Herpesvirus entry requires the coordinated action of at least four viral glycoproteins. Virus-specific binding to a cellular receptor triggers a membrane fusion cascade involving the conserved gH/gL complex and gB. Although gB is the genuine herpesvirus fusogen, it requires gH/gL for fusion, but how activation occurs is still unclear. To study the underlying mechanism, we used a gL-deleted pseudorabies virus (PrV) mutant characterized by its limited capability to directly infect neighboring cells that was exploited for several independent serial passages in cell culture. Unlike previous revertants that acquired mutations in the gL-binding N-terminus of gH, we obtained a variant, PrV-ΔgLPassV99, that unexpectedly contained two amino acid substitutions in the gH transmembrane domain (TMD). One of these mutations, I662S, was sufficient to compensate for gL function in virus entry and in in vitro cell–cell fusion assays in presence of wild type gB, but barely for cell-to-cell spread. Additional expression of receptor-binding PrV gD, which is dispensable for cell–cell fusion mediated by native gB, gH and gL, resulted in hyperfusion in combination with gH V99. Overall, our results uncover a yet-underestimated role of the gH TMD in fusion regulation, further shedding light on the complexity of herpesvirus fusion involving all structural domains of the conserved entry glycoproteins.

African swine fever virus (ASFV) causes a highly fatal disease in domestic pigs, resulting in substantial economic losses to the global swine industry. Vaccine development continues to be hindered by limited characterization of viral proteins and their functional redundancies. In this study, we employ combined experimental and computational approaches to characterize the ASFV I73R protein (pI73R), which contains a Z-DNA binding domain and plays a critical role in ASFV virulence and pathogenesis. We demonstrate that pI73R shares significant structural similarity with transcription factors of the forkhead box (FOX) protein family. Overexpression of pI73R results in downregulation of Crooked neck-like protein 1 (CRNKL1), a core spliceosome component, suggesting a potential mechanism by which pI73R modulates host protein synthesis. Using high-resolution mass spectrometry, we map the pI73R interactome and identify the host protein Guanine nucleotide-binding protein subunit beta-1 (GNB1) as a novel direct interactor of pI73R which may facilitate its nuclear transport. Furthermore, we show that pI73R exhibits consistent oligomerization and expression across different ASFV genotypes, highlighting its functional importance. Taken together, these results provide new insights into pI73R function, ASFV–host dynamics, and offer promising directions for antiviral strategy development.