A putative leucine zipper within the herpes simplex virus type 1 UL6 protein is required for portal ring formation.
The herpes simplex virus type 1 UL6 protein forms a 12-subunit ring structure at a unique capsid vertex which functions as a conduit for encapsidation of the viral genome. To characterize UL6 protein domains that are involved in intersubunit interactions and interactions with other capsid proteins, we engineered a set of deletion mutants spanning the entire gene. Three deletion constructs, D-5 (Delta 198-295), D-6 (Delta 322-416), and D-LZ (Delta 409-473, in which a putative leucine zipper was removed), were introduced into the viral genome. All three mutant viruses produced only B capsids, indicating a defect in encapsidation. Western blot analysis showed that the UL6 protein was present in the capsids isolated from two mutants, D-6 and D-LZ. The protein encoded by D-5, on the other hand, was not associated with capsids and was instead localized in the cytoplasm of the infected cells, indicating that this deletion affected the nuclear transport of the portal protein. The UL6 protein from the KOS strain (wild type) and the D-6 mutant were purified from insect cells infected with recombinant baculoviruses and shown to form ring structures as assessed by sucrose gradient centrifugation and electron microscopy. In contrast, the D-LZ mutant protein formed aggregates that sedimented throughout the sucrose gradient as a heterogeneous mixture and did not yield stable ring structures. A mutant (L429E L436E) in which two of the heptad leucines of the putative zipper were replaced with glutamate residues also failed to form stable rings. Our results suggest that the integrity of the leucine zipper region is important for oligomer interactions and stable ring formation, which in turn are required for genome encapsidation.
Nellissery
Jacob K
JK
University of Connecticut Health Center, 263 Farmington Ave., Farmington, CT 06030, USA.
Szczepaniak
Renata
R
Lamberti
Carmela
C
Weller
Sandra K
SK
D021581:Active Transport, Cell Nucleus; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D002213:Capsid / Q000737:chemistry; D036022:Capsid Proteins / Q000737:chemistry / Q000378:metabolism* / Q000502:physiology*; D002467:Cell Nucleus / Q000378:metabolism; D002522:Chlorocebus aethiops; D003593:Cytoplasm / Q000737:chemistry; D018259:Herpesvirus 1, Human / Q000502:physiology*; D007313:Insecta / Q000166:cytology; D016350:Leucine Zippers / Q000235:genetics*; D046911:Macromolecular Substances / Q000302:isolation & purification; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D025941:Protein Interaction Mapping; D016415:Sequence Alignment; D017384:Sequence Deletion; D014709:Vero Cells; D014764:Viral Proteins; D019065:Virus Assembly / Q000502:physiology*
D036022:Capsid Proteins; D046911:Macromolecular Substances; D014764:Viral Proteins; C069185:DNA cleavage and packaging proteins, Herpesvirus