title: Live visualization of herpes simplex virus type 1 compartment dynamics.
abstract: We have constructed a recombinant herpes simplex virus type 1 (HSV-1) that simultaneously encodes selected structural proteins from all three virion compartments-capsid, tegument, and envelope-fused with autofluorescent proteins. This triple-fluorescent recombinant, rHSV-RYC, was replication competent, albeit with delayed kinetics, incorporated the fusion proteins into all three virion compartments, and was comparable to wild-type HSV-1 at the ultrastructural level. The VP26 capsid fusion protein (monomeric red fluorescent protein [mRFP]-VP26) was first observed throughout the nucleus and later accumulated in viral replication compartments. In the course of infection, mRFP-VP26 formed small foci in the periphery of the replication compartments that expanded and coalesced over time into much larger foci. The envelope glycoprotein H (gH) fusion protein (enhanced yellow fluorescent protein [EYFP]-gH) was first observed accumulating in a vesicular pattern in the cytoplasm and was then incorporated primarily into the nuclear membrane. The VP16 tegument fusion protein (VP16-enhanced cyan fluorescent protein [ECFP]) was first observed in a diffuse nuclear pattern and then accumulated in viral replication compartments. In addition, it also formed small foci in the periphery of the replication compartments which, however, did not colocalize with the small mRFP-VP26 foci. Later, VP16-ECFP was redistributed out of the nucleus into the cytoplasm, where it accumulated in vesicular foci and in perinuclear clusters reminiscent of the Golgi apparatus. Late in infection, mRFP-VP26, EYFP-gH, and VP16-ECFP were found colocalizing in dots at the plasma membrane, possibly representing mature progeny virus. In summary, this study provides new insights into the dynamics of compartmentalization and interaction among capsid, tegument, and envelope proteins. Similar strategies can also be applied to assess other dynamic events in the virus life cycle, such as entry and trafficking.
authors.affiliation: Institute of Virology, University of Zurich, Winterthurerstrasse 266a, CH-8057 Zurich, Switzerland.
authors.forename: Anna Paula
authors.initials: AP
authors.lastname: de Oliveira
authors.forename: Daniel L
authors.initials: DL
authors.lastname: Glauser
authors.forename: Andrea S
authors.initials: AS
authors.lastname: Laimbacher
authors.forename: Regina
authors.initials: R
authors.lastname: Strasser
authors.forename: Elisabeth M
authors.initials: EM
authors.lastname: Schraner
authors.forename: Peter
authors.initials: P
authors.lastname: Wild
authors.forename: Urs
authors.initials: U
authors.lastname: Ziegler
authors.forename: Xandra O
authors.initials: XO
authors.lastname: Breakefield
authors.forename: Mathias
authors.initials: M
authors.lastname: Ackermann
authors.forename: Cornel
authors.initials: C
authors.lastname: Fraefel
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D002460:Cell Line; D002462:Cell Membrane / Q000737:chemistry; D002467:Cell Nucleus / Q000737:chemistry; D002522:Chlorocebus aethiops; D006224:Cricetinae; D003593:Cytoplasm / Q000737:chemistry; D005455:Fluorescent Antibody Technique; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D018259:Herpesvirus 1, Human / Q000235:genetics / Q000254:growth & development*; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D009685:Nuclear Envelope / Q000737:chemistry; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D014764:Viral Proteins / Q000096:biosynthesis* / Q000235:genetics; D000097573:Red Fluorescent Protein
chemical_list: D001426:Bacterial Proteins; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; D000097573:Red Fluorescent Protein; C527701:enhanced cyan fluorescent protein; C054562:yellow fluorescent protein, Bacteria