title: The putative herpes simplex virus 1 chaperone protein UL32 modulates disulfide bond formation during infection.
abstract: During DNA encapsidation, herpes simplex virus 1 (HSV-1) procapsids are converted to DNA-containing capsids by a process involving activation of the viral protease, expulsion of the scaffold proteins, and the uptake of viral DNA. Encapsidation requires six minor capsid proteins (UL6, UL15, UL17, UL25, UL28, and UL33) and one viral protein, UL32, not found to be associated with capsids. Although functions have been assigned to each of the minor capsid proteins, the role of UL32 in encapsidation has remained a mystery. Using an HSV-1 variant containing a functional hemagglutinin-tagged UL32, we demonstrated that UL32 was synthesized with true late kinetics and that it exhibited a previously unrecognized localization pattern. At 6 to 9 h postinfection (hpi), UL32 accumulated in viral replication compartments in the nucleus of the host cell, while at 24 hpi, it was additionally found in the cytoplasm. A newly generated UL32-null mutant was used to confirm that although B capsids containing wild-type levels of capsid proteins were synthesized, these procapsids were unable to initiate the encapsidation process. Furthermore, we showed that UL32 is redox sensitive and identified two highly conserved oxidoreductase-like C-X-X-C motifs that are essential for protein function. In addition, the disulfide bond profiles of the viral proteins UL6, UL25, and VP19C and the viral protease, VP24, were altered in the absence of UL32, suggesting that UL32 may act to modulate disulfide bond formation during procapsid assembly and maturation.
  
  
  OBJECTIVE
  Although functions have been assigned to six of the seven required packaging proteins of HSV, the role of UL32 in encapsidation has remained a mystery. UL32 is a cysteine-rich viral protein that contains C-X-X-C motifs reminiscent of those in proteins that participate in the regulation of disulfide bond formation. We have previously demonstrated that disulfide bonds are required for the formation and stability of the viral capsids and are also important for the formation and stability of the UL6 portal ring. In this report, we demonstrate that the disulfide bond profiles of the viral proteins UL6, UL25, and VP19C and the viral protease, VP24, are altered in cells infected with a newly isolated UL32-null mutant virus, suggesting that UL32 acts as a chaperone capable of modulating disulfide bond formation. Furthermore, these results suggest that proper regulation of disulfide bonds is essential for initiating encapsidation.
authors.affiliation: Department of Molecular Biology and Biophysics and the Molecular Biology and Biochemistry Graduate Program, University of Connecticut Health Center, Farmington, Connecticut, USA.
authors.forename: Brandon S
authors.initials: BS
authors.lastname: Albright
authors.affiliation: Department of Molecular Biology and Biophysics and the Molecular Biology and Biochemistry Graduate Program, University of Connecticut Health Center, Farmington, Connecticut, USA.
authors.forename: Athena
authors.initials: A
authors.lastname: Kosinski
authors.affiliation: Department of Molecular Biology and Biophysics and the Molecular Biology and Biochemistry Graduate Program, University of Connecticut Health Center, Farmington, Connecticut, USA.
authors.forename: Renata
authors.initials: R
authors.lastname: Szczepaniak
authors.affiliation: MRC-University of Glasgow Centre for Virus Research (CVR), Glasgow, United Kingdom.
authors.forename: Elizabeth A
authors.initials: EA
authors.lastname: Cook
authors.affiliation: MRC-University of Glasgow Centre for Virus Research (CVR), Glasgow, United Kingdom.
authors.forename: Nigel D
authors.initials: ND
authors.lastname: Stow
authors.affiliation: Department of Structural Biology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA.
authors.forename: James F
authors.initials: JF
authors.lastname: Conway
authors.affiliation: Department of Molecular Biology and Biophysics and the Molecular Biology and Biochemistry Graduate Program, University of Connecticut Health Center, Farmington, Connecticut, USA weller@nso2.uchc.edu.
authors.forename: Sandra K
authors.identifier: http://orcid.org/0000-0002-4519-6276
authors.initials: SK
authors.lastname: Weller
mesh_terms: D000818:Animals; D002522:Chlorocebus aethiops; D004220:Disulfides / Q000378:metabolism*; D017353:Gene Deletion; D020869:Gene Expression Profiling; D018259:Herpesvirus 1, Human / Q000235:genetics / Q000502:physiology*; D014709:Vero Cells; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*; D019065:Virus Assembly
chemical_list: D004220:Disulfides; D014764:Viral Proteins; C069185:DNA cleavage and packaging proteins, Herpesvirus