(a) The UL25 structure and a diagram of domain organization is shown along with a multiple sequence alignment of UL25 residues 45–74 from five alphaherpesviruses. Sequence alignment was generated using Clustal Omega45 and displayed using ESPript 3.046. Identical residues are shown as white letters on a red background. Similar residues are shown as red letters in a blue box. Secondary structure derived from the cryoEM reconstruction of capsid-bound HSV-1 UL25 is shown above the alignment. The following herpesvirus sequences were used (GenBank GeneID numbers in parentheses): HSV-1, herpes simplex virus type 1, strain 17 (2703377); HSV-2, herpes simplex virus type 2, strain HG52 (1487309); BHV-1, bovine herpesvirus-1 (4783418); EHV-4, equine herpesvirus-4, strain NS80567 (1487602); and VZV, varicella-zoster virus, strain Dumas (1487687). (b) SDS-PAGE of purified UL25 constructs: UL25∆44 (cleaved product; 57 kDa), UL25∆44 Q72A (single product; 57 kDa), UL25∆58 Q72A (56 kDa) and UL25∆73 (54 kDa). (c) UL25∆44 Q72A inhibits NEC budding, whereas other UL25 constructs do not. For each condition, NEC-mediated budding was tested at 1:1, 1:6, 1:8, 1:10, and 1:20 NEC:UL25 molar ratios. Each construct was tested in at least two biological replicates, consisting of three technical replicates. Symbols show average budding efficiency of each biological replicate relative to NEC220 (100%; pink). Error bars represent the standard error of measurement for at least two individual experiments. Significance compared to NEC220 was calculated using an unpaired t-test against NEC220. *p-value<0.1. The source file with all raw data values is provided in Figure 1—source data 1. (d) UL25∆44 Q72A does not bind to acidic lipid membranes.