Functional comparison of the two gene products of Thogoto virus segment 6

This study examined the functional differences between the two proteins encoded by segment 6 of Thogoto virus (THOV), the matrix protein (M) and the accessory protein ML. Although these proteins share nearly identical sequences, the authors aimed to determine whether they perform similar or distinct roles during infection. Using minireplicon assays, mutational analysis, and reporter-based experiments, the study showed that M is essential for viral replication and assembly. M strongly inhibited the viral RNA-dependent RNA polymerase (RdRP), thereby suppressing viral transcription. This inhibitory activity was localized to the C-terminal region, while the full-length protein was required for the formation of virus-like particles (VLPs). In contrast, ML did not affect polymerase activity and could not support particle formation. Instead, ML functioned as an interferon (IFN) antagonist, efficiently blocking IFN-β induction in response to viral infection or double-stranded RNA. This activity was associated with the C-terminal region of ML but required more than just the unique 38-amino-acid extension. Additionally, ML was found to be incorporated into virions, suggesting a role early in infection. Overall, the study demonstrates that M and ML have clearly distinct functions, with M involved in replication and assembly, and ML specialized in immune evasion.
(TMR)

Experimental and evolutionary evidence for horizontal transfer of an envelope fusion protein gene between thogotoviruses and baculoviruses

This study investigated the evolutionary relationship between thogotoviruses and baculoviruses by examining whether thogotovirus envelope fusion proteins (EFPs) can functionally substitute the baculovirus GP64 protein. The authors combined bioinformatic analyses, phylogenetics, recombinant virus construction, and cell culture experiments to test the hypothesis that baculoviral gp64 originated from a horizontal gene transfer (HGT) event involving thogotoviruses. Through RNA-seq data mining of lepidopteran transcriptomes, the researchers identified a novel virus named Melitaea didyma thogotovirus 1 (MediTHOV-1). Phylogenetic and phylodynamic analyses of glycoproteins from baculoviruses and thogotoviruses suggested that the HGT event giving rise to gp64 likely occurred during the Mesozoic era. To experimentally test functional compatibility, recombinant Autographa californica multiple nucleopolyhedrovirus (AcMNPV) lacking its native gp64 gene was engineered to express EFPs from MediTHOV-1 or Apis thogotovirus 1 (ATHOV-1). Infection assays showed that the MediTHOV-1 glycoprotein could not restore viral infectivity. In contrast, the ATHOV-1 EFP partially rescued infectivity, allowing viral replication and cell-to-cell spread in insect cells, although with delayed kinetics and significantly lower viral titers compared with GP64-containing viruses. Structural analysis using cryo-electron microscopy revealed reduced incorporation of ATHOV-1 EFP into viral envelopes, explaining the lower infection efficiency. Overall, the findings provide experimental evidence supporting the hypothesis that baculovirus GP64 originated from thogotovirus glycoproteins through horizontal gene transfer and highlight functional constraints that shaped the evolution of viral envelope fusion proteins.
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The Antiviral Activity of Equine Mx1 against Thogoto Virus Is Determined by the Molecular Structure of Its Viral Specificity Region

This study investigated the antiviral activity of mammalian Mx1 proteins against Thogoto virus, focusing on the molecular determinants responsible for viral restriction. Using polymerase minireplicon assays, infection experiments, mutational analyses, and protein interaction studies, the authors compared Mx1 proteins from different mammalian species. Among them, equine Mx1 (eqMx1) exhibited strong antiviral activity comparable to human MxA, whereas most other mammalian Mx1 proteins were inactive. In contrast, none of the tested Mx1 proteins inhibited the related Dhori virus, indicating virus-specific differences in susceptibility to Mx-mediated restriction. Detailed mutational analyses identified the flexible loop L4 in the stalk domain of eqMx1 as the key determinant of antiviral specificity. In particular, two adjacent residues, tryptophan (W562) and glycine (G563), were essential for THOV inhibition. Substitution of either residue abolished antiviral activity, while maintaining a bulky aromatic residue at position 562 together with glycine at position 563 preserved restriction. Importantly, introducing this motif into otherwise inactive bovine Mx1 conferred antiviral activity, demonstrating its functional sufficiency. Overall, the findings reveal that a minimal structural motif within loop L4 governs the antiviral activity of eqMx1 and highlight this region as an evolutionary hotspot shaping host–virus interactions in thogotoviruses.
(TMR)

Pseudotyped zoonotic thogotoviruses exhibit broad entry range in mammalian cells

This study investigated the entry mechanisms and cell tropism of zoonotic thogotoviruses by characterizing the envelope glycoproteins (GPs) of Thogoto virus (THOV), Dhori virus (DHOV), Bourbon virus (BRBV), and Sinu virus (SINV). Using a combination of cell–cell fusion assays and three complementary pseudotyping systems such as lentiviral, influenza A virus–based, and vesicular stomatitis virus (VSV)-based vectors, the authors systematically evaluated GP functionality and host range. All four thogotovirus GPs mediated pH-dependent membrane fusion, consistent with endosomal entry via class III fusion proteins. Lentiviral and influenza-based pseudotypes demonstrated that these GPs support entry into a broad spectrum of mammalian cell lines from multiple species, indicating the likely use of a conserved or ubiquitous receptor. Replication-competent VSV pseudotypes bearing DHOV or THOV GPs enabled multi-round infection and revealed efficient entry into human hepatocyte, neuronal, alveolar epithelial, and intestinal epithelial models, including primary and stem cell-derived cultures. Although pseudotyping efficiencies varied among vector systems and GPs, the overall findings highlight the promiscuous entry capacity of thogotoviruses. Collectively, the study establishes robust pseudotyping platforms for thogotovirus research and underscores the substantial zoonotic and tissue tropism potential of this underexplored group of orthomyxoviruses.
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Mx1 in Hematopoietic Cells Protects against Thogoto Virus Infection

