A UL47 Gene Deletion Mutant of Bovine Herpesvirus Type 1 Exhibits Impaired Growth in Cell Culture and Lack of Virulence in Cattle

Tegument protein VP8 encoded by the U_L47 gene of bovine herpesvirus type 1 (BHV-1) is the most abundant constituent of mature virions. In the present report, we describe the characterization of U_L47 gene-deleted BHV-1 in cultured cells and its natural host. The U_L47 deletion mutant exhibited reduced plaque size and more than 100-fold decrease in intracellular and extracellular viral titers in cultured cells. Ultrastructural observations of infected cells showed normal maturation of BHV-1 virions in the absence of VP8. There was no evidence for a change in immediate-early gene activator function of VP16 in the U_L47 deletion mutant virus-infected cells, since bovine ICP4 mRNA and protein levels were similar to those in the wild-type and revertant virus-infected cells throughout the course of infection. Whereas VP16, glycoprotein C (gC), gB, and VP5 were expressed to wild-type levels in the U_L47 deletion mutant-infected cells, the gD and VP22 protein levels were significantly reduced. The reduction in gD protein was associated with increased turnover of the protein. Furthermore, some of the analyzed early and late proteins were expressed with earlier kinetics in the absence of VP8. Extracellular virions of the U_L47 deletion mutant contained reduced amounts of gD, gB, gC, and VP22 but similar amounts of VP16 compared to those of wild-type or revertant virus particles. In addition, the U_L47 gene product was indispensable for BHV-1 replication in vivo, since no clinical manifestations or viral shedding were detected in the U_L47 deletion mutant-infected calves, and the virus failed to induce significant levels of humoral and cellular immunity.Bovine herpesvirus type 1 (BHV-1) is an alphaherpesvirus which is an important pathogen of cattle, causing a variety of clinical manifestations in its natural host (46). BHV-1 virions have a typical herpesvirus structure characterized by the presence of a double-stranded DNA genome enclosed in an icosahedral capsid, the tegument surrounding the capsid, and the outer host-derived lipid envelope bearing virus-encoded glycoproteins. While the major constituents of the viral envelope have been extensively studied (reviewed in reference 17), the proteins present in the tegument and nucleocapsid of BHV-1 have been poorly characterized. Compositionally, the tegument is the most complex compartment of the virion, containing more than 15 viral gene products (32). In addition to their structural role, various regulatory functions, including modulation of transcription (34, 47), kinase activity (39), RNase activity (41), and DNA packaging (43), have been assigned to some tegument proteins, suggesting that these virion constituents function at several stages during virus infection, establishing conditions for efficient viral replication and promoting virus assembly and egress.Although the U_L47 gene product, tegument protein VP8, is the most abundant component of mature BHV-1 virions (5), its function is unknown. Like its herpes simplex virus type 1 (HSV-1) homologue (31), VP8 is posttranslationally modified by phosphorylation (5, 23) and by the addition of O-linked carbohydrates (49). Both HSV-1 and BHV-1 U_L47 homologues possess nuclear localization and nuclear export signatures (7, 51, 53, 56), enabling them to shuttle between the nucleus and cytoplasm when expressed in transiently transfected cells (51, 56) or during viral infection (52, 53). Furthermore, both proteins exhibit a steady-state nuclear localization at early stages of infection and during transient expression (6, 35, 49, 51, 52, 56), suggesting a functional role for these homologues in the nucleus. Nucleocytoplasmic shuttling of VP8 is sensitive to treatment with a RNA polymerase II inhibitor, actinomycin D (52). This observation coupled with recently demonstrated RNA binding activity of the HSV-1 and BHV-1 U_L47 counterparts suggests that like RNA binding proteins encoded by other viruses (12, 44), these U_L47 gene products may be involved in promotion of the export of virus-encoded transcripts from the nucleus to the cytoplasm (8). Early genetic studies suggested a modulatory role of the HSV-1 U_L47 gene product in stimulation of viral immediate-early gene transcription, which is primarily mediated by VP16 (54, 55). However, these potential functions are not absolutely required for HSV-1 propagation in cell culture, as corresponding U_L47 deletion mutants have been produced using noncomplementing cells (2, 55). The U_L47 gene products of Marek''s disease virus serotype 1 (9), avian infectious laryngotracheitis virus (ILTV) (13), and pseudorabies virus (PrV) (20) were also considered nonessential in cell culture. A common feature of all U_L47-null herpesviruses characterized is a comparatively mild growth defect, which is an approximately 10-fold decrease in final titers. U_L47-deleted PrV exhibited normal replication in the nasal epithelium and only a slight delay in neuroinvasion in a mouse model (19). An in vivo phenotype was apparent during infection of a natural host with the U_L47 gene-deleted ILTV, which was significantly attenuated in chickens but conferred protection against subsequent challenge with a virulent strain of the virus (13). VP8 was also determined to be nonessential for viability of BHV-1 in cell culture (42). However, the phenotype of a U_L47-null BHV-1 has not been characterized either in cell culture or in vivo.In this report, we describe the construction and functional analysis of a BHV-1 mutant defective in expression of the U_L47 gene. This is the first phenotypic characterization of U_L47 gene-deleted BHV-1 both in vitro in permissive cells and in vivo in its natural host.