ABSTRACT. Using polymerase chain reaction, a 1050-bp sequence of the US1 gene was amplified from the pseudorabies virus (PRV) Becker strain genome; identification of the US1 gene was confirmed by further cloning and sequencing. Bioinformatics analysis indicated that the PRV US1 gene encodes a putative polypeptide with 349 amino acids. The encoded protein, designated PICP22, had a conserved Herpes_IE68 domain, which was found to be closely related with the herpes virus immediate early regulatory protein family and is highly conserved among the counterparts encoded by Herpes_IE68 genes. Multiple nucleic acid sequence and amino acid sequence alignments suggested that the product of PRV US1 has a relatively higher homology with ICP22-like proteins of genus Varicellovirus than with those of other genera of Alphaherpesvirinae. In addition, phylogenetic analysis showed that PRV US1 has a close evolutionary relationship with members of the genus Varicellovirus, especially Equid herpes virus 1 (EHV-1), EHV-4 and EHV-9. Antigen prediction indicated that several potential B-cell epitopes are located in PICP22. Also, subcellular localization analysis demonstrated that PICP22 is predominantly located in the cytoplasm, suggesting that it might function as a cytoplasmic-targeted protein.