An acidic cluster of human cytomegalovirus UL99 tegument protein is required for trafficking and function.
The human cytomegalovirus (HCMV) virion is comprised of a linear double-stranded DNA genome, proteinaceous capsid and tegument, and a lipid envelope containing virus-encoded glycoproteins. Of these components, the tegument is the least well defined in terms of both protein content and function. Several of the major tegument proteins are phosphoproteins (pp), including pp150, pp71, pp65, and pp28. pp28, encoded by the UL99 open reading frame (ORF), traffics to vacuole-like cytoplasmic structures and was shown recently to be essential for envelopment. To elucidate the UL99 amino acid sequences necessary for its trafficking and function in the HCMV replication cycle, two types of viral mutants were analyzed. Using a series of recombinant viruses expressing various UL99-green fluorescent protein fusions, we demonstrate that myristoylation at glycine 2 and an acidic cluster (AC; amino acids 44 to 57) are required for the punctate perinuclear and cytoplasmic (vacuole-like) localization observed for wild-type pp28. A second approach involving the generation of several UL99 deletion mutants indicated that at least the C-terminal two-thirds of this ORF is nonessential for viral growth. Furthermore, the data suggest that an N-terminal region of UL99 containing the AC is required for viral growth. Regarding virion incorporation or UL99-encoded proteins, we provide evidence that suggests that a hypophosphorylated form of pp28 is incorporated, myristoylation is required, and sequences within the first 57 amino acids are sufficient.
Jones
Thomas R
TR
Infectious Disease Section, Wyeth Research, Pearl River, New York 10965, USA. jonestr@optonline.net
Lee
Shi-Wu
SW
D000595:Amino Acid Sequence; D002478:Cells, Cultured; D003587:Cytomegalovirus / Q000235:genetics / Q000502:physiology*; D003586:Cytomegalovirus Infections / Q000821:virology; D003593:Cytoplasm / Q000378:metabolism*; D005347:Fibroblasts / Q000821:virology; D049452:Green Fluorescent Proteins; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013329:Structure-Activity Relationship; D014764:Viral Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; C090378:UL99 protein, cytomegalovirus; D014764:Viral Proteins; D049452:Green Fluorescent Proteins