid injected fish, low protection of the IFNb plasmid injected fish and no protection of the IFNa1 plasmid injected fish. Clues to the difference in protection obtained with IFNb and IFNc plasmids were found by immunohistochemical and immunoblot studies of Mx protein, which indicated that IFNc plasmid stimulated stronger Mx protein expression in heart tissues and liver endothelial cells than IFNb plasmid. Taken together, these data suggest that i.m. injection of the IFNc expression plasmid may be a new method for protecting Atlantic salmon against virus infection.

Engrailed-2 (EN2) is a homeodomain-containing transcription factor that has roles in boundary formation and neural guidance in early development, but which is also expressed in a range of cancers. In addition to transcriptional regulation, it is secreted by cells and taken up by others through a mechanism that is yet to be fully elucidated. In this study, the distribution of EN2 protein in cells was evaluated using immunofluorescence with a set of antibodies raised against overlapping epitopes across the protein, and through the use of an EN2-GFP construct. MX2 expression in primary prostate tumors was evaluated using immunohistochemistry. We showed that EN2 protein is present in the cell membrane and within microvesicles that can be secreted from the cell and taken up by others. When taken up by normal cells from the stroma EN2 induces the expression of MX2 (MxB), a protein that has a key role in the innate immune response to viruses. Our findings indicate that EN2 secretion by tumors may be a means of preventing viral-mediated immune invasion of tissue immediately adjacent to the tumor.

The antiviral cytokine interferon activates expression of interferon-stimulated genes to establish an antiviral state. Myxovirus resistance 2 (MX2, also known as MxB) is an interferon-stimulated gene that inhibits the nuclear import of HIV-1 and interacts with the viral capsid and cellular nuclear transport machinery. Here, we identified the myosin light chain phosphatase (MLCP) subunits myosin phosphatase target subunit 1 (MYPT1) and protein phosphatase 1 catalytic subunit-β (PPP1CB) as positively-acting regulators of MX2, interacting with its amino-terminal domain. We demonstrated that serine phosphorylation of the N-terminal domain at positions 14, 17 and 18 suppresses MX2 antiviral function, prevents interactions with the HIV-1 capsid and nuclear transport factors, and is reversed by MLCP. Notably, serine phosphorylation of the N-terminal domain also impedes MX2-mediated inhibition of nuclear import of cellular karyophilic cargo. We also found that interferon treatment reduces levels of phosphorylation at these serine residues and outline a homeostatic regulatory mechanism in which repression of MX2 by phosphorylation, together with MLCP-mediated dephosphorylation, balances the deleterious effects of MX2 on normal cell function with innate immunity against HIV-1. Dual functions of MX2 in innate immunity against HIV-1 and nuclear import of cellular proteins are regulated by phosphorylation.

The Bowman-Birk inhibitor (BBI), a soybean-derived protease inhibitor, is known to have anti-inflammatory effect in both in vitro and in vivo systems. Macrophages play a key role in inflammation and immune activation, which is implicated in HIV disease progression. Here, we investigated the effect of BBI on HIV infection of peripheral blood monocyte-derived macrophages. We demonstrated that BBI could potently inhibit HIV replication in macrophages without cytotoxicity. Investigation of the mechanism(s) of BBI action on HIV showed that BBI induced the expression of IFN-β and multiple IFN stimulated genes (ISGs), including Myxovirus resistance protein 2 (Mx2), 2′,5′-oligoadenylate synthetase (OAS-1), Virus inhibitory protein (viperin), ISG15 and ISG56. BBI treatment of macrophages also increased the expression of several known HIV restriction factors, including APOBEC3F, APOBEC3G and tetherin. Furthermore, BBI enhanced the phosphorylation of IRF3, a key regulator of IFN-β. The inhibition of IFN-β pathway by the neutralization antibody to type I IFN receptor (Anti-IFNAR) abolished BBI-mediated induction of the anti-HIV factors and inhibition of HIV in macrophages. These findings that BBI could activate IFN-β-mediated signaling pathway, initialize the intracellular innate immunity in macrophages and potently inhibit HIV at multiple steps of viral replication cycle indicate the necessity to further investigate BBI as an alternative and cost-effective anti-HIV natural product.