Epstein-Barr Virus Thymidine Kinase Is a Centrosomal Resident Precisely Localized to the Periphery of Centrioles

Abstract

The thymidine kinase (TK) encoded by Epstein-Barr virus (EBV) differs not only from that of the alphaherpesviruses but also from that of the gamma-2 herpesvirus subfamily. Because cellular location is frequently a determinant of regulatory function, to gain insight into additional role(s) of EBV TK and to uncover how the lymphocryptovirus and rhadinovirus enzymes differ, the subcellular localizations of EBV TK and the related cercopithecine herpesvirus-15 TK were investigated. We show that in contrast to those of the other family members, the gamma-1 herpesvirus TKs localize to the centrosome and even more precisely to the periphery of the centriole, tightly encircling the tubulin-rich centrioles in a microtubule-independent fashion. Centrosomal localization is observed in diverse cell types and occurs whether the protein is expressed independently or in the context of lytic EBV infection. Surprisingly, analysis of mutants revealed that the unique N-terminal domain was not critical for targeting to the centrosome, but rather, peptide sequences located C terminal to this domain were key. This is the first herpesvirus protein documented to reside in the centrosome, or microtubule-organizing center, an amembranous organelle that regulates the structural biology of the cell cycle through control of chromosome separation and cytokinesis. More recently, proteasome-mediated degradation of cell cycle regulatory proteins, production and loading of antigenic peptides onto HLA molecules, and transient homing of diverse virion proteins required for entry and/or egress have been shown to be coordinated at the centrosome. Potential implications of centrosomal localization for EBV TK function are discussed.

Human Papillomavirus Type 16 E7 Oncoprotein Associates with the Centrosomal Component γ-Tubulin

“…As mentioned above, regulators of centrosome duplication often show at least partial centrosomal colocalization. Furthermore, proteins encoded by other viruses, including the Gag proteins of foamy virus, human immunodeficiency virus type 1, Jaagsiekte sheep retrovirus, and MasonPfizer monkey virus as well as human T-cell lymphotropic virus type 1 Tax and Epstein-Barr virus thymidine kinase, have also been shown to localize to the centrosome, although the consequences of these interactions have not been fully delineated (1,14). Hence, we evaluated the subcellular localization of HPV16 E7.…”

Hsp90 Inhibitor 17-DMAG Decreases Expression of Conserved Herpesvirus Protein Kinases and Reduces Virus Production in Epstein-Barr Virus-Infected Cells

“…A primary antibody raised against HCMV-UL97 in rabbits was a gift from Donald Coen at Harvard University (66) and was used at a 1:1,000 dilution. Rabbit anti-EBV TK (used at a 1:200 dilution) was prepared and analyzed as described previously (67). Briefly, full-length recombinant EBV TK was expressed in bacteria and purified as described previously (68), and the GST moiety was removed as described previously for HHV8 TK (69).…”

“…Preimmune rabbit serum was collected, and rabbits were immunized with purified recombinant EBV TK that was then provided to a commercial vendor (Harlan Bioproducts) for antibody production. Serum was harvested at different time intervals, and antibodies from both preimmune and immune sera were purified by protein A chromatography (Pierce) and analyzed for titer and specificity (67). The secondary antibodies used were horseradish peroxidase-conjugated secondary antimouse IgG (catalog number 31430, 1:10,000; Pierce Biotechnology), antirabbit IgG (catalog number 31460, 1:5,000; Pierce Biotechnology), and anti-rat IgG (catalog number 31230, 1:5,000; Pierce Biotechnology).…”

KSHV ‐ TK is a tyrosine kinase that disrupts focal adhesions and induces Rho‐mediated cell contraction

“…They are also very inefficient thymidine kinases (Gustafson et al, 1998(Gustafson et al, , 2000. Of those studied to date, the TKs encoded by gammaherpesviruses, unlike that of HSV1, are also not found in the nucleus (Degreve et al, 1998;Gill et al, 2005Gill et al, , 2007. Of those studied to date, the TKs encoded by gammaherpesviruses, unlike that of HSV1, are also not found in the nucleus (Degreve et al, 1998;Gill et al, 2005Gill et al, , 2007.…”