title: Fluorescent tagging and cellular distribution of the Kaposi's sarcoma-associated herpesvirus ORF45 tegument protein.
abstract: Kaposi's sarcoma-associated herpesvirus (KSHV), also known as human herpesvirus 8 (HHV-8), is a cancer-related human virus, classified as a member of the Gammaherpesvirinae subfamily. We report here the construction of a dual fluorescent-tagged KSHV genome (BAC16-mCherry-ORF45), which constitutively expresses green fluorescent protein (GFP) and contains the tegument multifunctional ORF45 protein as a fusion protein with monomeric Cherry fluorescent protein (mCherry). We confirmed that this virus is properly expressed and correctly replicates and that the mCherry-ORF45 protein is incorporated into the virions. Using this labeled virus, we describe the dynamics of mCherry-ORF45 expression and localization in newly infected cells as well as in latently infected cells undergoing lytic induction and show that mCherry can be used to monitor cells undergoing the lytic viral cycle. This virus is likely to enable future studies monitoring the dynamics of viral trafficking and tegumentation during viral ingress and egress.
  
  
  OBJECTIVE
  The present study describes the construction and characterization of a new recombinant KSHV genome BAC16 clone which expresses mCherry-tagged ORF45. This virus enables the tracking of cells undergoing lytic infection and can be used to address issues related to the trafficking and maturation pathways of KSHV virions.
authors.affiliation: Mina and Everard Goodman Faculty of Life Sciences, Bar Ilan University, Ramat-Gan, Israel.
authors.forename: Shir
authors.initials: S
authors.lastname: Bergson
authors.affiliation: Mina and Everard Goodman Faculty of Life Sciences, Bar Ilan University, Ramat-Gan, Israel.
authors.forename: Inna
authors.initials: I
authors.lastname: Kalt
authors.affiliation: Mina and Everard Goodman Faculty of Life Sciences, Bar Ilan University, Ramat-Gan, Israel.
authors.forename: Inbal
authors.initials: I
authors.lastname: Itzhak
authors.affiliation: Department of Molecular Microbiology and Immunology, Keck School of Medicine, University of Southern California, Los Angeles, California, USA.
authors.forename: Kevin F
authors.initials: KF
authors.lastname: Brulois
authors.affiliation: Department of Molecular Microbiology and Immunology, Keck School of Medicine, University of Southern California, Los Angeles, California, USA.
authors.forename: Jae U
authors.initials: JU
authors.lastname: Jung
authors.affiliation: Mina and Everard Goodman Faculty of Life Sciences, Bar Ilan University, Ramat-Gan, Israel saridr@mail.biu.ac.il.
authors.forename: Ronit
authors.initials: R
authors.lastname: Sarid
mesh_terms: D019677:Artificial Gene Fusion; D002460:Cell Line; D003600:Cytosol / Q000737:chemistry* / Q000821:virology*; D020869:Gene Expression Profiling; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000032:analysis / Q000235:genetics; D019288:Herpesvirus 8, Human / Q000502:physiology*; D006801:Humans; D017874:Immediate-Early Proteins / Q000032:analysis* / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000235:genetics; D013194:Staining and Labeling / Q000379:methods; D017735:Virus Latency; D014779:Virus Replication
chemical_list: D017874:Immediate-Early Proteins; C456286:ORF45 protein, Kaposi's sarcoma-associated herpesvirus; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins