User:ManVhv/Sandbox

• "We report that the transcript start site of UL3 mRNA isolated from HSV-1 infected cells maps to a position downstream of the predicted translation start site."
• Constructed recombinant virus CB8116

• Site mutation at predicted UL-3 transcript start site
• "UL3 protein translation initiates at the second in-frame start codon of the UL3 ORF."

• "U_L-3 encodes a 224 amino acid protein"

• pU_L-3 Localizes to the nucleolus 4 to 6 hours post infection (h.p.i.)

• 4 h.p.i. Localization to nucleosomes has begun
• 5 h.p.i. Accumulations at nucleosomes has grown
• 6 h.p.i. Intranuclear "donut" formation of pU_L-3

• Colocalizes with ICP8 coincidentally with donut formation/6 h.p.i.

• Nucleolar localization requies amino acids 100 through 164^*

• Putative Nuclear Locating Sequence (1 of 2) predicted at aa 143-147.
• M100/164-GFP (Green Flouresecent Protein) mutant UL3 localized to the nucleolus.

• Putative phosphorylation site (1 of 2) in amino acids in 1-99^*

• Sequence anaysis predictions on U_L-3 ORF

• to contain an N-glycosylation site
• to be a glycoprotein

• UL-3 expressed in baculovirus system

• Four electrophoretic bands:

• Two major bands at 30kDa and 31kDa
• Two minor bands at 29kDa and 33kDa

• 33kDa form contained marked 32P isotope, after 32P labelling
• "None of the expressed UL3 protein species were susceptible to tunicamycin treatment, suggesting that they were not N-linked glycosylated."
• Cell fractionation showed localization in the cytoplasm and nucleus but not cell membrane, again suggesting a lack of N-linked glycosylation.

• Mouse antibodies v. pU_L-3 created by vaccination with baculovirus UL-3 proteins

• Reacted with two (2) HSV-1 proteins

• 27kDa and 33kDa forms
• "presumably represent the unphosphorylated and phosphorylated forms of UL3"

PubMed (1996, Cedars-Sanai Medical Center Research Institute)

• Predicted molecular weight 29681 Da.
• HSV-2 UL-3 75% homologous to HSV-1.
• Three electrophoretic bands: 28000, 30500, 33000 Da.

• 30500 and 33000 Da forms contained marked 32P isotope after 32P labelling
• 30500 (but not 33000) Da form convertable to 28000 form via alkaline phosphatase

• Immunohistochemical cell staining to locate protein

• Early stage perinuclear association
• Late stage associated with nucleus in discrete particles

PubMed (1993, Department of Molecular Biology, University of Medicine and Dentistry of New Jersey, Stratford )