This study examined the contribution of the interferon-induced restriction factor Mx1 in hematopoietic versus nonhematopoietic compartments during viral infection, with a particular focus on Thogoto virus (THOV). Using reciprocal bone marrow chimera mice carrying functional or nonfunctional Mx1 alleles, the authors compared outcomes following infection with influenza A virus (IAV) and THOV. While resistance to IAV infection depended exclusively on Mx1 expression in nonhematopoietic (stromal) cells, THOV infection revealed a distinct requirement for Mx1 in bone marrow-derived cells. Mice lacking stromal Mx1 but reconstituted with Mx1-competent bone marrow showed delayed morbidity, reduced liver pathology, and significantly lower viral dissemination to spleen, lung, and serum compared with mice receiving Mx1-deficient bone marrow. Histopathology, serum ALT/AST measurements, viral titration, and immunohistochemical analyses demonstrated that Mx1 expression in CD45⁺ hematopoietic cells, likely myeloid cells, restricted THOV replication and delayed liver necrosis, although it did not fully prevent hepatic infection in the absence of stromal Mx1. Collectively, the results establish that, unlike influenza A virus, effective control of THOV infection critically involves Mx1 expression in hematopoietic cells, highlighting virus- and cell type–specific roles of intrinsic antiviral immunity.
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In Vivo Conditions Enable IFNAR-Independent Type I Interferon Production by Peritoneal CD11b1 Cells upon Thogoto Virus Infection

This study investigated how type I interferon (IFN-I) can be induced independently of IFNAR-mediated feedback during Thogoto virus infection. Wild-type and innate immune-deficient mice (IFNAR⁻/⁻, MyD88⁻/⁻, TRIF⁻/⁻, MyD88⁻/⁻TRIF⁻/⁻, and MAVS⁻/⁻) were infected intraperitoneally in vivo, and IFN-β production was assessed in serum, peritoneal exudates, and organs by ELISA. To define cellular sources and signaling requirements, bone marrow-derived myeloid and plasmacytoid dendritic cells, as well as peritoneal exudate cells, were analyzed using in vitro infection, ex vivo culture, flow cytometry, reporter mice, MACS enrichment, and quantitative RT-PCR. THOV infection induced robust systemic IFN-β responses in vivo even in IFNAR-deficient mice, whereas comparable in vitro infections failed to do so, demonstrating a strict dependence on in vivo conditions. IFN production localized primarily to the peritoneal cavity and later the spleen. Using replication-incompetent but transcriptionally active THOV-derived virus-like particles, the authors showed that IFNAR-independent IFN induction required viral polymerase activity but not productive viral replication, as UV-inactivated particles were inactive. Genetic ablation revealed that IFN induction depended on MAVS-mediated RIG-I-like helicase signaling and was independent of TLR pathways. Reporter mouse analyses, supported by flow cytometry and transcript quantification, identified CD11b⁺ F4/80⁺ peritoneal myeloid cells as the dominant source of IFN-I. Collectively, the results define an alternative, tissue-restricted pathway enabling strong IFN-I production in vivo without IFNAR amplification.
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Thogoto Virus Infection Induces Sustained Type I Interferon Responses That Depend on RIG-I-Like Helicase Signaling of Conventional Dendritic Cells

This study investigated the mechanisms underlying type I interferon (IFN) induction during Thogoto virus (THOV) infection using a recombinant virus lacking the IFN-antagonistic ML protein [THOV(ΔML)]. Mice, including wild-type and innate immune signaling–deficient strains (IFNAR⁻/⁻, MyD88⁻/⁻TRIF⁻/⁻, and IPS-1⁻/⁻), were infected in vivo, and serum IFN levels, viral loads, and survival were assessed. In parallel, bone marrow–derived myeloid dendritic cells (BM-mDC) and plasmacytoid dendritic cells (BM-pDC) were infected in vitro to identify cellular sources of IFN. THOV(ΔML) infection induced unusually high and sustained systemic IFN-α/β responses lasting up to 72 h, even in the absence of IFNAR signaling. Genetic ablation studies showed that early IFN induction partially involved Toll-like receptor pathways, whereas sustained IFN production strictly depended on the RIG-I–like helicase adaptor IPS-1. Viral replication was required for IFN induction, as UV-inactivated virus failed to elicit responses. Unexpectedly, BM-mDC, but not BM-pDC, were the dominant producers of type I IFN, and this response was IPS-1 dependent. Collectively, the results identify myeloid dendritic cells and IPS-1–mediated sensing as central drivers of sustained type I IFN responses during THOV infection.
(TMR)