Bovine Herpesvirus 1 UL3.5 Interacts with Bovine Herpesvirus 1 α-Transinducing Factor

Virions of alphaherpesviruses are structurally composed of membrane, tegument, nucleocapsid, and core (24). The tegument is an amorphous structure between the nucleocapsid and membrane. In addition to being essential virion structural components, tegument proteins are important for releasing viral genomic DNA early in infection, nucleocapsid formation, viral DNA packaging, and regulation of viral gene expression (24). However, the process of tegument assembly and the precise functions of most tegument proteins are still unclear. Analysis of the genome of Bovine herpesvirus 1 (BHV-1), an alphaherpesvirus, reveals that there are at least 16 proteins known or presumed to be present in the tegument. A short open reading frame (ORF) in the BHV-1 genome designated the UL3.5 gene encodes a 13-kDa tegument protein expressed late in infection (23). Unlike most alphaherpesviral proteins, UL3.5 is not conserved throughout the alphaherpesvirus family. Homologs have been found only in Pseudorabies virus (PrV) (6), Varicella zoster virus (VZV) (5), Equine herpes virus 1 (EHV-1) (25), and Infectious laryngotracheitis virus (11). Herpes simplex virus type 1 (HSV-1) and HSV-2 do not have UL3.5 homologs (18, 19). Moreover, homologs of UL3.5 differ in size (from 71 amino acids [aa] for VZV to 220 aa for PrV) (5, 6, 13) and have overall 20 to 30% amino acid sequence homology that is restricted mostly to the N-terminal 50 aa (13). The roles of UL3.5 homologs in virus replication are apparently different. PrV UL3.5 is required for virus egress. A PrV mutant lacking UL3.5 replicates very poorly in the one-step replication and plaque assays (10). On the other hand, VZV lacking gene ORF57, the homolog of the UL3.5 gene, grows in cell culture at a same rate as wild-type virus (3). Neither the need for nor the function of UL3.5 in BHV-1 replication has been determined. However, BHV-1 UL3.5 rescued a PrV UL3.5 deletion mutant (9) implying that BHV-1 UL3.5 may also participate in virus egress. BHV-1 α-transinducing factor (αBTIF), encoded by the UL48 gene, is a virion component that transactivates immediate-early gene promoters during viral lytic infection (20). Homologs are present in HSV-1, EHV-1, and VZV (1, 5, 15, 22) and probably all other Alphaherpesviruses. In addition to being a transcription factor, the HSV homolog, αTIF, also known as VP16 or Vwm65, is a tegument protein indispensable for virion assembly (27). No structural function of other αTIF homologs has been reported. One approach to understanding tegument assembly and the function of tegument proteins is to identify and characterize proteins that associate with previously characterized tegument proteins. In this study, we sought to identify proteins that interact with BHV-1 UL3.5. Five methods showed a specific interaction between BHV-1 UL3.5 and αBTIF. (i) αBTIF specifically attached to His-tagged BHV-1 UL3.5 in an in vitro pull-down assay. (ii) A complex containing UL3.5 and αBTIF immunoprecipitated from BHV-1-infected cell lysates with either anti-UL3.5 or anti-αBTIF polyclonal antibodies. (iii) Confocal microscopy showed that BHV-1 UL3.5 and αBTIF colocalized in a Golgi-like subcellular compartment late in infection. (iv) In a transient expression system, UL3.5 and αBTIF colocalized in the nucleus. (v) Deletion of the N-terminal 20 aa, but not the C-terminal 40 aa, of UL3.5 abolished the binding of αBTIF in the pull-down assay and the colocalization in the transient expression system. The interaction between UL3.5 and αBTIF may have roles in virion assembly and regulation of αBTIF transactivation activity.