Signals That Dictate Nuclear, Nucleolar, and Cytoplasmic Shuttling of the γ134.5 Protein of Herpes Simplex Virus Type 1

The γ134.5 gene of herpes simplex viruses (HSVs) is located in the inverted repeats of the viral genome flanking the unique long sequence and is present in two copies per genome (1, 13, 14). In HSV type 1 (HSV-1) strain F, the γ134.5 gene encodes a protein of 263 amino acids consisting of an amino-terminal domain, a linker region of three amino acid repeats (Ala-Thr-Pro), and a carboxyl-terminal domain (13). The triplet repeats are a constant feature of the γ134.5 protein in HSV-1, but the number of repeats varies from strain to strain (3, 13, 16). In HSV-2, these triplet repeats are not present in the γ134.5 protein, as determined by nucleotide sequence analysis (32). Interestingly, the carboxyl-terminal domain of the γ134.5 protein is partially homologous to the corresponding domains of the murine myeloid differentiation primary-response protein MyD116 (28; D. J. McGeoch and B. C. Barnett, Letter, Nature 353:609, 1991), the human and hamster growth arrest and DNA damage response protein GADD34 (46), and virulence factor NL/I14L of African swine fever virus (18, 47). The γ134.5 protein is essential for HSV to display neurovirulence in experimental animal models (10, 29, 44). Deletion or nonsense mutation in the γ134.5 gene abates the ability of HSV to replicate in the central nervous system neurons, and therefore, the mutant is incapable of causing encephalitis (10, 37). In human neuroblastoma cell lines infected with HSV-1, the γ134.5 protein is expressed to prevent the shutoff of protein synthesis mediated by the double-stranded-RNA-dependent protein kinase (PKR) (9, 11). This function requires the carboxyl terminus of the γ134.5 protein to recruit cellular protein phosphatase 1 (PP1), forming a high-molecular-weight complex that dephosphorylates the α subunit of the translation initiation factor 2 (eIF-2α) (8, 12, 21, 22). In virus-infected cells, the γ134.5 protein-mediated eIF-2α dephosphorylation contributes to HSV resistance to the antiviral effect of alpha/beta interferon (7). The carboxyl-terminal domains of the γ134.5 protein and GADD34/MyD116 are functionally interchangeable in the context of the HSV genome (20). GADD34/MyD116 belongs to a family of proteins induced under conditions of genotoxic stress, growth arrest, differentiation, and apoptosis (23, 28, 46). GADD34 promotes apoptosis induced by ionizing radiation or methyl methanesulfate, and this activity is negatively regulated by Src kinase Lyn (19, 23). It is also involved in the negative regulation of a stress-inducible gene, CHOP (36). Like the γ134.5 protein, GADD34 complexes with proliferating cell nuclear antigen (PCNA) through its carboxyl-terminal domain (6). It has been proposed that the interaction of GADD34 or the γ134.5 protein with PCNA may release cells from growth arrest and facilitate viral replication in HSV-infected cells (6). Studies show that the γ134.5 protein is required for viral glycoprotein processing and maturation from infected cells (3, 5). In mouse 3T6 cells, the γ134.5 deletion mutant is defective in viral egress and the growth of the mutant is severely affected in resting but not in actively dividing cells (4, 5). Early studies indicate that the γ134.5 protein of HSV-1(F) is a soluble protein that accumulates in both the nucleus and cytoplasm late in infection (1). However, studies with the γ134.5 protein of HSV-1(17+) suggest that it is a cytoplasmic protein (33, 34). In analyzing the domain function of the γ134.5 protein, we found that although it is predominantly found in the cytoplasmic fraction, the γ134.5 protein is also present in the nuclear fraction (20). Interestingly, deletion of the carboxyl terminus or substitution of it with the corresponding region of MyD116 leads to an increased accumulation of the γ134.5 protein in the nuclear fraction (20). These observations raise the possibility that the γ134.5 protein may contain cis elements that determine its subcellular localization. In the present study, we further examined the cellular localization of the γ134.5 protein derived from strain HSV-1(F). We show that in transfected cells the γ134.5 protein is distributed in the nucleus, nucleolus, and cytoplasm. We demonstrate, by the use of deletions and site-specific mutations, that the γ134.5 protein contains nuclear import and export signals that control its nuclear, nucleolar, and cytoplasmic accumulation. We also find that leptomycin B inhibits the cytoplasmic localization of the γ134.5 protein. These findings suggest that the γ134.5 protein continuously shuttles between the nucleus, nucleolus, and cytoplasm. While this work was in progress, it was reported that the cellular localization of the γ134.5 protein is affected by the number of ATP repeats and the arginine-rich cluster in the amino terminus (31).