The highly conserved alphaherpesvirus inner tegument protein UL37 has diverse functions in viral replication and manipulation of the host innate immune response. Understanding species differences among alphaherpesviruses may help elucidate conserved functions of the inner tegument protein UL37. To compare functional differences of UL37 among alphaherpesviruses, we used bovine herpesvirus type 1 (BoHV-1), as a representative of genus Varicellovirus, to determine the role of UL37 in intracellular transport and innate immune regulation. The UL37 gene was modified by double-red recombination in conjunction with the viral genome cloned as a bacterial artificial chromosome (BAC). Insertion of premature double
stop codons within amino acids 34-35 resulted in a lack of UL37 expression as detected by western immunoblots using anti-BoHV-1 UL37-specific antibodies. Replication kinetics and plaque morphology analysis showed slightly delayed replication and decrease in plaque size for
BoHV-1 ΔUL37 mutant virus as compared to wild-type (WT). In MDBK cells, a slight delay in capsid transport to the nucleus was observed at 1 hpi in BoHV-1 ΔUL37 as compared to the WT, indicating the facilitative role of BoHV-1 UL37 in efficient intracellular transport. To analyze the role of BoHV-1 in inhibiting cGAS and type 1 interferon response, we used a FLAG-tagged BoHV-1 UL37 (FLAG-UL37) co-expressed with eGFP-tagged bovine cGAS (bocGAS) in 293 cells. Co-immunoprecipitation assay showed that FLAG-UL37 interacts with bocGAS in 293 cells. Under confocal microscopy, bocGAS appeared to be sequestered in perinuclear zones when co-expressed with FLAG- UL37. Analysis of 2’3’-cGAMP levels indicate that BoHV-1 UL37 targets cGAS function. Infection with BoHV-1 ΔUL37 resulted in significant increase in relative mRNA expression levels of cGAS, interferon alpha (IFNα) and interferon beta (IFNβ) in MDBK cells. This study shows that BoHV-1 UL37 is important in facilitating intracellular virion capsid transport and innate immune regulation in MDBK cells.