The UL48 Tegument Protein of Pseudorabies Virus Is Critical for Intracytoplasmic Assembly of Infectious Virions

Abstract

The pseudorabies virus (PrV) homolog of the tegument protein encoded by the UL48 gene of herpes simplex virus type 1 (HSV-1) was identified by using a monospecific rabbit antiserum against a bacterial fusion protein.UL48-related polypeptides of 53, 55, and 57 kDa were detected in Western blots of infected cells and purified virions. Immunofluorescence studies demonstrated that the PrV UL48 protein is predominantly localized in the cytoplasm but is also found in the nuclei of infected cells. Moreover, it is a constituent of extracellular virus particles but is absent from primary enveloped perinuclear virions. In noncomplementing cells, a UL48-negative PrV mutant (PrV-⌬UL48) exhibited delayed growth and significantly reduced plaque sizes and virus titers, deficiencies which were corrected in UL48-expressing cells. RNA analyses indicated that, like its HSV-1 homolog, the PrV UL48 protein is involved in regulation of immediate-early gene expression. However, the most salient effect of the UL48 gene deletion was a severe defect in virion morphogenesis. Late after infection, electron microscopy of cells infected with PrV-⌬UL48 revealed retention of newly formed nucleocapsids in the cytoplasm, whereas enveloped intracytoplasmic or extracellular complete virions were only rarely observed. In contrast, capsidless particles were produced and released in great amounts. Remarkably, the intracytoplasmic capsids were labeled with antibodies against the UL36 and UL37 tegument proteins, whereas the capsidless particles were labeled with antisera directed against the UL46, UL47, and UL49 tegument proteins. These findings suggested that the UL48 protein is involved in linking capsid and future envelope-associated tegument proteins during virion formation. Thus, like its HSV-1 homolog, the UL48 protein of PrV functions in at least two different steps of the viral life cycle. The drastic inhibition of virion formation in the absence of the PrV UL48 protein indicates that it plays an important role in virion morphogenesis prior to secondary envelopment of intracytoplasmic nucleocapsids. However, the UL48 gene of PrV is not absolutely essential, and concomitant deletion of the adjacent tegument protein gene UL49 also did not abolish virus replication in cell culture.Pseudorabies virus (PrV, suid herpesvirus 1), the causative agent of Aujeszky's disease of pigs (41), has been classified as a member of the Varicellovirus genus within the Alphaherpesvirinae subfamily of Herpesviridae (49). Although the DNA sequence of the ca. 150-kbp genome of PrV has not yet been elucidated completely, gene content and arrangement appear to be widely similar to those of other alphaherpesviruses including herpes simplex virus type 1 (HSV-1) (38), and therefore the gene nomenclature established for HSV-1 has been adopted (41).PrV virions exhibit the typical morphology of herpesvirus particles (26). The nucleoprotein core containing the DNA genome is enclosed in an icosahedral capsid shell. The capsid is surrounded by a protein layer named the tegument a...

Vesicle formation from the nuclear membrane is induced by coexpression of two conserved herpesvirus proteins

“…Rabbit kidney cells (RK13) were transfected and selected for G418 resistance. Cell clones were screened for pUL31 and pUL34 expression by indirect immunofluorescence and for complementation of respective virus mutants (8,11). Three cell clones yielding the highest virus titers in the complementation assays expressed both proteins in comparable amounts, as tested by Western blot (Fig.…”

“…Rabbit sera against PrV pUL31 and pUL34 have been described (8,11). To generate monospecific anti-PrV pUL34 mouse serum, two mice were immunized with 20 g of GST-UL34 fusion protein (8).…”

Determination of Interactions between Tegument Proteins of Herpes Simplex Virus Type 1

“…Most of the tegument proteins are present in HSV-1 light particles, including UL36 (37) and UL37 (25). In contrast, UL36 and UL37 are not required for the assembly of pseudorabies virus (PRV; a closely related alphaherpesvirus) light particles (11). Evidence for UL48 in both the inner and outer tegument layers has come from detergent solubilization/salt extractions of purified virions (35,40).…”

“…In PRV, there is evidence for UL49 interacting with glycoproteins E/I and M (13) and for UL36 interacting with UL37 (20,21), suggesting superficial and deep positions within the tegument, respectively. Studies with PRV mutants suggest that UL37, UL46, UL47, and UL48 are important structural components of the tegument (11,12,21,23,28,29). Those tegument proteins that are present in high amounts (1,000 to 2,000 copies per virion), such as UL46, UL47, UL48, and UL49, are likely to play a structural role (17,40).…”

Cell biological and molecular characteristics of pseudorabies virus infections in cell cultures and in pigs with emphasis on the respiratory tract

“…Early (E) genes are the next group of genes that are active. Like IE180, several of them (EP0, UL54 and UL48) are regulating the expression of genes of PRV and the cell [13,45,59]. Other E genes are producing proteins which are important for nucleotide synthesis (UL23, UL39/UL40, UL50) and DNA replication (UL5, UL8, UL9, UL29, UL30, UL42, UL52).…”