Human cytomegalovirus protein kinase UL97 forms a complex with the tegument phosphoprotein pp65.
UL97 is a protein kinase encoded by human cytomegalovirus (HCMV) and is an important target for antiviral drugs against this ubiquitous herpesvirus, which is a major cause of life-threatening opportunistic infections in the immunocompromised host. In an effort to better understand the function(s) of UL97 during HCMV replication, a recombinant HCMV, NTAP97, which expresses a tandem affinity purification (TAP) tag at the amino terminus of UL97, was used to obtain UL97 protein complexes from infected cells. pp65 (also known as UL83), the 65-kDa virion tegument phosphoprotein, specifically copurified with UL97 during TAP, as shown by mass spectrometry and Western blot analyses. Reciprocal coimmunoprecipitation experiments using lysates of infected cells also indicated an interaction between UL97 and pp65. Moreover, in a glutathione S-transferase (GST) pull-down experiment, purified GST-pp65 fusion protein specifically bound in vitro-translated UL97, suggesting that UL97 and pp65 do not require other viral proteins to form a complex and may directly interact. Notably, pp65 has been previously reported to form unusual aggregates during viral replication when UL97 is pharmacologically inhibited or genetically ablated, and a pp65 deletion mutant was observed to exhibit modest resistance to a UL97 inhibitor (M. N. Prichard, W. J. Britt, S. L. Daily, C. B. Hartline, and E. R. Kern, J. Virol. 79:15494-15502, 2005). A stable protein-protein interaction between pp65 and UL97 may be relevant to incorporation of these proteins into HCMV particles during virion morphogenesis, with potential implications for immunomodulation by HCMV, and may also be a mechanism by which UL97 is negatively regulated during HCMV replication.
Kamil
Jeremy P
JP
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, 250 Longwood Ave., SGMB 304A, Boston, MA 02115, USA.
Coen
Donald M
DM
D000595:Amino Acid Sequence; D015153:Blotting, Western; D022202:Chromosomes, Artificial, Bacterial / Q000235:genetics; D003587:Cytomegalovirus / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D006801:Humans; D047468:Immunoprecipitation; D008969:Molecular Sequence Data; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism*; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000235:genetics / Q000378:metabolism*; D025941:Protein Interaction Mapping; D014763:Viral Matrix Proteins / Q000235:genetics / Q000378:metabolism*; D014779:Virus Replication
D010750:Phosphoproteins; D014763:Viral Matrix Proteins; C065418:cytomegalovirus matrix protein 65kDa; D017853:Phosphotransferases (Alcohol Group Acceptor); C075365:ganciclovir kinase