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

Baculoviruses are insect-specific viruses with large, double-stranded DNA genomes classified into four genera. Alphabaculoviruses, which infect lepidoptera, are further divided into group I (G1-α) and group II (G2-α). The GP64 protein, essential for cell attachment and viral entry in G1-α baculoviruses, is thought to have originated through horizontal gene transfer (HGT) from thogotoviruses (family ). This study investigates the functional substitution of GP64 by thogotovirus fusion proteins. Through RNA-seq data mining, we identified a novel thogotovirus, Melitaea didyma thogotovirus 1 (MediTHOV-1), in lepidopteran hosts. Phylodynamic analysis of G1-α baculovirus and thogotovirus glycoproteins suggests that the HGT event occurred during the Mesozoic era. To test functional substitution, we constructed recombinant Autographa californica multiple nucleopolyhedrovirus (AcMNPV) carrying either the envelope fusion protein (EFP) genes from MediTHOV-1 or Apis thogotovirus 1 (ATHOV-1), while deleted for its native gene. Our results show that, while the MediTHOV-1 glycoprotein failed to rescue AcMNPV infectivity, the ATHOV-1 fusion protein (EFP) partially restored infectivity, albeit with reduced efficiency. Cryo-electron microscopy revealed lower incorporation of ATHOV-1 EFP into viral envelopes compared to GP64. The recombinant AcMNPV carrying ATHOV-1 EFP (Ac-ATHOVGPgp64Δ) displayed delayed replication kinetics and lower viral titers. Interestingly, ATHOV-1 EFP significantly enhanced baculovirus entry and gene transduction in mosquito cells. These findings provide experimental support for the HGT hypothesis, demonstrating the functional incorporation of a thogotovirus glycoprotein into a baculovirus. This study sheds light on the evolutionary relationship between baculovirus GP64 and glycoproteins, offering insights into viral evolution and potential biotechnological applications in gene delivery and protein expression.IMPORTANCEBaculoviruses are widely utilized for the biological control of insect pests and as versatile biotechnological tools, with their effectiveness largely dependent on the activity of their envelope fusion proteins (EFPs). Thogotoviruses, in contrast, are emerging vector-borne pathogens of significant concern. In this study, we present the first successful functional substitution of the baculovirus GP64 protein with a thogotovirus EFP, alongside the identification of what appears to be a lepidopteran-associated thogotovirus, Melitaea didyma thogothovirus 1. Our work provides functional and phylogenetic insights into the evolutionary relationship between these distantly related viral groups, particularly the hypothesized horizontal gene transfer event that gave rise to baculoviral gene. These findings offer a deeper understanding of the determinants underlying the adaptation of baculoviral glycoproteins to novel hosts. Furthermore, the discovery of novel viral genes highlights promising opportunities for biotechnological advancements, including the development of enhanced baculovirus-based gene delivery systems and tools for protein expression.