Nuclear trafficking of the human cytomegalovirus pp71 (ppUL82) tegument protein.
The human cytomegalovirus tegument protein pp71 localizes to the nucleus immediately upon infection, and functions to initiate viral gene expression. Analysis of a series of random insertion mutations revealed that sequences within the mid region (MR) of pp71 are important for localization to the nucleus. Fusion of MR sequences with eGFP revealed that amino acids 94 to 300 were sufficient to target proteins to the nucleus. Random substitution mutagenesis within this domain resulted in two double substitution mutants, pp71P203T/T223M and pp71T228M/L275Q, with a predominantly cytoplasmic localization. Disruption of nuclear targeting resulted in relocalization of the fusion proteins to a distinct perinuclear region. Using tandem mass spectrometry, we determined that threonine 223 can be phosphorylated. Mutation of this residue to a phosphomimetic amino acid resulted in abrogation of nuclear targeting. These results strongly suggest that the intracellular trafficking of pp71 is regulated by phosphorylation.
Shen
Weiping
W
Department of Microbiology and Molecular Cell Biology, Eastern Virginia Medical School, Norfolk, VA 23507, USA.
Westgard
Elizabeth
E
Huang
Liqun
L
Ward
Michael D
MD
Osborn
Jodi L
JL
Chau
Nha H
NH
Collins
Lindsay
L
Marcum
Benjamin
B
Koach
Margaret A
MA
Bibbs
Jennifer
J
Semmes
O John
OJ
Kerry
Julie A
JA
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D019677:Artificial Gene Fusion; D002467:Cell Nucleus / Q000737:chemistry*; D003587:Cytomegalovirus / Q000502:physiology*; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D020125:Mutation, Missense; D019913:Nuclear Localization Signals; D010766:Phosphorylation; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D053719:Tandem Mass Spectrometry; D013912:Threonine / Q000378:metabolism; D014763:Viral Matrix Proteins / Q000737:chemistry / Q000378:metabolism*
D019913:Nuclear Localization Signals; D011993:Recombinant Fusion Proteins; D014763:Viral Matrix Proteins; C047015:cytomegalovirus matrix protein, pp 64-69; D049452:Green Fluorescent Proteins; D013912:Threonine