Formation of virus-like particles from cloned cDNAs of Thogoto virus

This study investigated whether virus-like particles (VLPs) of Thogoto virus (THOV) can be generated entirely from cloned cDNAs and whether these particles are functionally competent. Using a plasmid-based reverse genetics system, the authors co-expressed all six structural proteins of THOV (PB1, PB2, PA, NP, GP, and M) along with a minireplicon encoding a reporter gene flanked by viral promoter sequences. The viral polymerase complex and NP successfully reconstituted functional ribonucleoprotein complexes capable of replicating and transcribing the minireplicon RNA. Upon inclusion of GP and M, these artificial nucleocapsids were efficiently packaged into VLPs and released into the supernatant. The VLPs were able to transfer the minireplicon into indicator cells, although detectable reporter expression required prior infection with helper virus, indicating limited intrinsic polymerase activity. Importantly, all six structural proteins were necessary for the formation of infectious VLPs, as omission of either GP or M abolished particle infectivity. Neutralization assays confirmed that the VLPs structurally resembled authentic virions. Overall, this study establishes a functional THOV VLP system and provides a foundation for full virus rescue and detailed analysis of viral replication and assembly mechanisms.
(TMR)

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.
(TMR)

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)