Identification and characterization of pseudorabies virus dUTPase

Area of Science:

• Virology
• Molecular Biology
• Biochemistry

Background:

• The pseudorabies virus (PrV) genome contains numerous genes with unknown functions.
• Deoxyuridine triphosphate nucleotidohydrolase (dUTPase) is a crucial enzyme in DNA synthesis and repair across various organisms.
• Herpesvirus dUTPases play roles in viral replication and pathogenesis.

Purpose of the Study:

• To identify and characterize the dUTPase gene in the pseudorabies virus (PrV) genome.
• To investigate the function and localization of the PrV dUTPase.
• To determine the necessity of PrV dUTPase for viral replication.

Main Methods:

• Sequence analysis of the PrV genome to identify open reading frames.
• Antiserum production against a bacterially expressed PrV UL50 fragment.
• Construction and characterization of a UL50-negative PrV mutant (PrV UL50-) and its rescuant (PrV UL50resc).
• Enzyme activity assays and subcellular localization studies.

Main Results:

• An open reading frame, designated UL50, was identified in the PrV genome, encoding a protein homologous to herpesvirus dUTPases.
• A 33 kDa protein, corresponding to the predicted size of PrV UL50, was detected in infected cell lysates.
• The UL50 gene disruption abolished the 33 kDa protein, while its restoration re-established its presence.
• Enzyme assays confirmed dUTPase activity for the PrV UL50 protein, which copurifies with infected cell nuclei.
• The PrV UL50- mutant showed only slightly reduced replication efficiency in cultured epithelial cells compared to wild-type PrV.

Conclusions:

• The PrV UL50 gene encodes a 33 kDa dUTPase localized to the nuclei of infected cells.
• This dUTPase is not essential for PrV replication in cultured epithelial cells, suggesting alternative pathways or redundancy in host cells.
• Further studies are warranted to elucidate the precise role of PrV dUTPase in the viral life cycle and pathogenesis.